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Charvin Smocks
…in this smock! Look stylish and keep your clothes free of wayward paint in these new and stylish linen artist smocks from Charvin. Made from comfortable and breathable linen, these finely made artist smocks are an excellent choice to keep your clothes clean while painting or sculpting. Each smock…
Artist Apron Design No 37
Made of comfortable lightweight denim fabric, this machine washable apron has two large front pockets for holding paint brushes and other gear. This apron is knee-length and has an unique adjustable neck strap and drawstring waist. Affordable too! One size fits all.
First Impressions Kids Art Smock And Brush Set
Kids Art Smock - Cleanliness and creativity in one convenient package! This handy Kids Art Smock and Brush set is the perfect introduction to art tools for your own petite Picasso! With a child-size, water-resistant nylon smock with pockets and 3 quality assorted paint brushes, your young artist…
Wipe-Tex Plastic Aprons
Perfect for Painting Parties and Groups Wipe-Tex Disposable Plastic Aprons can protect clothing and skin from paints and inks such as oils, acrylic and most other media. Made of nonporous 1.25 ml white plastic, these aprons are great for painting groups, workshops, and painting parties. At 28"…
Faber-Castell Young Artist Smock
…child clean with an all-covering smock with sleeves Does your child love art? Perfectly sized for preschoolers, this young artist smock features long sleeves and waterproof nylon fabric. This makes it perfect for even the messiest of art projects. This child's smock also has a hook and loop in the…


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Banded Axe Blind Leg Kit, 4-Pack
Prices, promotions, styles and availability may vary by store and online.
Converts your boat blind into a shore blind
Holds to mud, sand, gravel, or hard ground
Adjusts from the ground level up to 15"
The Axe Blind Leg Kit from Banded is specifically designed to convert your Axe Combo Boat Blind into a Shore Blind. The leg feet attach easily in seconds and securely hold each corner of your blind in mud, sand, gravel, or hard ground. In addition, the legs can be adjusted from ground level to 15" in height, making it perfect for dry ground, shorelines, and even shallow water. Each kit includes four legs.

subset: commoncrawl


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Brighton & Hove Chamber of Commerce
British Parking Association
Elwood Wines
Grassroots Suicide Prevention
Historic Jewellery Reproductions
Lewes Old Grammar School
MIME Consulting
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Outstanding Cufflink Designs
Scouts Association/Scout Shops
Sussex Payroll Services
Tennyson Suite
The Gym Group
The Wellbeing Practice
Wandsworth Children's Uni
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Logo and branding, marketing collateral, website design
We were delighted to be asked to design and produce a logo and branding for local music and arts festival Seaford Live!, as well as producing a website and various marketing collateral such as printed posters, leaflets, banners and signage. The resulting logo design and branding has become recogniseable over the last six years throughout the local community and across the south east with this successful and much loved festival. We also helped the festival trademark their logo to help protect the brand as it grows each year.
We designed a simple functional website with a simple, updateable layout to keep visitors informed of latest nes, booked artists and information on the organisation itself.
We have also designed a number of printed posters, leaflets, programmes, banners and signage in line with their branding and that help to promote the festival each year.


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- Social:
Oprah Winfrey & George C. Wolfe: The Immortal Life of Henrietta Lacks Actress Oprah Winfrey and director George C. Wolfe join Elvis Mitchell to discuss The Immortal Life of Henrietta Lacks.
Bassem Youssef and Sara Taksler on 'Tickling Giants' Known as the "Jon Stewart of Egypt," Bassem Youssef hosted a satirical news show that was the first of its kind in the Middle East. The show was immensely popular, until the military-backed government forced Youssef off the air and out of the country. Youssef and director Sara Taksler tell us about their documentary Tickling Giants, which profiles Youssef's leap from heart surgeon to super star satirist.
In 'Free Fire,' Ben Wheatley wants to "meet the audience halfway" British filmmaker Ben Wheatley has built up a cult following with his hyper-violent, darkly funny movies. His newest film Free Fire is an action comedy starring Brie Larson, Armie Hammer, and a whole lot of guns. The movie has the broadest commercial appeal of any of his work to date, but it's still a Ben Wheatley film, which means, spoiler alert...a lot of people die.
North Korea tests more missiles, Turkey's president gains more power Early Tuesday morning, North Korea tested another intercontinental ballistic missile. It blew up shortly after take-off. But North Korea keeps working on a nuclear missile that could reach the U.S. Also in Turkey, a close vote has given sweeping new powers to President Recep Tayyip Erdoğan. Turkey is an important Western ally in the region, but its leader is becoming increasingly authoritarian.


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Attention Vancouver hackers!
Shopify will be attending Hack Vancouver on August 20th as an API provider. If you're a developer in the area I highly recommend signing up, tickets are $10 which is worth it just for the food that's laid on (Breakfast + lunch), not to mention getting to hack on/mash up a host of brilliant APIs. So far the list of providers looks like this:
Representing Shopify will be myself and Joey deVilla, who will be rocking his accordion as always. We're bringing prizes and swag so there's really no reason not to come.
I've attended two of these events in the past (Ottawa and Waterloo) as a participant and had a fantastic time at both events. Now I get to see things from the other side of the fence and I'm really excited. If you've not been to a HackDay before, I recommend reading this article that I wrote after Hack Waterloo: How To Write An App In 8 Hours (Or Die Trying)


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Gross domestic product is a monetary measure of the value of all final goods and services produced in a period. Nominal GDP estimates are commonly used to determine the economic performance of a whole country or region, and to make international comparisons. Nominal GDP, however, does not reflect differences in the cost of living and the inflation rates of the countries; therefore using a GDP PPP per capita basis is arguably more useful when comparing differences in living standards between nations.
Modern economies are run by complex systems of formulas and methods. Since the birth of the Industrial Revolution, countries throughout the world have been turning to one particular means of measuring their overall economic growth.
This measurement has most famous come in the form of the Gross Domestic Product (GDP). A nation's Gross Domestic Product is simple the monetary value of all the goods and services it produces within its borders in a defined period of time. Most often, GDP is measured by "quarters" in the business world, and annually on a national level.
For a government, it is important to measure one's own government outlays, private and public consumption, investments, and amount of exports incurred on a yearly basis.
By using the formula GDP = C + G + I + NX, any person may calculate the Gross Domestic Product of a country or another situation that they desire.
Without this formula, or the measure of GDP itself, all nations throughout the world would be unable to indicate the economic health of their country. In almost every single major survey or evaluation used, Gross Domestic Product is used to measure the standard of living within a nation's borders. These results are fairly accurate, as the measure of GDP is uniform from country to country.
Besides from evaluating the standard of living, Gross Domestic Product measurements are also sure ways to discover the production rates for a nation during any given year. These evaluations are highly accurate, and can even be adjusted to current inflation levels to compare current GDP years with those of the past. This allows for GDP's to be compared by percentages to any given quarter or period of time.
Recessions are often discovered using the measurement that a GDP provides. By comparing a succession of years, or another similar long span length of time, a nation's level of economy and productivity can be ascertained. Those nations whose comparisons show a increasingly developed downward slope, or stagnation, are most likely entering into a period of recession.
Gross Domestic Products do not come without their critics, however.
Many critics state that a measure of GDP only takes official statistics into account, and never any unofficial, or "underground", economy that might be taking place within a nation's borders. These unofficial economies can be in the form of black-markets, under-the-table employments, but also any transactions that might take place without the knowledge of the government itself (such as selling an item online). Ever since GDP first came into use in the year 1937, it has been an integral part of our modern system of economics. Although it has had its critics and its skepticisms, the measure of Gross Domestic Product as an official evaluation of a nation's overall standard of living and production rates is here to stay. With innovations each year taken by governments throughout the world, the measure of a country's Gross Domestic Product has increased in accuracy and specificity.
Financial education is so important, but barely taught at all in our schools. Having resources online is great, but not if they are inaccessible to so many. Thanks 508!


