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Musashi-1 maintains blood–testis barrier structure during spermatogenesis and regulates stress granule formation upon heat stress

Msi-1 knockdown disrupts blood-testis barrier structure and the continuous process of spermatogenesis. A role for Msi-1 in regulating Sertoli cell fate following heat-induced injury is noted.


INTRODUCTION
The blood-testis barrier (BTB) is an important ultrastructure composed of coexisting tight junctions (TJs), basal ectoplasmic specialization and gap junctions, and desmosomes between adjacent Sertoli cells in the seminiferous epithelium near the basement membrane (Mok et al., 2012(Mok et al., , 2013. The functional BTB structure is of significant importance for the initiation of spermatogenesis, in particular in the differentiation of spermatogonia beyond type A and in meiosis (Mok et al., 2012). The BTB undergoes extensive restructuring at stages VIII-IX of the epithelial cycle to permit the transit of preleptotene spermatocytes (Cheng and Mruk, 2010). However, the underlying mechanisms that regulate BTB permeability and the participating molecules that are critical for constructing the BTB structure are largely unknown.
In mammals, normal spermatogenesis occurs continuously in the scrotum at a lower temperature than core body temperature (Danno et al., 2000). Raised testicular temperature has a detrimental effect on mammalian spermatogenesis and increases the risk of male infertility (Durairajanayagam et al., 2015). Clinical cryptorchidism or experimental hyperthermia leads to spermatogenesis failure by inducing apoptosis in spermatocytes nearing meiosis and in spermatids (Hutson et al., 1997;Lue et al., 1999). In contrast, spermatogonia and early spermatocytes are more tolerant of heat-induced injury (Paul et al., 2008(Paul et al., , 2009. Sertoli cells are known to provide germ cells with structural and nutritional support (Griswold, 1998) and are able to survive heat stress (Jegou et al., 1984;Chen et al., 2008;Cai et al., 2011). However, the underlying mechanism of these testicular cells' distinct responses to heat-induced stress remains to be discovered.
Heat stress is also a common inducer of the formation of stress granules, which are cytoplasmic mRNA-silencing transient structures formed in response to a variety of environmental stresses (Moore, In this paper, we demonstrate that the knockdown of Msi-1 by RNA interference (RNAi) in Sertoli cells perturbs cell-cell junction via changes in the protein levels and distribution of BTB-associated proteins in vitro and disrupts BTB structure and spermatogenesis in vivo. Moreover, we observe a novel function for Sertoli cell Msi-1 following heat-induced stress. Msi-1-containing stress granules in Sertoli cells have a protective function against apoptosis upon heat stress and likely through activating p-ERK1/2 signaling.

Msi-1 is a Sertoli cell protein in mouse testes
In the present study, various tissue distributions of Msi-1 protein were detected by immunoblotting in mice. Endogenous Msi-1 expression was almost exclusively detected in the brain and testis, and lower levels of Msi-1 were detected in the spleen and pancreas ( Figure 1A). Immunoblotting for Msi-1 in lysates of adult mouse testes, Sertoli cells, germ cells, and Thy1 + spermatogonia further revealed that Msi-1 expression was restricted to Sertoli cells in the testes ( Figure  1B). In immature testes, before the formation of a lumen within the seminiferous tubule, Sertoli cell cytoplasm in the center of the tubule was immunopositive, and Sertoli cell nuclei were clearly stained, whereas germ cells were clearly immunonegative ( Figure 1C, b). In adult testes, Msi-1 protein was detected in the cytoplasmic and nuclear compartments of Sertoli cells ( Figure 1C, c, asterisks) rather than in germ cells. Thus we speculate that Msi-1 may play an important role in supporting spermatogenesis.

