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Proteasome α6 Subunit Negatively Regulates the JAK/STAT Pathway and Blood Cell Activation in Drosophila melanogaster

JAK/STAT signaling regulates central biological functions such as development, cell differentiation and immune responses. In Drosophila, misregulated JAK/STAT signaling in blood cells (hemocytes) induces their aberrant activation. Using mass spectrometry to analyze proteins associated with a negative regulator of the JAK/STAT pathway, and by performing a genome-wide RNAi screen, we identified several components of the proteasome complex as negative regulators of JAK/STAT signaling in Drosophila. A selected proteasome component, Prosα6, was studied further. In S2 cells, Prosα6 silencing decreased the amount of the known negative regulator of the pathway, ET, leading to enhanced expression of a JAK/STAT pathway reporter gene. Silencing of Prosα6 in vivo resulted in activation of the JAK/STAT pathway, leading to the formation of lamellocytes, a specific hemocyte type indicative of hemocyte activation. This hemocyte phenotype could be partially rescued by simultaneous knockdown of either the Drosophila STAT transcription factor, or MAPKK in the JNK-pathway. Our results suggest a role for the proteasome complex components in the JAK/STAT pathway in Drosophila blood cells both in vitro and in vivo.


INTRODUCTION
Regulation of blood cell differentiation and function is a central aspect in immune responses in animals. In addition to the effective activation of the immune cells, controlled silencing of activation is equally important; constant immune cell activity consumes energy and leads to detrimental processes such as autoimmune reactions (1,2). The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling is involved in controlling and regulating biological functions including cell differentiation, developmental processes and immune responses (3). In humans, JAK/STAT is central for blood cell homeostasis, and its aberrant activation can lead to myeloproliferative neoplasms (4,5). In Drosophila, misregulated JAK/STAT signaling in blood cells leads to blood cell activation and formation of tumor-like melanotic blood cell clusters (6)(7)(8). The Drosophila larval blood cell system consists of three main types of hemocytes: plasmatocytes, lamellocytes and crystal cells (9,10). Plasmatocytes, the main blood cell type, are round, macrophage-like cells found in all developmental stages in the circulation and in reservoir compartments (11)(12)(13). Plasmatocytes are responsible for the phagocytosis of pathogens and apoptotic cells (12,14,15). Lamellocytes are a specialized hemocyte type, produced as a response to infection by parasitoid wasps and other pathogens that cannot be phagocytosed (16). They encapsulate the intruder and produce melanin to seal the capsule (17). The third group is crystal cells, which function in the melanization response that is essential for wound healing (18,19). The activation of several signaling pathways, including the JAK/STAT, Toll, Ras-MAPK and the c-Jun N-terminal kinase (JNK) signaling pathways, is known to induce lamellocyte formation (6,20).
Drosophila JAK/STAT signaling is simpler than its human counterpart, and therefore Drosophila offers a prominent model for studying the JAK/STAT pathway, particularly in vivo (21). Instead of tens of ligands, four JAKs and seven STATs in humans (22), Drosophila only has one JAK (Hopscotch), one STAT (STAT92E) (23,24) and three cytokine-like ligands (Upd1, Upd2 and Upd3) (25,26). The signaling pathway is activated when one of the three ligands binds to the receptor Domeless (Dome), a Drosophila homolog to the human GP130 receptor (27). Ligand binding leads to the dimerization of Dome and thus, activation of the tyrosine kinase Hopscotch (Hop) (28). Hop activation induces the phosphorylation of the intracellular part of Dome, transphosphorylation of Hop and enables the SH2 domain of the STAT92E transcription factor to attach to the receptor. Hence, Hop phosphorylates STAT92E which dimerizes and translocates to the nucleus where it activates the target genes of the JAK/STAT pathway (29). Similarly to its mammalian counterpart, the Drosophila JAK/STAT pathway is involved in several processes including development, stem cell maintenance, immune and stress responses and larval hematopoiesis (25,30).
Due to its key role in regulating blood cells both in mammals and in insects, it is important to understand the negative and positive regulatory events controlling the activity of the JAK/ STAT pathway. We aimed at identifying novel factors that would interact with a known negative regulator of Drosophila JAK/ STAT signaling, the Eye transformer (ET/CG14225) (31,32), and therefore participate in the regulation of JAK/STAT activity. In our mass spectrometry study in S2 cells harboring a hemocyte-like identity, we identified several members of the proteasome complex as interactors of ET. Furthermore, we show that silencing Proteasome a6 subunit in vivo in Drosophila hemocytes induces JAK/STAT activation in them, leading to typical JAK/STAT induced hemocyte phenotypes, the appearance of melanotic nodules and the formation of different types of hemocytes usually present after an immune challenge. Hence, we show that proper functioning of the proteasome complex is crucial for keeping JAK/STAT signaling at bay in hemocytes in healthy animals.