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|"Dennis E. Hamilton" <orc...@apache.org>
|RE: Volunteers needed to pickup some tasks
|Sat, 08 Sep 2012 17:10:07 GMT
I will soon need to be replaced as a moderator on ooo-users@ and ooo-private@ (requires [P]PMC member) as I unwind activities that require daily attention. So two volunteers would be good for those list. Then there can be overlap while they get the hang of it. - Dennis -----Original Message----- From: Rob Weir [mailto:email@example.com] Sent: Friday, September 07, 2012 22:51 To: email@example.com Subject: Volunteers needed to pick up some tasks [ ... ] 1. List moderation. I intend to stop moderating dev, users, marketing, qa and private. [ ... ]
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To try the thousands of add-ons available here, download Mozilla Firefox, a fast, free way to surf the Web!Close
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Choose from thousands of extra features and styles to make Firefox your own.Close
Reviews for Whirling Leaves & Trees
2 reviews for this add-on
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A nice impression of a landscape from an abstract image, and the leaves add impact. Well done! It may be worth trying this with fall-colored leaves, which should be easy to convert.
To create your own collections, you must have a Mozilla Add-ons account.


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Eren yeager an 18 year old his dad sold him after doing alot of experiments then was found by hanji he lived with hanji for a long time he was her maid always cleaning until it was clearly sparkling Hanji loves her maid eren because he has a good personality and is good at cooking,cleaning, and is mostly doing what she says also tries on anything hanji tells him to wear
Levi Ackerman a boss of a really famous company, erwin and hanji, his friends since elementary until hanji asked him to come to her house for dinner levi nods his head prepared to go in a house that is really filthy what happens when he finds out the house is really clean? What happens when hanji calls her favorite brunette? What will hanji plan to make her ship sail? And the biggest question of all will levi and eren fall in love?
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No need to apologize it's a cute ship, kinda looks steampunk
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RiggerDom Jan 15
Ciel: Earl grey?! *peeks head out of bedroom* Sebastian: Don't worry, my lord, it's most likely not as good as what I would make for you.


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I did a review on the Hemingway Classic a little while ago...the reason I bring that up is because the 8-5-8 reminded me of it. Even though the 8-5-8 is not as exquisite as the Hemingway series, it is still an excellent cigar. I loved everything about it, so much in fact, that I am looking forward to buying myself a box...along with a box of Hemingways. It's burn is very good, with a smooth draw, and holds together nicely. The taste is very nutty, with a hint of pepper. The left over aroma is very annoying...only because it gives you the urge to open that humidor and grab another.
Used to smoke 858's on a fairly regular basis. Not any more! They are wrapped to tight with a poor draw which makes them burn hot. Will not waste money on them again


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Leafs Lounge V104: A great man once said, FIRE BURN THE ICE.
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LL Genius
Join Date: Jan 2012
Location: London
Posts: 14,157
vCash: 50
Originally Posted by
I just got home from a night out where I spent from 1am to 3:30am with two homeless elderly men looking for something to eat. Neither of them smoke, drink, or do illegal drugs, and were part of a london radio show in the past. I spent about 110$ on random food articles for them to put in their fridges, and when they hugged me goodbye and walked away I cried like a little baby. Saddest thing in the world watching these old men waddling away from me on their way to find a warm place to sleep. And then we have useless artists like Lil Wayne, 2 Chainz and Birdman buying 1,800,000$ Bugatti's so that they can look good on their way to the liquor store. Today's society is so based on popularity we forget that there are people in need of help and love in the world. Just imagine somebody you love losing their mind and having to sleep under newspaper in an alley. I had an eye opening experience tonight, and I honestly couldn't care less about my past. Can we please find a way to help those people / animals in need instead of wasting our mental ability on **** that doesn't mean a single thing? Tonight is the best night I've ever had and I didn't even talk to a single attractive girl. YOLO?! SO DO THEY. Spread your love, forgive and forget. ♥ Wishing you all a happy, comfortable Saturday. Please try to at least research something where somebody or something needs your help. Goodnight friends, much love.
That's one of my friends' status' from last night. Wow.
I read all of that thinking it was you who did it. Then I got to the end
Reminds me that I should start looking/thinking about where I want to volunteer this summer. Haven't in the past, but usually I don't get as many work hours as I would like so I figure this year I will try and do something useful. Need it for my resume as well... My resume isn't looking so great
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Winter 2016 - Volume: 22 Issue: 4
Audri Beugelsdijk, Surviving spouse of Navy Seaman Jason Clay Springer
Coping with grief and loss is one of life's greatest challenges. Sometimes the task is so monumental that it feels impossible, and yet here we are doing the impossible, surviving the loss of someone who is inextricably a part of our heart, existence and routine. During the holidays, the loss is illuminated as we witness people making plans with their families, buying gifts, or engaging in long-standing traditions. In the midst of millions of people giving thanks for another year with their families, we are still left with the reality that this holiday will be different for us, if we observe it at all.
Some of us have endured this season without our loved one for many years and have developed strategies for coping through this time. For others, this will be our first holiday without the person who has always been an integral part of our holiday memories. Some may feel lost or struggle with indecision about how to mark the season. The change being thrust upon you can be disorienting and can create inner conflict as we may have competing demands - our own need to reflect and find how this loss has changed the way we do holidays, and the needs others have to engage with them at a time of year when people come together to celebrate. This can be difficult to balance, especially when "celebration" is not in our current vocabulary. The conflicting needs or demands, along with our own holiday disorientation, can lead to a need for reflection. Taking time for solitude may be the best gift you give yourself this year.
When we are grieving, there is often concern when we choose to be alone. Family, friends and others may be concerned this is unhealthy, especially at the holidays - and yet there may be times when you feel the need to have solitude as a respite from the bustling activity of the season. Solitude can offer a time of reflection that allows us to explore our thoughts and synthesize information, provides a time for rest, and can allow for growth as we still ourselves. While solitude can be refreshing, too much time alone can lead to social isolation and feelings of loneliness. When we become isolated, the walls can begin to close in on us, feeling more like a prison as we avoid others.
Like many things in life, striking a balance is important. Whether choosing a temporary period of solitude or struggling with isolation, reflections are likely to cause a mix of emotions as we engage memories of our loved one and "feel the feels." While solitude may be preferable for certain periods, surrounding ourselves at various times with others can provide a different kind of respite from our grief. Engaging with family, friends, coworkers and neighbors can bring feelings of social connectedness, which can yield a greater sense of well-being and have positive health effects, all of which are important to offset the effects of grief on our minds and bodies.
If your time of quiet solitude has turned to social avoidance, feelings of loneliness and isolation, and is draining you of energy instead of restoring you, it is time to reach out for support. Reach out to the person or people who have been helpful and encouraging during this season of your life. If it is difficult to identify who that might be, there are actions you can take to develop helpful supports for your journey: join a care group in your local community, reach out to your faith community, re-engage with others in an activity you have set aside, explore ways to honor your loved one during the holiday season, and reach out to your TAPS family. The Survivor Care Team is always here to help you explore your supports and encourage you on your journey.
This article is copyrighted by the Tragedy Assistance Program for Survivors (TAPS). This article may not be reproduced in whole or in part by any means, electronic or otherwise, without prior written approval. It is permissible for an individual reader to view, reproduce or store a copy of this article, provided it is used only for their own personal and non-commercial use. Uses beyond that allowed by the "Fair Use" limitations (sections 107 and 108) of the U.S. Copyright law require permission from TAPS. Please contact email@example.com to request permission. All other rights reserved.
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By Laura Morrison, July 2014
Summer is drawing to a close, and for soon-to-be students everywhere that means one thing: back-to-school time is here. Not even graduate students are immune to the frantic shopping that seems to permeate the air at this time of year. Indeed, it's more important than ever that future graduates pay close attention to their shopping lists and habits, as budget should be a primary concern. There are a few things to keep in mind so that you're able to make the grade without breaking the bank.
Know the basics...
The key to a successful and financially friendly shopping experience is knowing exactly what you're going to need in advance. Making a list and doing some cursory research with different retailers online can give you a rough idea of what you can expect to spend, which is a huge help for those adhering to a strict budget. Every program is different, but in general, there are a few staples each student might be interesting in purchasing in advance of the first day of class: Pens, notebooks, Post-it notes and even a laptop or tablet are all practically required for keeping up with notes and reading. Get a handle on what you absolutely need, and try not to get side-tracked by the extraneous things that may jump out at you as you peruse the aisles.
...But don't forget the finishing touches
Of course, graduate school is about more than hitting the books. You're there to form relationships, make connections and network as much as you are to learn. This gives new meaning to the idea of the back-to-school wardrobe. For some fields - MBA candidates, for example – this may be of particular importance. But in general, you should make sure you're able to look professional, in case you ever find yourself in a situation that demands it. According to Experience, one cardinal sin of prospective graduate students is not realizing just how demanding their new studies can be on their wardrobes. From research fellowships to job interviews, you never know when you'll need to break out a power suit.
Become a hunter - of bargains
Regardless of what field your graduate studies are in, when it's time to shop for back to school, everyone is a hunter. Scoping out deals, promotions and sales is one of your major priorities in this endeavor, as anywhere you can save a couple extra bucks will add up down the road. As Fastweb pointed out, there are many helpful tools to make use of, from price comparison apps for your smartphone to good old-fashioned coupon clipping.
About the Author: Laura Morrison is the Web Content Manager for GradSchools.com. She earned an MBA from the Rutgers School of Business in 2010.