Knockdown of Msi-1 by RNAi in Sertoli cells perturbs Sertoli cell-cell junctions via changes in BTB-associated proteins
Msi-1 was knocked down by transfecting Sertoli cells with Msi-1specific small interfering RNA (siRNA) duplexes. Endogenous Msi-1 was knocked down by ∼60% in Sertoli cells (Figure 2, B and C). Sertoli cells cultured in vitro established a functional Sertoli cell-cell junction that mimicked the BTB structure in vivo. Compared with nontargeting siRNA duplexes, Msi-1-specific siRNA duplexes were found to partially perturb the Sertoli cell TJ barrier, as indicated by significantly reduced transepithelial electrical resistance (TER) across the epithelium (Figure 2A). The levels of TJ proteins, such as zo-1 and occludin, were not obviously changed. However, steady-state levels of claudin-11 were significantly reduced after Msi-1 knockdown. In addition, significant changes in the protein levels of the basal ectoplasmic specialization protein β-catenin, but not of E-cadherin and N-cadherin, were observed ( Figure 2, B and C). Furthermore, immunostaining results indicated that claudin-11 and βcatenin signals were considerably reduced in Msi-1-silenced cells (Figure 2, E and F), consistent with the immunoblotting data. Although the protein levels of occludin and E-cadherin were not significantly changed, their cell-cell interface distributions were abnormal in some areas (Figure 2, D and E, arrows). components of stress granules include polyadenylated mRNAs (poly-(A) + RNAs); 40S ribosomal subunits; translation initiation factors, such as the eukaryotic translation initiation factors eIF3, eIF4E, eIF4G, and poly(A)-binding protein (PABP); and several RNA-binding proteins, such as TIA1, HuR, and G3BP (Kedersha et al., 1999(Kedersha et al., , 2002Gallouzi et al., 2000;Tourriere et al., 2003). It is accepted that the phosphorylation of eIF2α prevents the assembly of the eIF2-GTP-Met tRNAi (inhibitor tRNA) translation initiation complex (Kedersha et al., 1999) and acts as an inducer of stress granule assembly. Stress granules function as a transient place of messenger ribonucleoprotein remodeling for storage, degradation, or reinitiation of translation during environmental stress and rapid recovery from stress. Recent studies have demonstrated that stress granule formation is a protective event in cells, as disrupting stress granule formation leads to the release of apoptosis-inducing components from stress granules and triggers cell death (Arimoto et al., 2008;Tsai and Wei, 2010).
Musashi-1 (Msi-1) is predominantly expressed in CNS stem cells and contributes to the maintenance of the self-renewal activity of CNS stem cells as a marker of the CNS (Okano et al., 2005). Global Msi-1 knockout mice can survive up to 1-2 mo; a fertility issue in these mice has not been mentioned in any published papers . In addition to the high level of Msi-1 expression in CNS stem cells, Msi-1 is specifically expressed in the cytoplasm and nuclei of Sertoli cells in rat testis from the fetal stage to adulthood (Saunders et al., 2002). However, the function and molecular regulation of Msi-1 in mouse Sertoli cells are still undefined. and basal compartment but was excluded from the adluminal compartment. By contrast, damage to the BTB was visualized by the influx of biotin through the BTB into the apical compartment of the seminiferous epithelium after 7-d postintratesticular injection of Msi-1 siRNA ( Figure 3F). Twelve days after Msi-1 knockdown, the continuous process of spermatogenesis was severely disrupted ( Figure 3G).