MATERIALS AND METHODS
dsRNA Synthesis dsRNAs were produced as described previously (31). The primers used for dsRNA synthesis are listed in Supplementary Table 1.
The S2 cells were transiently transfected using Fugene6 reagent (Roche, Basel, Switzerland) following the manufacturer's instructions. The JAK/STAT pathway was activated by transfection with either the constitutively active pMT-hop Tum-l or with the pMT-upd1-myc plasmid. To measure the JAK/STAT pathway activation, a Turandot M-luciferase (TotM-luc) reporter construct, a kind gift from Professor Jean-Luc Imler (University of Strasbourg, France), was used. For studying the effect of RNAi knockdown of selected genes on the expression of ET-V5, S2 cells were transfected with either the pMT-empty or pMT-Upd1 plasmids together with the pMT-ET-V5 plasmid and selected dsRNAs. For studying the effect of RNAi knockdown of selected genes on the expression of endogenous STAT92E, S2 cells were transfected with the pMT-Upd1 plasmid for induction of the JAK/STAT pathway. The pMT-empty plasmid was used as a control for uninduced conditions. The protein production from the pMT plasmids was induced 24-48h post transfection by adding CuSO 4 to each well to a final concentration of 250-500 µM. For Toll and Imd pathway reporter assays, a Spätzle or Imd overexpressing plasmid was used to induce the pathway and Drosomycin-luc or Attacin-luc was used as the reporter, respectively (33,34). In all reporter assays, an Actin5C-b-galactosidase reporter plasmid was used to normalize the results for transfection efficiency and cell numbers as described previously (31). Luciferase and b-galactosidase reporter assays were carried out as previously described in (31).

Stable Transfection of S2 Cells With Selected Plasmids
The S2 cells were stably transfected with 9.5 µg of each plasmid construct (empty pMT-vector or pMT-ET-V5 ± pMT-upd1myc) together with 0.5 µg of the coHygro plasmid to enable Hygromycin-B selection of transfected cells. Transfections were carried out on 6-well plates using the Fugene6 transfection reagent (Roche, Basel, Switzerland). Three days post-transfection the transfected cells were selected by adding medium containing 150-300 µg/ml of Hygromycin-B (HyClone Laboratories, Inc. USA). The Hygromycin-B concentration was increased in every passaging up to 300 µg/ml. Under Hygromycin-B selection, only cells carrying the coHygro plasmid survive. From here onwards, the cells were grown under Hygromycin selection.

Cell Lysis, Protein Extraction and Protein Concentration Measurement
24-48h after the protein production from the pMT-plasmids was induced with CuSO 4 , the cells from three pooled wells, each containing 3 ml of S2 cell suspension, were collected per sample and centrifuged at 5000 x g for 3 min. The supernatants were discarded and 900 µl of lysis buffer (