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Future Unknown
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Scaffolds printed in 3d
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wires connecting buildings
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Augmented reality on a mobile phone
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Long Exposure of a girl dancing
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Light Trails as Vehicles Communicate
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Studio lit still life of a rat with woodpecker's wings.
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Digital Image
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man designing a car using augmented reality
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text:
The Scottish champions have tried to turn up the psychological pressure on the other half of the Old Firm by ensuring a helicopter is kept on stand-by today to whisk them back to Glasgow if they beat Dundee United and Celtic lose, a combination of results which which would present Rangers with a record 10th title in a row.
Ibrox is playing host to 32,000 fans watching events from Tannadice unfold on giant screens and one-upmanship has enticed them into their bold aerial gesture.
However, Gould, whose meagre total of just 24 goals conceded has played a crucial part in Celtic being in the brink of their first title since 1988, believes that a victory for the Premier Division leaders against St Johnstone at Parkhead will bring their eternal rivals crashing back to earth.
"You have to be very careful when you do things the way Rangers have this week," bristled Gould, leaving no doubt that it has supplied extra motivation, if any more were needed. "Put it this way. I would be pretty disappointed if I didn't have a championship medal by Saturday night."
More than 52,000 fans will pack Parkhead to see if Wim Jansen's team can protect their two-point advantage over Rangers. The Dutch coach remains unfazed by all the hype, saying: "We know what we have to do. We don't have to rely on anyone else, only ourselves."
Rangers believed their championship ambitions were dead after they lost to Kilmarnock last week until Dunfermline offered them a reprieve 24 hours later. Now they are desperate to take advantage.
Walter Smith has a string of injury problems for his final league match in charge of the club, including Jonas Thern and Andy Goram who have hamstring injuries. Smith said: "Celtic have the destiny of the title in their hands. But this has been a strange season. All we can hope for is a result and await the outcome of Celtic's game."
Kilmarnock will clinch the last remaining Uefa cup place if they defeat Hibernian at home and finish in fourth place. But St Johnstone, who are a point behind, have plenty of incentive to spoil the Parkhead party and replace it with one of their own. Hearts, already assured of a place in Europe, will rest some players ahead of next week's Scottish Cup final, for their home match with Dunfermline, while Motherwell will bring down the curtain at home to Aberdeen.
- More about:
- Celtic F.c.
- Kilmarnock F.c.
- Motherwell F.c.
- Rangers Fc


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There's a little storm coming my way, so I'm posting this a bit early. It's hard telling when the electricity will go out. Hopefully this little storm will pass right by my area and not hit us, but if it does, since it is so weak, hopefully it won't take out the power. Just in case, though, here y'all go:
This one is slow-paced and pretty direct. It might take a particularly slow person to figure out who is doing what, but it's actually very straightforward. Normally I love Asian horror movies because they tend to do creepy very, very well. I can't say that this one was creepy at all, though. It was really more of a tragedy than a horror.
It is the prequel to Carved: Slit-Mouthed Woman. There wasn't much similarity between the two movies, though. The predecessor was a supernatural tale. This is a mix of true story and urban legend surrounding the true story. Many of the events did occur, some were creative license.
Still, the story is very emotional and while slow, it never lost my interest. I've seen better, but I have most definitely seen much worse. I give this movie 5/10 kupos.
Since October is also Adopt a Shelter Dog Month, I am begging people to go to this link http://www.shelterchallenge.com/ and vote for Tangi Humane Society Hammond LA 70401. They really, really need the money. Please vote every day until the contest ends (the 14th, I believe) to help.


text: More pictures of Matt Bomer please! * big smile * Even hidden under a cap and aviator glasses, I can still see his sexiness shine through. I can't get enough of this gorgeous talented man. Get more pictures of this man when he's out on celebrity events or filming because I just love, love, love Matt Bomer


text:
The rich have become much richer and the poor relatively poorer - 40 per cent of British people exist on income levels below the average wage - while directors of public utility companies shamelessly utilise what were public assets for private gain. Despite promises to the contrary, there have been 22 increases in taxation, public debt has doubled and public facilities in education, hospitals and housing are falling to new levels of insufficiency. The Government's claim to be the party of efficient economic management is exposed for the charade that it is.
Government inefficiencies, ministerial mistakes and political scandals - such as the ERM debacle, the Pergau Dam, defence sales to Iran and Iraq, mad cow disease, Gulf war syndrome and cash-for-questions - combine to show a lack of concern for morality, justice and humanity in public life. It seems incapable of recognising that the systems of government evolved to manage a hierarchical 19th-century imperial power are wholly inappropriate for the management of a modern, participative industrial democracy.
By weakening local government, refusing to accept limited devolution in Scotland and Wales, and failing to recognise the diminished power of the nation state within an increasingly interdependent world economy, the Government has forfeited any claim to be capable of leading the UK into the 21st century.
Unrestrained by a largely irrelevant and unrepresentative Parliament, individual ministers have ceased to recognise or serve the public interest. Yet they remain determined to hang on to office at any price. Their cynical contempt for the public has contributed in no small measure to the general culture of selfishness and disrespect which exists in Britain today. It really is time for a change.
Although they may win some seats in the South-west, the Liberal Democrats cannot defeat the Government in a general election. In any event, the public good requires opinion formers across the political spectrum to vote for change. Only new Labour has the combination of moral purpose and potential electoral support nationally to form a government committed to change.
Under Tony Blair's leadership the Labour Party has the vision to implement the reforms necessary to secure a prosperous future for all our people. My decision to join the Labour Party is prompted by Tony Blair's determination to pursue the constitutional reform, efficient economic management and fairer social provision that alone can restore One Nation.
Christopher Brocklebank-Fowler
- More about:
- Iraq
- Liberal Democrat Party
- Management
- Morality