Endogenous Msi-1 protein is accumulated in stress granules in heat-treated Sertoli cells
Heat-induced stress is both a harmful factor for spermatogenesis and a known inductor of stress granules in various cell lines. In addition to its regulation of the permeability of BTB and spermatogenesis, we discovered a novel function of Msi-1 in Sertoli cells upon heat stress. We performed double-staining analyses to In vivo knockdown of Msi-1 by RNAi disrupts BTB structure and spermatogenesis Because knockdown of Msi-1 in Sertoli cells was shown to damage the Sertoli cell-cell junction in vitro, we sought to examine whether Msi-1 knockdown in vivo would disrupt BTB functional structure and the continuous process of spermatogenesis. In vivo knockdown of Msi-1 was performed by the intratesticular injection of siRNA duplexes specifically targeting Msi-1. After 3-d postintratesticular injection of Msi-1 siRNA, the expression ( Figure  3A) and protein levels ( Figure 3B) of endogenous Msi-1 protein in testes were considerably diminished. As shown in Figure 3, C-E, the β-catenin and claudin-11 signals at the BTB were significantly down-regulated after knockdown of endogenous Msi-1 after 5-d postintratesticular injection of Msi-1 siRNA. In nontargeting control testes, the biotin tracer was observed in the interstitial spaces FIGURE 2: Msi-1 knockdown by RNAi in Sertoli cells perturbs Sertoli cell-cell junctions via changes in BTB-associated proteins. (A) On day 3, Sertoli cells were transfected with Msi-1-specific siRNA duplexes and nontargeting control siRNA duplexes. The TER was detected until day 7, and each data point is the mean ± SD of n = 5 replicates from a representative experiment. *, p < 0.05. (B) Immunoblot showing the steady-state levels of Msi-1, basal ectoplasmic specialization (ES) proteins, and TJ proteins in lysates of Sertoli cells lysed 48 h after transfection; β-tubulin served as a loading control. (C) Histogram summarizing selected immunoblotting results in B and normalized against β-tubulin. Each bar represents the mean ± SD of n = 3 experiments. *, p < 0.05. (D-F) Changes in the localization of TJ proteins, basal ES proteins, and the framework protein vimentin at Sertoli cell-cell interfaces after Msi-1 knockdown were assessed 48 h after transfection. Sertoli cell nuclei were stained with DAPI. Note that considerably less β-catenin and claudin-11 were detected, and the distributions of occludin and E-cadherin (white arrows) were changed at Sertoli cell-cell interfaces after Msi-1 knockdown. Scale bars: 20 μm.
FIGURE 3: An in vivo study assessing the role of Msi-1 in BTB function and spermatogenesis by RNAi intratesticular injection. Msi-1-targeting siRNA duplexes and nontargeting duplexes were administered to each testis in adult mice as a single treatment. The knockdown efficiency, BTB-associated protein expression, biotin tracer experiment, and hematoxylin-eosin (HE) staining at 3, 5, 9, and 12 d postintratesticular injection, respectively. (A) Considerably more intense Msi-1 staining was observed in the Sertoli cells from testes transfected with nontargeting control (a) versus testes transfected with Msi-1-specific siRNA duplexes (b). Scale bars: 20 μm. (B) Histogram summarizing immunoblotting results of Msi-1 and normalized against β-tubulin. Each bar represents the mean ± SD of n = 3 experiments. *, p < 0.05. (C) Paraffin sections were used to study changes in the expression of β-catenin (a and b) and claudin-11 (c and d) after Msi-1 knockdown in vivo. The boxed area was randomly selected. Scale bars: 20 μm. (D) The protein levels of β-catenin and claudin-11 in nontargeting control and Msi-1 knockdown testes. β-tubulin served as a loading control. (E) Histogram summarizing selected immunoblotting results in D and normalized against β-tubulin. Each bar represents the mean ± SD of n = 3 experiments. *, p < 0.05. (F) We injected a biotin tracer into the testes of live anesthetized mice (n = 3) and examined the subsequent changes in BTB integrity after siRNA duplexes injection. (G) HE staining showed that the continuous process of spermatogenesis was seriously disrupted after Msi-1 knockdown in vivo. Scale bars: 80 μm.
confirming that Msi-1 was recruited to stress granules following heat-induced stress.

Msi-1 is essential for heat-induced stress granule formation
To explore the involvement of endogenous Msi-1 in stress granule formation in Sertoli cells, we silenced Msi-1 by expressing siRNA duplexes. In the untreated Sertoli cells, Msi-1 and TIA-1 were localized throughout the cytoplasm ( Figure 5, A-C). As expected, examine the possibility that endogenous Msi-1 protein is recruited to stress granules upon heat stress (Figure 4, A-D). Stress granulespecific markers, including TIAR (Kedersha et al., 1999), eIF3 (Kedersha et al., 1999), eIF4G (Kedersha and Anderson, 2009), and HuR (Gallouzi et al., 2000), together with Msi-1 were dispersed throughout the cytoplasm in untreated Sertoli cells. In heat-treated Sertoli cells, however, these stress granule-specific markers were assembled into Msi-1-positive cytoplasmic granules,  The nuclei were visualized with DAPI (blue). Scale bars: 10 μm. (M) p-eIF2α was not activated in Msi-1-silenced Sertoli cells exposed to heat treatment. Compared with the group treated with heat alone, p-eIF2α was not activated after Msi-1 knockdown. Histograms summarize the data shown in the panel below after normalizing each data point to β-tubulin. Each bar represents the mean ± SD of n = 3 experiments. *, p < 0.05.
Next we observed that p-ERK1/2 was instantaneously activated in the cytoplasm of Sertoli cells after heat treatment ( Figure 7A). p-ERK1/2 could be induced by stress granule formation upon exposure to various stress granule stimulators, including heat stress, NaAsO 2 , and MG132 ( Figure 7B). By contrast, the formation of stress granules in Sertoli cells was not affected by inhibiting ERK1/2 phosphorylation, as shown by using the specific inhibitor U0126 ( Figure  7C). Figure 7, A-C, suggests that the p-ERK1/2 pathway is activated by stress granule formation and acts as a downstream effector of stress granules during heat resistance. When p-ERK1/2 signaling was blocked by U0126, the percentage of apoptotic Sertoli cells was significantly increased after exposure to heat stress ( Figure 7D). In addition, we found that p-ERK1/2 was not activated in Msi-1 knockdown Sertoli cells upon heat stress ( Figure 7E). Altogether these results suggest that p-ERK1/2 signaling mediates the effects of Msi-1 in stress granule formation and Sertoli cell survival upon heat treatment.