Co-Immunoprecipitation of Protein Complexes With the V5 Antibody, SDS-PAGE and Silver Staining
Putative ET-interaction partners were co-immunoprecipitated from S2 cell protein lysates using Anti-V5 Agarose Affinity Gel (Sigma, #A7345, Israel) according to the manufacturer's instructions. The following lysates were used: S2 cells (neg. ctrl 1), empty pMT-vector (neg. ctrl 2), pMT-ET-V5 and pMT-ET-V5 + pMT-upd1-myc. 900 µl of each lysate, containing approximately 2.5 mg of protein, was incubated overnight with the Anti-V5 Agarose Affinity Gel, after which the complexes were washed 6 x 10 minutes with PBS containing protease and phosphatase inhibitor cocktail. All incubations and treatments were carried out at 4°C or on ice. Finally, loading buffer was added on the washed affinity gel containing protein complexes and the samples were boiled to release the proteins. Purified protein complexes were analyzed by SDS-PAGE and silver-staining.
For SDS-PAGE, Novex 10% NuPAGE Bis-Tris (Life Technologies, #NP0301BOX, USA) gels were used. Precision Plus Protein ™ Dual Xtra Standard (Bio-Rad, #161-0377, USA) was used as a marker. The electrophoresis was carried out for 45 min using MOPS buffer and NuPAGE Gel program (200 V, 120 mA, 25 W). To stain the protein bands on the gel, the Pierce ® Silver Stain for Mass Spectrometry kit (Thermo Scientific, #24600, USA) was used according to the manufacturer's instructions.

Mass Spectrometry and Identification of Protein Complex Components
The anti-V5 antibody purified samples were analyzed by mass spectrometry with the help of Tuula Nyman at the University of Helsinki. To identify the proteins in complex with ET, the anti-V5 purified lysates were separated on SDS-PAGE gels and silver stained as described above. The whole sample lanes on SDS-PAGE gel were cut into equal sized pieces after which an in-gel trypsin digestion was performed. The digested peptides were analyzed as described in (35). Flybase Query and Blast tools were used to process the obtained data. The data from the LC-MS/MS was searched against the SwissProt database through ProteinPilot with MASCOT. The following MASCOT search parameters were used: Drosophila melanogaster as species, carbamidomethyl modification of cysteine as a fixed modification and oxidation of methionine as a variable modification, trypsin digestion allowing one missed cleavage, 50 ppm mass tolerance and 0.2 Da fragment tolerance for peptides. False discovery rates for the LC-MS/MS data were 1.5-4%.

SDS-PAGE, Western Blotting and Protein Band Intensity Analysis
For studying the effect of RNAi knockdown of selected genes on the expression of pMT-ET-V5, protein lysates were prepared as above and the production of the ET-V5 protein was analyzed by SDS-PAGE and Western blotting. The total protein content of the lysates was set to 50 µg/lane. For SDS-PAGE, Biorad TGX 4-20% mini gels (Bio-Rad, #456-1093, USA) were used. PageRuler ™ Prestained protein ladder (Thermo Scientific #26616, Lithuania) was used as a marker. The electrophoretic separation of proteins was carried out for 20-25 min with 300 V using the Tris/Glycine/SDS buffer (Bio-Rad #1610732, USA).
For Western blotting, proteins were transferred from the SDS-PAGE gel either onto 0.45 µm nitrocellulose membrane (Hybond-C extra; Amersham Biosciences, RPN203E, United Kingdom) using the wet transfer technique with an in-house transfer buffer (25 mM Tris, 192 mM glycine, 8% methanol) and the NuPAGE Blot program (25 V, 160 mA, 1 h 15 min), or onto 0.2 µM nitrocellulose membrane using Trans-Blot turbo transfer packs (Bio-Rad, #1704158, USA) and equipment according to the manufacturer's instructions for TGX mini-gels. The membranes containing the transferred proteins were first incubated with blocking buffer (5% skimmed milk powder in PBS + 0.05% Tween 20 [PBST]) for 2 h at RT or overnight at 4°C. After blocking, the membranes were incubated with primary antibodies in blocking buffer for either 1-2h at RT or overnight at 4°C. The following antibodies were used: anti-V5-HRP conjugate (1:3000, Invitrogen ™ , Life Technologies, P/N 46-0708), anti-V5 antibody (1:3000, Invitrogen ™ , Life Technologies P/N 46-0705) and atubulin antibody (1:1000, clone DM1A, Sigma-Aldrich). After incubation, the membranes were rinsed three times with PBST and further washed with PBST 4 x 5 min at RT. Goat anti-mouse IgG (H+L) HRP secondary antibody (1:5000, ThermoFisher #G-21040) was used for detecting the mouse monoclonal antibodies (V5 antibody, a-tubulin antibody). Secondary antibody was incubated for 1 h at RT, after which the membranes were washed as before. Immunostained proteins were visualized using an ECL Western Blotting Detection Reagent (either with #RPN2232 by GE Healthcare Amersham ™ , UK or Westernbright, Advansta, USA) and imaged with the BioRad ChemiDoc ™ MP imager using the BioRad ImageLab software (Bio-Rad, California, USA) or by developing the signal on an X-ray film (Fuji Super RX-N film, Japan).
To estimate the amounts of the proteins of interest produced, band intensities on exposed films or imager images were analyzed with the FiJi-ImageJ-64-bit (ver1.51) software. Briefly, images were saved in grayscale in 32-bit mode, after which the bands to be analyzed were selected and their intensities plotted. The intensity of the ET-V5 protein band from the cell lysate was normalized to a-tubulin signal from that lysate. Plotted values were normalized to the control (GFP dsRNA-treated) protein band value. Per phenotype, 6-7 replicates were analyzed.
For detecting the JAK/STAT activity in vivo, the 10xStat92E-GFP [BL #26197 (40)], reporter combined with He-GAL4 to get 10xSTAT92E-GFP; He-GAL4 was used (a gift from Prof. Dan Hultmark). To activate JAK/STAT signaling, the constitutively active form of Drosophila JAK, UAS-hop Tum-l (41) was expressed in hemocytes. 15-20 females of a driver or genetic background control line were crossed with 10 males from transgenic construct lines and kept at 25°C and 12:12 light:dark cycle (LD12:12). Flies were placed daily in fresh vials, and vials with eggs were transferred to 29°C and LD12:12.