text:
By: Michael Kaplan, author of "7 Pillars of an Unshakable Christian Faith"
The path of Christian discipleship is not an easy one; Jesus made that clear to the multitudes and His disciples. He did teach, however, that the reward of Eternal Life in the presence of God the Father made the cost worthwhile.
As you prepare to pursue your own path - as one who disciples, or one who is being discipled – there are steps you can take to overcome many common obstacles you'll encounter during the journey. These seven practices apply to all Christian disciples, in any capacity they serve.
1. Share the Gospel at every opportunity: Sharing the Gospel of Christ is the hallmark of making disciples, being a disciple, and attracting potential disciples. It fulfills the command of Scripture, increase the faith of those sharing the message, and shows by example your willingness to be a leader in your faith.
Preach the word; be ready in season and
out of season; reprove, rebuke, exhort,
with great patience and instruction.
~ 2 Timothy 4:2 ~
2. Practice radical obedience to Christ: The Scripture is your instruction manual to a life centered around Christ; read it, use it, and follow it. A disciple isn't a believer who shows up to church once a week and puts faith on like a sweater when it's needed. Radical obedience to Christ guides your actions from the time you wake up until the time you go to bed for the evening. It shows God you take Him as seriously as He takes Himself.
Let your light shine before men in such a
way that they may see your good works,
and glorify your Father who is in heaven.
~ Matthew 5:16 ~
3. Live like Christ in word and deed: Jesus taught His disciples to follow Him meant to practice radical purity, love, sacrifice and obedience. That's not something a disciple can fake; it requires an inner change, a change of the heart. The indwelling of the Holy Spirit works in the believer as a progressive process of Sanctification, but actively imitating Christ – as did the apostle Paul – helps to guide and shape the transformation.
Imitate me, just as I also imitate Christ. Now
I praise you, brethren, that you remember
me in all things and keep the traditions
just as I delivered them to you.
~ 1 Corinthians 11:1-2 ~
4. Practice spiritual disciplines daily: Nothing in the human experience is accomplished without training and practice. The same holds true for spiritual disciplines. They help to develop the believer's new inner being, and support the transformation that accompanies Salvation in Christ. Each of these disciplines – such as Abstinence, Confession, Fasting, Fellowship, Prayer, Sacrifice, Service, Study, and Worship – are found in Scripture, and were practiced by Jesus and His disciples.
For the Spirit God gave us does
not make us timid, but gives us
power, love and self-discipline.
~ 2 Timothy 1:7 ~
5. Serve as the standard, model, and example: Credible testimony happens when a believers words of faith match their deeds in the world. Excellent leaders and teachers like Jesus lead from the front; they set the standard, model the action, and serve as an example to those who follow. By the end of His earthly ministry, Jesus' disciples trusted that He was the Christ, and therefore believed in Him. That level of trust – the type of trust for which a person would give their life – doesn't come from words or doctrine. It comes from showing and doing. Rule #1: set the standard even when no one is watching. Rule #2: someone is always watching – see Rule #1.
Remember your leaders, who spoke the word
of God to you. Consider the outcome of
their way of life and imitate their faith.
~ Hebrews 13:7 ~
6. Be a humble servant, prepared to serve: Jesus made His mission clear to His disciples: "For even the Son of Man did not come to be served, but to serve, and to give His life a ransom for many" (Mark 10:45). In any field, the best leaders began as excellent followers; their leadership style reflects their commitment to serve. Jesus gave God the Father glory for everything, and kept no glory for Himself. A faithful disciple of Christ does the same.
Seek the Lord, all you humble of the land, you who
do what he commands. Seek righteousness, seek humility;
perhaps you will be sheltered on the day of the Lord's anger.
~ Zephaniah 2:3 ~
7. Practice full transparency and accountability: During Jesus' earthly ministry, He had nothing to hide, nothing to hold back, from His disciples. A faithful disciple of Christ cannot be double-minded, engaged in sin in the darkness, while practicing faith in the light. That type of duality leads to fear, guilt and weakness. Every believer will sin; that's a certainty. Once those sins are confessed they are removed, the slate is wiped clean, and a clear conscious is left in its place. Habitual and intentional sin, however, cannot be hidden forever, nor should it be. Transparency and accountability builds the level of trust needed for others to believe in the Gospel of Christ, and to follow it as Jesus' disciples did: without questioning, wavering or quitting under pressure.
Everyone who does evil hates the light, and
will not come into the light for fear that their
deeds will be exposed. But whoever lives by
the truth comes into the light, so that it may
be seen plainly that what they have done
has been done in the sight of God.
~ John 3:20-21 ~
I pray that the Holy Spirit guides you in your studies, and brings you to a more intimate relationship with Jesus Christ. Be Blessed.