DISCUSSION
Msi-1, an mRNA-binding protein, is considered a marker of neural stem cells (Imai et al., 2001;Okano et al., 2005). In this study, we discovered that Msi-1 protein was detected in the cytoplasm and nuclei of Sertoli cells rather than germ cells in mice, which is consistent with a previous study showing that Msi-1 is specifically expressed in Sertoli cells in rat testes (Saunders et al., 2002). Herein we demonstrated a critical role of Msi-1 in supporting spermatogenesis in mice. Msi-1 knockdown by RNAi duplexes in Sertoli cells perturbed the Sertoli cell-cell junction that mimicked the BTB structure in vivo via changes in the expression of the BTB-associated proteins claudin-11 and β-catenin and in the distribution of occludin and E-cadherin at Sertoli cell-cell interfaces. Msi-1 Msi-1 was restricted to TIA-1-positive stress granules in heat-treated Sertoli cells ( Figure 5, D-F). However, stress granule formation was significantly affected in Msi-1-silenced cultures upon the induction of heat stress ( Figure 5, G-I). To test whether stress granule formation was initiated, we evaluated p-eIF2α levels in the Msi-1-silenced heat-treated Sertoli cells ( Figure 5M). Compared with the single heat-treated group, eIF2α was not significantly activated after Msi-1 knockdown, confirming that Msi-1 is essential for p-eIF2α-dependent stress granule formation.

Msi-1 mediates Sertoli cell survival upon heat treatment via p-ERK1/2 activation
Testicular heat stress alters the expression of junction-associated molecules and upstream transcription factors; however, it does not induce Sertoli cell apoptosis (Chen et al., 2008), indicating that undiscovered mechanisms exist in Sertoli cells to survive heat stress. Using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay, we investigated whether Msi-1 had an effect on regulating Sertoli cell survival upon heat stress. As shown in Figure 6A, heat stress induced a significant increase in the number of apoptotic Sertoli cells in the Msi-1 siRNA-treated group but not in the control siRNA-treated group. To further confirm the above results, we examined the activation of proapoptotic markers, such as Fas, FasL, Bax, caspase-3, caspase-8, and caspase-9. The levels of these proapoptotic markers in the cells treated with heat or Msi-1 siRNA alone were not different from those of the untreated Sertoli cells ( Figure 6B, lanes 1, 2, and 3). However, the protein levels of these proapoptotic markers were significantly increased in Msi-1 siRNA-treated Sertoli cells upon heat stress ( Figure 6B, lane 4). In the single-treatment groups (heat stress or Msi-1 siRNA), the protein levels of Fas, FasL, Bax, caspase-3, caspase-8, and caspase-9 were not increased and were similar to those of untreated Sertoli cells. However, these cell proapoptotic markers were significantly increased in Msi-1-knockdown Sertoli cells upon exposure to heat stress. The protein levels were normalized to β-tubulin.