Flow Cytometry Analysis of Larval Hemocytes
Late 3rd instar larvae were washed in water with a brush until clean, placed in a 20 µl drop of 8% bovine serum albumin (BSA) in 1 x phosphate buffered saline (PBS) and carefully ripped open using forceps. The hemolymph was allowed to bleed out and the carcass was removed. The hemolymph sample was pipetted into a 1.5 ml Eppendorf tube with 80 µl of 8% BSA in PBS. 30 µl of the sample was run with a BD Accuri C6 flow cytometer (Becton, Dickinson & Company). Each genotype was analyzed in triplicate (3 x 10 larvae). For the eaterGFP and msnCherry reporter analysis, a 488 nm 50 mW solid-state laser and 510 +/-15 nm (FL1, GFP) and 610 +/-20 nm (FL3, mCherry) optical filters were used to capture the fluorescence signal. GFP-only, mCherry-only and non-fluorescent hemocytes were used to check for the location of these hemocytes and to deduct fluorescence spill over into a wrong channel. By using these reporters, five hemocyte populations can be detected, consisting of single and double positive hemocytes. Following the naming strategy presented in (42), these hemocyte are: plasmatocytes (GFP high ), activated plasmatocytes (GFP high ,mCherry low ), lamelloblasts (GFP low ), prelamellocytes (GFP low ,mCherry low ) and mature lamellocytes (mCherry high ). The complete procedure of the hemocyte flow cytometry with gating strategies is described in (42).
For the 10xStat92E-GFP reporter fluorescence measurement, the hemocyte population was separated from the debris based on forward scatter (FSC-A) and side scatter (SSC-A). This population was gated and used in subsequent analyses. A 488 nm 50 mW solid-state laser was used to excite the green fluorescent protein (GFP) and the emission was detected using a 510 +/-15 nm (FL1) optical filter. Non-fluorescent hemocytes (w GD ) were used as a negative control and hemocytes with hop Tum-l overexpression (10xStat92E-GFP;He-GAL4/UAShop Tum-l ) as a positive control. Hemocytes with a fluorescence intensity above the autofluorescence of the hemocytes in the FL1 channel were considered GFP-positive. A forward scatter area vs. height plot (FSC-A vs. FSC-H) was used to define the population of round hemocytes, representing the majority of hemocytes in a healthy Drosophila larva, the plasmatocytes. These hemocytes were gated, and the GFP fluorescence intensity was measured for all hemocytes gated in the FSC-SSC plot and separately only for the hemocytes falling into the plasmatocyte gate.
Hemocyte Imaging 3 rd instar larvae were bled in a drop of 1% BSA in PBS on a 12well glass slide, pooling hemocytes from three larvae per well. Hemocytes were allowed to attach and spread on the slide for 50 min, after which they were fixed for ten minutes using 20 µl of 3.7% paraformaldehyde. After fixation, wells were washed three times using cold 1 x PBS and permeabilized for 5 min using 0.1% Triton-X100. After washing three times using cold 1 x PBS, cells were incubated for 30 min with 20 µl of Alexa Fluor 680 -conjugated Phalloidin (Invitrogen), in a 1:50 dilution in 1% BSA in PBS, to stain filamentous actin. The wells were washed, and the samples mounted using ProLong Gold antifade reagent with DAPI (ThermoFisher Scientific) and Zeiss coverslips (thickness no. 1 ½, 18 x 18 mm). Slides were left to cure overnight at room temperature protected from light and imaged with Zeiss LSM 780 laser scanning confocal microscope using a 40x oil immersion objective at several random locations on the next day. 405 nm, 488 nm and 628 nm lasers were used to excite DAPI, GFP and AlexaFluor 688 nm, respectively, and emission was collected at 410-488 nm, 490-544 nm and 661-759 nm. Multiple layers were imaged in Z-plane at 0.36 µm intervals. ImageJ version 2.1.0/1.53 c was utilized to create stacked images. ImageJ and Adobe Photoshop (release 22.5.1) were used to enhance the fluorescence signal for better visibility, keeping modifications constant across the images.