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Simultaneous silencing of VEGF and KSP by siRNA cocktail inhibits proliferation and induces apoptosis of hepatocellular carcinoma Hep3B cells
© Doan et al.; licensee BioMed Central. 2014
Received: 10 September 2014
Accepted: 6 December 2014
Published: 15 December 2014
Vascular endothelial growth factor (VEGF) is involved in the growth of new blood vessels that feed tumors and kinesin spindle protein (KSP) plays a critical role in mitosis involving in cell proliferation. Simultaneous silencing of VEGF and KSP, an attractive and viable approach in cancer, leads on restricting cancer progression. The purpose of this study is to examine the therapeutic potential of dual gene targeted siRNA cocktail on human hepatocellular carcinoma Hep3B cells.
The predesigned siRNAs could inhibit VEGF and KSP at mRNA level. siRNA cocktail showed a further downregulation on KSP mRNA and protein levels compared to KSP-siRNA or VEGF-siRNA, but not on VEGF expression. It also exhibited greater suppression on cell proliferation as well as cell migration or invasion capabilities and induction of apoptosis in Hep3B cells than single siRNA simultaneously. This could be explained by the significant downregulation of Cyclin D1, Bcl-2 and Survivin. However, no sigificant difference in the mRNA and protein levels of ANG2, involving inhibition of angiogenesis was found in HUVECs cultured with supernatant of Hep3B cells treated with siRNA cocktail, compared to that of VEGF-siRNA.
Silencing of VEGF and KSP plays a key role in inhibiting cell proliferation, migration, invasion and inducing apoptosis of Hep3B cells. Simultaneous silencing of VEGF and KSP using siRNA cocktail yields promising results for eradicating hepatocellular carcinoma cells, a new direction for liver cancer treatment.
KeywordsVascular endothelial cell growth factor (VEGF) Kinesin spindle protein (KSP) siRNA cocktail Proliferation Apoptosis Hepatocellular carcinoma
Primary liver cancer, hepatoblastoma (HB) and hepatocellular carcinoma (HCC), is one of the most common solid tumors, ranking the fifth in most common malignancy worldwide and the second cause of cancer-related deaths. The major therapeutic strategies in solid tumors as well as HCC are excision of the primary tumor, followed by radiotherapy and chemotherapy. However, in some cases, this treatment still leaves some problems such as metastatic reactivation and subsequent tumor recurrence[1]. Recently, following the rapid advances in molecular biology, many new therapeutic strategies, including RNA interference (RNAi) technology for treating liver cancer at genetic level have been developed[2]. RNAi is a specific gene regulatory mechanism in which activation of an intracellular pathway triggered by small-interfering RNA (siRNA) of 21–23 nucleotides (nt), leading to gene silencing through degradation of a homologous target mRNA[3]. The selective and robust effect of RNAi on gene expression makes it become a valuable tool for basic research in biology, and thereby continue to have a major impact on medical science[4]. Another unique advantage of RNAi is that non-druggable protein targets can also be efficiently knocked-down and possibly achieve therapeutic effects[5]. Therefore, RNAi-based therapeutic strategy presents an effective and simple approach in new area of clinical therapy for HCC.
It has been known that human cancer is a gene-related disease involving abnormal cell growth. As a new member of the kinesin superfamily of microtubule-based motors, kinesin Eg5, also called kinesin spindle protein (KSP) or KIF11 participates in mitosis, by separating the microtubules that are attached to the two centrosomes, and contributing to the bipolar arrangement of the spindles[6]. Thus, inhibition of KSP may block the formation of bipolar mitotic spindles of mitotic cells, causing cell-cycle arrest, activation of the mitotic checkpoint, induction of apoptosis and eventually, to cell death[5, 7]. KSP gene was found to be lowly expressed in normal primary cells, but higher in transformed cells . Its expression was also higher in breast, colon, lung, ovary, and uterine carcinomas than in their adjacent tissues[8]. The overexpression of KSP as a transgene may cause genomic instability and tumor formation in mice[9]. In addition, KSP gene was also frequently expressed in HCC tissues and there was also a strong correlation between the level of KSP expression and HCC development[10]. These findings have indicated that the important role of KSP in mitotic progression makes it an significant candidate of anticancer therapy. Several KSP inhibitors have been studied in clinical trials and showed efficacy in preclinical models of human tumors[10, 11]. However, more trials must be studied to test their efficacy in clinic due to the toxicological side effects of KSP inhibitors, such as the observed neutropenia and leukopenia[12].
Additionally, the ability of the highly vascularized tumors, including HCC to attract blood vessels (tumor angiogenesis) is one of the rate-limiting steps for tumor progression[13]. Angiogenesis is governed differently by multiple factors, including growth factors, cytokines, chemokines, enzymes, and adhesion molecules, but the most important one is vascular endothelial growth factor (VEGF)[14]. Among all family members of VEGF, VEGF-A is the most potent and specific angiogenic factor. Many studies have shown that VEGF, mainly VEGF-A, is frequently expressed in HCC and increased VEGF levels correspond to increased tumor sizes[14, 15]. Another study reported that there was also a strong correlation between the level of VEGF expression and HCC pathological grading and clinical stages[16]. In addition, VEGF was identified as a key hypoxia-induced angiogenic stimulator in liver cancer[14]. It was suggested that the gene plays a critical role in the HCC progression of tumor growth. Therefore, VEGF is a logical target for HCC therapy. For the last decade, there have been several options of inhibiting VEGF binding to its receptors which have been developed as anticancer agents, such as soluble VEGF receptors, humanized anti VEGF monoclonal antibody (Bevacizumab; Avastin), various small molecules inhibiting VEGFR2 signal transduction[17, 18]. However, the use of anti VEGF antibodies or other inhibitors is responsible for unexpected toxic side effects, especially in terms of thromboembolic events and bleeding that require further investigation[18]. It is therefore a challenge to explore a new approach to inhibit VEGF expression in identification of novel druggable targets.
In this study, we aimed to use siRNA cocktail which targets VEGF-A (referred here as VEGF) and KSP gene as a therapy for HCC treatment. Pre-designed VEGF and KSP siRNAs were screened in Hep3B cell line, isolated from liver biopsy specimens with primary HCC and widely used as an experimental model. The best siRNA targets were used as cocktail to inhibit the growth, migration, invasion and induce apoptosis of Hep3B cells. The effect of siRNA cocktail on inhibiting in vitro angiogenesis ability of HUVECs induced by Hep3B cells was also evaluated.
Effects of pre-designed siRNAs on KSP and VEGF mRNA expression in Hep3B cells
Sequences of siRNAs targeting VEGF and KSP
Sequences (5'–3')
Sense: GCACAUAGGAGAGAUGAGCUUdTdT
Antisense: AAGCUCAUCUCUCCUAUGUGCUGdTdT
Sense: UGAAGUUCAUGGAUGUCUAdTdT
Antisense: UAGACAUCCAUGAACUUCAdTdT
Sense: GCCUUGCCUUGCUGCUCUAdTdT
Antisense: UAGAGCAGCAAGGCAAGGCdTdT
KSP-siRNA #1
Sense: CUGAAGACCUGAAGACAAUdTdT
Antisense: AUUGUCUUCAGGUCUUCAGdTdT
KSP-siRNA #2
Sense: UCGAGAAUCUAAACUAACUdTdT
Antisense: AGUUAGUUUAGAUUCUCGAdTdT
KSP-siRNA #3
Sense: CUGGAUCGUAAGAAGGCAGdTdT
Antisense: CUGCCUUCUUACGAUCCAGdTdT
Sense: GCGGAGAGGCUUAGGUGUAdTdT
Antisense: UACACCUAAGCCUCUCCGCdTdT
Effects of VEGF-siRNA#1, KSP-siRNA#2 and siRNA cocktail on KSP and VEGF expression in Hep3B cells
Similarly, KSP expression was effectively inhibited by KSP-siRNA#2 at both mRNA and protein levels by 75.07 ± 3.56% (p < 0.01, Figure 2B) and 53.48 ± 2.19% (p < 0.05, Figure 3C) by Real-time qRT-PCR analysis and Western blot analysis, respectively. These values indicated that the effective silencing of KSP-siRNA#2 on both mRNA and protein levels of KSP. As shown in Figures 2 and3, KSP-siRNA#2 did not produce any effect on the VEGF expression at the mRNA and protein levels.
Eventually, we examined siRNA cocktail on VEGF and KSP expressions respectively. As shown in Figures 2 and3, siRNA cocktail inhibited the VEGF and KSP expression at the mRNA and protein levels, obviously in comparison to the untreated ones. The results showed that VEGF mRNA was downregulated by 77.54 ± 3.22% (p < 0.01, Figure 2A) and VEGF protein level was downregulated by 59.42 ± 2.14% (p < 0.05, Figure 3B), which was also confirmed by ELISA analysis compared to untreated cells (Figure 3D). Downregulation of VEGF by siRNA cocktail was similar with that of VEGF-siRNA#1. When compared to VEGF-siRNA#1 or KSP-siRNA#2 alone, the siRNA cocktail showed higher inhibition on KSP mRNA expression up to 85.77 ± 1.78% (p < 0.01, Figure 2B) and protein level up to 69.42 ± 2.11% (p < 0.05, Figure 3C), indicating a significant effect of siRNA cocktail on KSP expression.
Effects of VEGF-siRNA#1, KSP-siRNA#2 and siRNA cocktail on cell proliferation in Hep3B cells
Effects of VEGF-siRNA#1, KSP-siRNA#2 and siRNA cocktail on cell migration ability in Hep3B cells
Effects of VEGF-siRNA#1, KSP-siRNA#2 and siRNA cocktail on cell invasion ability in Hep3B cells
Effects of VEGF-siRNA#1, KSP-siRNA#2 and siRNA cocktail on apoptosis in Hep3B cells
Inhibition of Cyclin D1, Bcl-2 and Survivin expression in Hep3B cells by VEGF-siRNA#1, KSP-siRNA#2 and siRNA cocktail
Effects of VEGF-siRNA#1, KSP-siRNA#2 and siRNA cocktail on tube formation in HUVECs
As tumor cells are characterised by multiple genetic and epigenetic alterations, the single inhibition of one tumour-associated gene as a therapeutic strategy may not be sufficient for inhibition of tumor development. It has been well known that gene therapy targeting either VEGF or KSP alone may cause inhibition of HCC growth[14, 19]. However, the current finding showed that siRNA cocktail silencing VEGF and KSP together could inhibit the proliferation, migration or invasion of HCC cells better than single siRNA simultaneously. On the other hand, the siRNA cocktail might also increase apoptosis induction in HCC cells. This is a better therapeutic strategy which could be adopted in clinics.
As one of the most important angiogenesis-stimulating factors, VEGF is correlated with liver cancer progression through its action of tumor neovascularization, tumor invasion and metastasis[14–16]. Some reports have shown that siRNA-mediated downregulation of VEGF expression results in decreased proliferation and induced apoptosis in colorectal cancer cells[20], prostate cancer cells[21], gastric cancer cells[22]. Our results also demonstrated that siRNA targeting VEGF could inhibit proliferation, migration, invasion and induce apoptosis in hepatocellular carcinoma Hep3B cells. Our observations were consistent with a previous report that also used VEGF-siRNA to suppress VEGF expression in liver cancer cells[14]. To elucidate its molecular mechanisms of VEGF inhibiting cell proliferation and inducing apoptosis, we have examined the expressions of the key regulators Cyclin D1, Bcl-2 and Survivin. Our results demonstrated that the expression levels of Cyclin D1, Bcl-2 and Survivin were significantly decreased in Hep3B cells upon cell transfection with VEGF-siRNA. Cyclin D1 is known to accumulate during the G1 phase of the cell cycle. Overexpression of Cyclin D1 may be a frequent event in hepatocarcinogenesis and therefore plays an important role in growth of liver tumors[23]. In contrast, Bcl-2 and Survivin are thought to be very important anti-apoptotic proteins in cells. They are identified to be one of the mechanisms involved by cancer cells to evade apoptosis. Bcl-2, a prominent member of the Bcl-2 family proteins, is responsible for governing the release of cytochrome c from the mitochondrial membrane, the activation of caspase cascade, the execution of apoptosis, and finally, to the prevention of death in cancer cells[24]. Overexpression of Bcl-2 also may protect human hepatoma cells from antibody mediated apoptosis[25]. Similarly, Survivin, belong to the inhibitors of apoptotic proteins (IAPs), has been implicated in both cell division and inhibition of apoptosis. By inhibiting apoptosis and promoting mitosis, Survivin may confer cancer cell survival and growth. Unlike other members of IAP family, Survivin is lowly or not expressed in normal tissues, but highly in tumor tissues. The induction of apoptosis is generally associated with suppression of Survivin within tumor cells[26]. The overexpression of Survivin in the majority of human tumor types, including liver cancer, can prevent apoptosis by binding and inhibiting pro-apoptotic caspases as a microtubule stabilizer during mitosis, and promote cell cycle progression[27].