two classes of somatic cells, namely Sertoli cells and Leydig cells.
During spermatogenesis, different cells undergo distinct cellular responses to heat stress. Previous studies have suggested that a single exposure to heat induces apoptosis in spermatocytes nearing meiosis and in spermatids (Hutson et al., 1997;Lue et al., 1999). In contrast, spermatogonia and early spermatocytes are not susceptible to heat stress (Paul et al., 2008). Previous studies reveal that forced heat stress on the testes perturbs the inter-Sertoli BTB assembly without any apparent cytotoxicity (Chen et al., 2008;Cai et al., 2011). Once perturbed, the inter-Sertoli BTB is capable of resealing after the heat treatment is removed (Cai et al., 2011), indicating that Sertoli cells can survive heat stress. Leydig cells are found adjacent to the seminiferous tubules in the testes and produce testosterone to support spermatogenesis (Parvinen et al., 1984;Sharpe et al., 1988). The cellular response of Leydig cells is extremely different from the responses of germ cells and Sertoli cells. Local heat stress on the testes leads to Leydig cell hyperplasia (Jegou et al., 1984). However, the mechanism behind these distinct cellular responses in testes exposed to local heat stress remains unknown.
RNA-binding proteins in germ cells are necessary for fertility. Previous studies have suggested that loss of the RNA-binding protein DAZL results in infertility in both males and females (Ruggiu et al., 1997;Saunders et al., 2003). A novel function of DAZL in germ cell fate has been investigated by a recent study, which indicated that endogenous DAZL protein is essential for the stress granule formation functions as an mRNA-binding protein through sequence-specific interactions with the 3′-untranslated regions (UTRs) of various target mRNAs (Imai et al., 2001) and inhibits translation initiation of its target mRNAs by competing with eIF4G for PABP (Kawahara et al., 2008). Gene regulation data for several organs and mammalian cell lines suggest that the best-established targets of Msi-1 are Notch signaling regulators, such as Numb (Imai et al., 2001;Battelli et al., 2006). We find there are putative Msi-1-binding sites ((G/A)U n AGU) in the 3′UTRs of cldn11 (claudin11-coding gene) and ctnnb1 (β-catenin-coding gene) mRNA. Further studies are needed to identify whether cldn11 and ctnnb1 are targets of Msi-1. Our in vitro result was further supported by findings using an in vivo RNAisilencing model, and we further assessed that the permeability of BTB and the process of spermatogenesis were seriously disrupted after Msi-1 knockdown. Further studies using conditional geneknockout methods to identify the in vivo functions of Msi-1 in the permeability of BTB and the continuous progress of spermatogenesis are essential. Although we show that Msi-1 is localized to Sertoli cells rather than germ cells, we could not exclude the possibility that there are trace amounts of Msi-1 expression in germ cells.
In mammals, the testes are located in the scrotum and are maintained at a lower temperature than the body cavity (Lue et al., 1999). The low temperature is important for normal spermatogenesis, as significant germ cell loss is observed with cryptorchidism and local heat treatment of the testes (Yin et al., 1997;Lue et al., 1999;Rockett et al., 2001). Germ cells develop in association with FIGURE 7: p-ERK1/2 is activated by stress formation and is critical for protection against apoptosis. (A) p-ERK1/2 was activated in the cytoplasm of Sertoli cells (black arrows) after heat treatment. Scale bars: 40 μm. (B) ERK1/2 was phosphorylated as long as stress granules were formed upon treatment with various stress granule stimulators, including heat stress, NaAsO 2 , and MG132. (C) The formation of stress granules in Sertoli cells was not affected by inhibiting ERK1/2 phosphorylation using the specific inhibitor U0126. (D) The percentage of apoptotic Sertoli cells is counted from each treated group, based on the TUNEL staining. Each bar represents the mean ± SD of n = 3 experiments. *, p < 0.05. (E) p-ERK1/2 was not activated in Msi-1-silenced Sertoli cells upon heat treatment. (F) Schematic presentation of two distinct roles of Msi-1 in the BTB regulation and Sertoli cell fate upon heat stress.
Sertoli cells were transfected with Msi-1-specific duplexes and nontargeting control duplexes. The TER was detected until day 7, and each data point is the mean ± SD of n = 5 replicates from a representative experiment.

Biotin tracer experiment
The integrity of the BTB was assessed using a biotin tracer, as previously described (Meng et al., 2005). Briefly, male mice were anesthetized, and ∼50 μl of EZ-Link Sulfo-NHS-LC-Biotin (10 mg/ml; 21335; Pierce Chemical) freshly diluted in phosphate-buffered saline (PBS) containing 1 mM CaCl 2 were injected into one testis. The other testis was injected with 50 μl of 1 mM CaCl 2 in PBS as an internal control. The mice were killed 30 min after the injections, and their testes were removed and immediately embedded in OCT (Sakura, Tokyo, Japan). Cryosections (7 μl thick) were prepared for further staining.