In contrast to microtubules which are also presented in post-mitotic cells, KSP is exclusively expressed in mitotic cells, which makes it an important target for anti-mitotics[6]. Therefore, inducing a degradation of KSP by siRNA was expected to lead to a novel approach for the control of cancer cells. In this study, the expression of KSP was downregulated at both mRNA and protein levels in Hep3B cells by KSP-siRNA transfection. This result was similar with reports using KSP-siRNA to monitor the expression of KSP in ovary cancer cells[5], cervical cancer cells, myeloma cells[7], lung carcinoma cells and breast carcinoma cells[8]. In addition, our study also indicated that KSP-siRNA could inhibit proliferation, migration/invasion and induce apoptosis of Hep3B cells. The expression of genes involved in anti-apoptosis (Bcl-2 and Survivin) and proliferation (Cyclin D1) was downregulated in KSP-siRNA transfected cells. From these results, we surmised that the downregulation of Cyclin D1, Bcl-2 and Survivin expressions by VEGF-siRNA or KSP-siRNA transfection in one of the important ways to induce cell apoptosis, subsequently leading cell death.
It has been reported that siRNA cocktail was composed of two different siRNA sequences showed more effective inhibition of the two corresponding target genes at one time than siRNA alone[28]. In present study, we prepared the siRNA cocktail of best siRNAs, analyzed the cell treated with siRNA cocktail and controls, including single siRNA targeting VEGF or KSP and negative control siRNA. Our results revealed that using the siRNA cocktail targeting VEGF and KSP to inhibit the proliferation, migration, invasion and induce apoptosis of Hep3B cells was better than each siRNA alone. This could be explained by the significant downregulation of Cyclin D1, Bcl-2 and Survivin following the treatment of siRNA cocktail as compared to single siRNA simultaneously. Our results corresponded with several previous studies reporting the influences of siRNA cocktail on cell growth and apoptosis of gastric cancer cells[28], pancreatic cancer cells[29] and colorectal cancer cells[30]. The siRNA cocktail exhibited specific and high efficiency on silencing multi genes simultaneously and would have great potential for therapeutic siRNA applications.
Interestingly, we found that VEGF-siRNA exhibited significant inhibition on KSP mRNA and protein levels, while the KSP-siRNA alone did not show any effect on VEGF expression. We assumed that VEGF might be acted as a KSP upstream regulator, which could probably lead to downregulation of KSP expression. The fact that VEGF-siRNA presented similar KSP-siRNA effects on proliferation, migration, invasion and apoptosis of Hep3B cells suggesting that one of inhibiting tumor growth mechanisms of VEGF-siRNA functioned through inhibition of KSP expression. Furthermore, we also found that the expression of KSP protein was suppressed to a greater extent in the siRNA cocktail treated group than that in the KSP-siRNA or VEGF-siRNA alone group, indicating a significant silencing effect of siRNA cocktail on KSP protein expression. The results were supported by the highest inhibition of proliferation, migration, invasion and induction of apoptosis on Hep3B cells by the siRNA cocktail as compared to each of siRNAs alone. In contrast, suppression on VEGF expression in Hep3B cells by the siRNA cocktail was similar to that by the VEGF-siRNA, which supported the observation that KSP-siRNA did not influence VEGF protein expression. This observation was also demonstrated by angiogenesis and ANG2 expression on the HUVECs induced by Hep3B cell culture media following siRNAs treatment. A previous study indicated that HUVECs was induced to form new blood vessels with higher VEGF concentration[31]. In addition, ANG2 which is expressed in the areas undergoing vascular remodeling and leads to decreased vessel maturation and enhanced vessel sprouting could be upregulated by VEGF in endothelial cells[32]. ANG2 inhibition prevents the growth of new vessels by endothelial sprout formation[33]. In our study, the results showed that inhibition of capillary tube-like structure formation and suppression of ANG2 expression in HUVECs by siRNA cocktail was relatively equal when compared to VEGF-siRNA. Meanwhile, KSP-siRNA showed no effect on inhibiting tube formation as well as ANG2 expression in HUVECs. These results demonstrated that VEGF acts as a KSP upstream regulator promoting the effect of KSP on Hep3B cells. More importantly, our estimates were strongly supported by another study reporting a strong upregulation of KSP or Eg5 expression after application of recombinant human VEGF on the differentiated day-13 chick chorio-allantoic membrane (CAM). The results showed that numerous known genes encoding mitotic kinesins were consistently upregulated by VEGF, including KIF4A, KIF11/Eg5, KIF15, KIF20A/Mklp2 and KIF23[34].
This study showed that the expression of KSP was inhibited by VEGF-siRNA, suggesting that VEGF serves as an upstream regulator of KSP gene expression. Although there were several reports using siRNA to inhibit VEGF or KSP, combined siRNA therapy to simultaneously reduce VEGF and KSP expression was proved to be an effective approach to inhibit cell growth and induce apoptosis of HCC cells. This could be a new targeted strategy to eradicate HCC cells.
This experimental study was approved by the Committee for Ethics in Research, University of Science, Vietnam National University.
Culture of cells
Hep3B cell line (hepatocellular carcinoma cells, HB-8064), HUVECs cell line (human umbilical vein endothelial cells, CRL-1730) were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Hep3B cells were thawed and cultured in DMEM-F12, supplemented with 10% FBS and 0.5% antibiotic-mycotic (All were bought from Sigma-Aldrich, St. Louis, MO, USA). HUVECs cells were thawed and cultured in endothelial cell growth medium-2 (EGM-2) (Lonza, Walkersville, MD, USA), supplemented with 10% FBS and 0.5% antibiotic-mycotic. All cells were maintained at 37°C, 5% CO2.
Transfection of siRNAs
The sequences of the siRNAs targeting VEGF-A (VEGF-A-siRNA, also simply referred to as VEGF-siRNA), siRNAs targeting KSP (KSP-siRNA) and mismatched siRNA (CONT-siRNA) were shown in Table 1. All siRNAs were synthesized by Bioneer (Daejeon, Republic of Korea). Each siRNA was resuspended in nuclease – free water and the stock solutions were stored at 4°C until use. The cells were divided into five groups with different treatments at final concentration of 20 nM: Group 1: untreated cell group, group 2: CONT-siRNA treated cell group, group 3: VEGF-siRNA treated cell group, group 4: KSP-siRNA treated cell group, group 5: siRNA cocktail treated cell group (VEGF-siRNA mixed with KSP-siRNA at equal concentration). All the above siRNAs were transiently transfected with a Lipofectamine RNAiMAX Transfection Reagent kit (Invitrogen Inc., Carlsbad, CA, USA) by reverse transfection protocol. Briefly, for each well of 24-well plate (Corning Costar Corp., Cambridge, MA), 3μl of siRNA duplex (20μM) was mixed with 1 μl transfection reagent and 100 μl Opti-MEM medium supplied with the kit. Then, the siRNA-transfection reagent complex was incubated with 500 μl of diluted cells (5 × 104 cells/ well) for 24-72 hours at 37°C, 5% CO2. Control cells (untreated with siRNAs) were also grown under the same condition. The siRNAs treated cells and control cells were harvested during time intervals for transfection efficiency analysis.
Real-time quantitative reverse transcription PCR (real-time qRT-PCR)
Sequences of the primers for Real-time qRT-PCR
Sequences (5'–3')
Product size (bp)
Cyclin D1
Western blot
After washing with cold PBS, the cells were lysed by a lysis buffer containing 0.01M Tris, pH 7.5, 0.1M NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS), with added protease inhibitors. Total proteins in cell lysates were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene fluoride (PVDF) blotting membrane (Sigma-Aldrich, St. Louis, MO, USA). The membranes were blocked in blocking solution BSA (Sigma-Aldrich, St. Louis, MO, USA) and incubated with mouse anti-Eg5/KSP monoclonal antibody (1:200), mouse anti-VEGF monoclonal antibody (1:200), mouse anti-Cyclin D1 monoclonal antibody (1:500), mouse anti-Bcl-2 monoclonal antibody (1:500), mouse anti-Survivin monoclonal antibody (1:500), mouse anti-ANG2 monoclonal antibody (1:200) (All were bought from Abcam, Cambridge, ENG, UK) for 1 hour at room temperature. After washing, the membranes were incubated for 45 minutes with horseradish peroxidase (HRP)-linked goat anti-mouse IgG (1:5000, Sigma-Aldrich, St. Louis, MO, USA). The protein bands were visualized by enhanced chemiluminescence (Sigma-Aldrich, St. Louis, MO, USA). Mouse monoclonal Ab against β-actin (Abcam, Cambridge, ENG, UK) was used as a housekeeping gene control. Band intensities were semi-quantitatively analyzed by Image J densitometer (NIH, Bethesda, MD, USA).
Enzyme linked immunosorbent assay (ELISA)
The amount of VEGF in cell supernatants was measured by using human VEGF ELISA kit (Life Technologies, Carlsbad, CA, USA) following the manual of the kit. The human VEGF ELISA kit is a "sandwich" enzyme immunoassay employing monoclonal and polyclonal antibodies. Quantitation can be determined by constructing an absolute standard curve using known concentrations of human VEGF proteins.
Cell proliferation assay
Cell proliferation was measured by WST-1 assay kit (Roche, Basel, Switzerland). Briefly, siRNAs transfected cells and control cells were seeded at a concentration of 3 × 103 cells per well in 96-well plates (Corning Inc., NY, USA). For indicated time, WST-1 solution was applied at 10 μl per well and incubated for 4 hours at 37°C, 5% CO2. The absorbance was measured with a microplate ELISA reader (BioTek, Winooski, VT, USA) at 450 nm.
Clonogenic survival assay
Cells were seeded at a density of 500 cells per well in 6-well plates (Corning Inc., NY, USA) contained complete medium followed by treatment with siRNAs. The medium was then replaced with fresh medium and incubated for an additional 10 days. Clones were fixed with 4% paraformaldehyde for 30 minutes and stained with Crystal Violet (Sigma-Aldrich, St. Louis, MO, USA) for about 15 minutes. Stained clones that had more than 50 cells were counted at low magnification.
Wound-healing assay
Cell migration was measured by wound-healing assay. Hep3B cells were seeded and transfected with siRNAs as described above in 24-well plates (Corning Inc., NY, USA) at the density of 5 × 104 cells per well. After 48 hours, wound was made through confluent monolayer cells with a pipette tip. Wounded monolayers were then washed with PBS, and incubated in DMEM-F12 without FBS. Photographs of cells were taken at 0, 24, 48, and 72 hours to monitor cell movements.
Transwell invasion assay
Cell invasion was carried out by transwell assays. The upper surface of the transwell filters (Corning Inc., NY, USA) was coated with matrigel (BD Biosciences, Franklin Lakes, NJ, USA). The siRNAs treated cells and control cells (1 × 105 cells) were suspended in 200 μl serum-free media, and then added to the chamber. The chamber afterthat was placed in a 24-well plate containing complete medium. After 48 hours of incubation at 37°C, the filters were gently taken out and matrigel on the upper surface of the filters was removed by cotton swabs. Cells on the underside of transwell filters were fixed with 4% paraformaldehyde for 30 minutes, stained with Crystal Violet (Sigma-Aldrich, St. Louis, MO, USA) for 10 minutes, and then photographed. For quantitative assessment, the number of invading cells was counted from five randomly selected visual fields per filter.
Apoptosis assay
Apoptosis was investigated by flow cytometry using annexin V and propidium iodide (PI) (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, the cell concentration was firstly adjusted to 1 × 106 cells/ml, and then 1 ml of the cell suspension was taken and centrifuged at 500 × g for 10 minutes at 4°C. The pellet was rinsed twice with PBS and then re-suspended in a proper volume of binding buffer so that the cell concentration was 5 × 104 cells/ml. After addition of 10 μl Annexin V-FITC and 5 μl PI followed by gentle mix, a 15 minute reaction was initiated at room temperature in darkness. After that, 300 μl binding buffer was added and flow cytometry (FACSCalibur) using CellQuest Pro software (BD Biosciences, Franklin Lakes, NJ, USA) was performed to detect cell apoptosis rate (%).
Tube formation assay
The ability of endothelial cells to sprout new blood vessels stimulated by pro-angiogenic factors released from Hep3B cells was examined in HUVECs angiogenesis in vitro model. Briefly, 6 × 104 HUVECs were collected, resuspended in a conditioned medium, which was the supernatant of siRNAs treated Hep3B cells, seeded in 24-well plates coated with 100 μl Matrigel (BD Biosciences, Franklin Lakes, NJ, USA), and cultured at 37°C, 5% CO2. After incubation for 24 hours, numbers of branching points were counted.
Statistical analysis
Each experiment was performed in triplicate for all data (n = 3). Data was expressed as mean ± standard error of the mean. Statistical comparisons were performed using the Student's t –test and ANOVA. p -values < 0.05 were considered to be statistically significant.
Bovine serum albumin
- EtdBr:
Ethidium bromide
Fluorescein isothiocyanate
Hepatocellular carcinoma
Horseradish peroxidase
Inhibitors of the apoptosis protein
Kinesin spindle protein
- mRNA:
Messenger RNA
Polyacrylamide gel electrophoresis
Propidium iodide
Polyvinylidene fluoride
- Real-time qRT-PCR:
Real-time quantitative reverse transcription PCR
- RNAi:
RNA interference
Reverse transcription PCR
Sodium dodecyl sulfate
- siRNA:
Small interfering RNA
Vascular endothelial growth factor.
This work was funded by grants from the National Product Development Program, Ministry of Science and Technology, Vietnam. We would like to thank to Dr. Bruce May, University of British Columbia for his technical support and useful discussion.
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.


text:
The media is in a lachrymose frenzy, perhaps induced by a sense of culpability. Every radio station, newspaper, and television station is torturing us with their exaggerated sorrow. There must be some other news worth reporting.
I hope that after the funeral we will all be permitted to get on with our own lives, and Diana will be allowed to rest in peace.
ManchesterReuse content


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Friedrich Theodor von Vischer, (born June 30, 1807, Ludwigsburg, Württemberg [Germany]—died Sept. 14, 1887, Gmunden, Austria), German literary critic and aesthetician known for his efforts to create a theoretical basis for literary realism.
Vischer's theories of aesthetics, based on ideas of G.W.F. Hegel, began to develop while he was teaching at the University of Tübingen, where he had studied. He became a professor at Tübingen in 1844 but was suspended for two years because of an outspokenly liberal inaugural address. His work was finally published in six volumes as Ästhetik, oder Wissenschaft des Schönen (1846–57; "Aesthetics, or Fine Arts"). In 1855 he became ... (100 of 158 words)


text:
SYNOPSIS: Several years after the tragic death of their little girl, a doll maker and his wife welcome a nun and several girls from a shuttered orphanage into their home, soon becoming the target of the dollmaker's possessed creation, Annabelle.Read More »
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Film Review: Don't Knock Twice (2016)
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Insidious: Chapter 4 (2017)
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SYNOPSIS: The action now takes place miles away from the original location and partly in broad daylight, giving the film an entirely fresh yet disturbing new reality. The infection has left the building. In a clever twist that draws together the plots of the first two movies, this third part of the saga also works as a decoder to uncover …Read More »