Induced formation of stress granules
Adherent cells were placed in a 42°C water bath. Sertoli cells were held at this temperature for 30 min before fixation and staining. Treatment with 250 μM NaAsO 2 (Sigma-Aldrich) in PBS for 30 min was used to induced stress granule formation (Detzer et al., 2011). Cells were incubated with the proteasome inhibitor MG132 (Sigma-Aldrich) at 100 μM for 1 h (Restelli et al., 2010).
ERK1/2 is activated in response to environmental stress and signals, and it promotes cellular responses to growth inhibition and survival (Cupp et al., 1999;Sinha et al., 2004;Lu and Xu, 2006). In this study, we show that ERK1/2 is specifically activated in the cytoplasm of Sertoli cells by various stress granule-inducible stimulators. On the other hand, stress granules assemble properly in Sertoli cells with inhibited ERK1/2 phosphorylation, suggesting that p-ERK1/2 acts downstream of stress granule formation upon environmental stress. When p-ERK1/2 signaling is blocked by the specific inhibitor U0126, apoptotic signals are clearly detected after heat treatment, suggesting that p-ERK1/2 has a protective role against apoptosis. In Msi-1-knockdown Sertoli cells, p-ERK1/2 is not activated upon heat stress, indicating that the p-ERK1/2 protective pathway is regulated by Msi-1 via the triggering of stress granule formation. Msi-1-deficient male mice may be more susceptible to heat stress than normal males, which may lead to male infertility.
In summary, this report demonstrates the involvement of Msi-1 in regulating the functional BTB structure and the continuous process of spermatogenesis in mouse testes. Our findings also provide a novel function of Msi-1 protein in regulating stress granule formation and Sertoli cell survival upon heat-induced stress via p-ERK1/2 signaling.

Animal studies
Animal experiments were performed according to the Guiding Principles for the Care and Use of Animals of the Second Hospital of Tianjin Medical University, China. The mice were fed ad libitum with a standard diet, were housed under temperature-and lightcontrolled conditions (12 h light:12 h darkness), and were killed by cervical dislocation. This study was specifically approved by the Committee of Animal Research, Second Hospital of Tianjin Medical Universtiy.

Germ cells and primary Sertoli cell isolations
Total germ cells were isolated from adult mice using a mechanically based protocol, as previously described (Aravindan et al., 1996). The glass-wool filtration step was omitted to retain elongating/elongated spermatids and spermatozoa. Digestion of testis and isolation of Thy1 + testis cell populations were carried out as previously described (Kubota and Brinster, 2008). A modified method was used to isolate primary Sertoli cells from adult mouse testes (Cai et al., 2011).

Immunohistochemistry and immunofluorescence
For immunohistochemistry, testes were dissected in PBS and fixed in 4% paraformaldehyde for up to 24 h, stored in 4% ethanol, and embedded in paraffin. Tissues sections (5 μm thick) were cut and mounted on glass slides. Sections were deparaffinized and rehydrated; this was followed by antigen retrieval in 10 mM sodium citrate buffer. After being blocked with 5% BSA for 1 h, the sections were incubated with primary antibodies at 4°C overnight. After washing with PBS, secondary antibodies were applied for 1 h; this was followed by washing in PBS. Staining was visualized using a DAB substrate kit (Zhong Shan Technology, Beijing, China). For immunofluorescence, Sertoli cells cultured on coverslips were rinsed in PBS and fixed in 4% paraformaldehyde for up to 20 min. The coverslips were blocked in blocking buffer (goat serum, 0.3% Triton X-100 in PBS) at room temperature for 1 h and then incubated with primary antibodies overnight at 4°C. Coverslips were washed in 0.3% Triton X-100 in PBS and incubated with fluorescein isothiocyanate (FITC)-or tetramethylrhodamine (TRITC)-conjugated secondary antibodies (Jackson ImmunoResearch) for 1 h. The coverslips were washed in 0.3% Triton X-100 in PBS and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for identification of nuclei.

Western blotting
Western blotting analysis was conducted as previously described (Chen et al., 2008). In brief, the proteins were electrophoresed under reducing conditions in 12% SDS-PAGE gels and transferred to nitrocellulose membranes. The blots were incubated with primary antibodies overnight at 4°C and then with the corresponding horseradish peroxidase-labeled secondary antibodies for 1 h at room temperature. Specific signals were detected using the enhanced chemiluminescence Western blotting detection system.

TUNEL assay
TUNEL assays were conducted with the In Situ Cell Death Detection Kit, Fluorescein (Promega), according to the manufacturer's recommendations. The coverslips were washed in 0.3% Triton X-100 in PBS and counterstained with DAPI (Sigma-Aldrich) to identify the nuclei. Images were acquired with a Nikon DMR Epifluorescence Microscope and were captured by a Hamamatsu CCD camera.

Statistical analysis
Experiments run in duplicate were repeated at least three times. Statistical analysis of the data derived from immunoblotting was performed with the two-way ANOVA followed by Student's t test. Each bar represents the mean ± SEM of n = 3; *, p < 0.05.