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With the advancement in telecommunication technology, you can locate a person anywhere in the world by calling an international operator. Each country is assigned an international dialling code. When you dial the code, you have the option of dialling the city code number to locate the correct person. However, dialling out to an international number may be expensive depending on the type of phone plan you have, so exercise caution when dialling.
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Other People Are Reading
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Dial "01" on your phone to reach an international line.
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Look for the country's code (see Resources). Dial the code followed by "01." For example, if you were looking for someone in Australia, you would dial "01" followed by the country code "61." If there is a city code, you may also dial it to better help the operator locate the person you wish to speak to.
- 3
Speak to the country's operator. Please note, that by dialling "01," your phone company will bill you for speaking to an operator.
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Come on in and rent a super fun eBike! We're located just a few blocks away from the Santa Cruz Wharf, The Boardwalk, and the spectacular West Cliff Drive multi-use path.
West Cliff Drive, among the most gorgeous pathways on the West Coast, runs 2.5 miles, overlooks beautiful Monterey Bay and ends directly at Natural Bridges State Park. Ride your bike, stop and enjoy the views, and maybe swing by Swift Street Courtyard (up Swift Street about six blocks from West Cliff Drive) for a snack or beverage.
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after many photos of dogs jumping outside (along with one goat), i have one more to offer. this was taken several years ago in burlington, vermont. i watched as this dog bounded down the pier and threw himself into lake champlain with happy abandon. he would swim back and repeat the process about four times before…Read more jump
dogs! jumping! outside!
Our very serious focus group determined that dog agility would make for a lovely series of photos. Behold, a lovely series of dog agility photos! um, what are you doing in this project? you're not a dog. baaa... er, bark? no, goat. bad goat. this is not for you. you're not the boss of…Read more dogs! jumping! outside!
focus group
after a bagful of crunchy treats and many hydrant breaks, the very serious research panel that i use for determining what posts i should add to my blog has determined a series of posts should be devoted to... dogs! jumping! outside!* thank you for your participation. ladies and gentlemen: SCIENCE. and two seriously enthusiastic pups. *of…Read more focus group
when it rains…
…cute pups like Guapa don morton salt girl raincoats and remind us that it can't be sunny ALL the time. thankfully, there are always cute things to make us smile when it's wet and icky outside.
since i've moved away from Boston, i am no longer able to photograph the wonderful pups available for adoption through Underdog ResQ. like Eve here, all swaddled up in a blanket because the world out there is really scary for a timid pup. look at that eyebrow! Barooooo! some days i feel like this too,…Read more burrito.
i say no to donuts.
there is something oddly therapeutic about sad, cute animals. they will plead at you with giant, watery eyes for that treat you have hidden behind your back. they will look askance at you before bounding off into their safe haven. they will baroo? at you until you give them that soft belly rub or scritches behind…Read more i say no to donuts.
in 2005 my husband and i visited St. Martin. We stayed at Baie Orientale, a beautiful beach with fine white sand and crystal water, protected by distant mountains. It was gentle. Calm. Two years later, I met a beautiful family with a collection of rescue pups (and rats, and hammies, and chickens!), one of which…Read more baie
yesterday i said goodbye to a close friend (i'm moving to washington, d.c. next week), and boarded the green line train to boston. exhausted from the stress of a divorce and moving to a new city, i sat down and closed my eyes for a while. when i opened my eyes, the train was full and…Read more cookie
Maverick wa such a patient model for the Underdog ResQ holiday shoot. Those EARS - you can't even see the reindeer antlers!
the holidays are better with sock monkeys.
Brandy seems to think so. You should get your own sock monkey! Broken ornaments? Sock monkey did it. All the brandy is gone? You know that cheeky little monkey did it. Really, they're all-purpose monkeys...
Continuing the parade of pups from the LaundroMutt photo shoot to benefit Underdog ResQ, I bring you... a fluffy Husky! Seriously. Have you ever SEEN a fluffy Husky? I certainly hadn't, until Meeka sauntered into the studio and had us all wrapped around her fluffy little paws in minutes. Isn't she gorgeous?
angie wishes you a happy Hanukkah
Angie, a very sweet English Mastiff, joined us last Sunday for my holiday photo fundraiser at LaundroMutt in Cambridge. We raised over $500 to benefit Underdog ResQ's dogs in need! Stay tuned: I'll be featuring more happy holiday pups until Christmas!
howloween: RAAAAR
photos from last week, raising some money for Underdog ResQ! I have another photo shoot fundraiser coming up on Sunday, 2 December at LaundroMutt at Fresh Pond in Cambridge, so save the date! Smokey, Rocky, Mia, Luna, Boulder & Pebbles, and Artie.
never underestimate the healing power of puppies. this is from a past photo shoot; i came across this image while searching for something to use for a flyer. i have wanted to Photoshop a cape into this photo for a while now. 🙂 i haven't been able to sort through photos and post to this…Read more superpuppy
among all of my pet photography projects, one of my most memorable shoots was a dog agility course at Guilford Fair (VT) in 2009. I enjoyed capturing all the happy faces of the pups in midair! I hope to have the opportunity to photograph another agility course. I have also seen cat agility videos on…Read more agile
today i had an Underdog ResQ photo shoot with Tessa, a gorgeous, happy girl who was very attentive to her people. i still haven't gone through the photos, but i felt that an outtake would be cute to share. this photo needs a funny caption. i shall soon be putting together the Underdog ResQ 2013…Read more tessa
i have many photos of pups at play. i'm going to use them to coerce people to send me various used dog toys, still in good condition, for a project. if you have gently-used dog toys, whether they are squeaky toys, balls, lone sneakers, or anything that can be considered a dog toy, please email…Read more ball!
On Wednesdays weekday woes can lurk, And witty writings will not work. I've got an acute need for perk, Thus: emergency Chihuahua. Scooby here was rescued through Underdog and its foster crew Of volunteers who rescue slews Of pit bulls and chihuahuas. But not just those two breeds, of course; From tiny pups to big-as-horse,…Read more scooby
underdogs: lucy & esmerelda
On Sunday, I photographed two lovely little Pittie-mix puppies who are currently up for adoption through Underdog ResQ. These two adorable girls are named Lucy and Esmerelda, and both were born in May of 2011. You can meet both Lucy and Esmerelda on Underdog's "Available Dogs" pages. They are both currently on page 2 of…Read more underdogs: lucy & esmerelda
....or it might be Renny; I have trouble telling the two Chihuahuas apart.


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Valentine Recital 2012
On Sunday, February 12th, students and faculty of Carondelet Music Center performed in a Valentine Recital, Love is in the Air, for an audience of nearly 100 family members and friends in the Chapel of St. Joseph's Provincial House in Latham. Preceding the event, the Board of Trustees for the Music Center made a special presentation to Sister Patricia St. John, Founder and Executive Director of the Center, and the faculty in celebration of the 20th Anniversary Year. The recital showcased the musical skill of twenty students who performed works for piano, violin and guitar, and included faculty performances by guitarist John Charles Cook who played a slick, jazz rendition of Misty, and violinist Barbara Brothers with guest violinist Michael Emery and accompanist Elizabeth O'Neil, performing Andante from Bach's Concerto in d minor for two violins. All present enjoyed Valentine treats at the reception immediately following the event.


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Benq Joybee 120 Driver Download
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After a terribly cold and gray day yesterday, the sun shone bright in Monterey today for Father's Day. It made for a perfect day to celebrate our love for Daddy!
We started off our day at church followed by lunch in Carmel.
It was then back home to change clothes before we headed out for a hike down at Elkhorn Slough.
Upon checking in at the visitors center, we were told that a mountain lion was spotted on the trails last week. Good thing we had two lookouts with us!
The trail was great.
We had "telescopes" to look through.
We also got to check out at couple of old barns from a former dairy farm that used to be on the property.
And with views like this, it's no wonder that happy cows come from California.
We also spotted some crabs from the boardwalk (that did not have any rides – Trenton was apparently expecting a Santa Cruz-ish type boardwalk).
Charlotte started getting worn out, and asked Daddy to carry her for awhile.
After about 5 minutes of Jarrod holding her, Charlotte started screaming and arching her back. Jarrod lifted her shirt up, and something large and grayish in color flew out. We took a look at Charlotte's back, and she had two sting marks. Note to self: start carrying Benadryl on hikes. Thankfully Charlotte didn't have any type of allergic reaction to the stings, but we did cut our hike short. We will definitely be going back again to check out the rest of the trail though!
We got home, had some play time in the sprinkler, ate dinner, and sat down with popcorn for a movie night. A great end to a great day!
Happy Father's Day, Daddy! And happy Father's Day to my Daddy too! I love both of you!