Pupation and Eclosion Success and Larval and Pupal Melanotic Nodules
The effect of silencing Prosa6 in hemocytes on the number of successfully pupated and eclosed animals was assessed in msnCherry,eaterGFP;Hml D -GAL4;He-GAL4 flies crossed with UAS-Prosa6 GD , UAS-Prosa6 KK and w GD flies. Three to four replicate crosses of each genotype were made, and adults were transferred to fresh vials on three subsequent days. Eggs were counted from each vial and the vials were monitored daily for pupated/eclosed animals. At the same time, the number of pupae with melanotic nodules was recorded. Examples of pupae with melanotic nodules were imaged with a Nikon DS-Fi2 camera attached to a Nikon SMZ745T microscope and a Nikon DS-L3 camera control unit.
The prevalence of larval melanotic nodules was assessed for the same genotypes in three replicate crosses (100 animals each) by inspecting the larvae under a stereomicroscope. Example images of larvae (20x magnification) were taken with a Deltapix Invenio 10EIII camera (DeltaPix, Denmark) attached to a Nikon SMZ745T stereomicroscope using the DeltaPix InSight software.

Lifespan Experiment
The effect of silencing Prosa6 in hemocytes on the viability of the flies was assessed by monitoring the lifespan of the flies compared to controls. UAS-Prosa6 GD and UAS-Prosa6 KK RNAi lines were crossed with the msnCherry,eaterGFP;Hml D -GAL4; He-GAL4 flies (described above) at 25°C. As a control, msnCherry,eaterGFP;Hml D -GAL4; He-GAL4 flies were crossed with w GD flies. The fly eggs were transferred to 29°C, and this temperature was used for the entire lifespan of the flies. Every 2-3 days the number of living flies was recorded, and the flies were transferred to fresh food.

RNA Extraction and qRT-PCR from S2 Cells and Hemocytes
Expression levels of selected genes in S2 cells and hemocytes were measured by quantitative reverse transcriptase PCR (qRT-PCR) from extracted RNA. For monitoring expression levels in S2 cells, the cells were grown on 24-well plates, treated with selected dsRNAs and transfected with selected plasmids as described above. For studying expression levels in hemocytes in vivo, hemocytes from 50 dissected larvae were collected for each replicate; three biological replicates from three independent crosses were used per genotype. The RNA extraction both from hemocytes and cultured cells was performed using the TRI reagent (MRC, Thermo Fisher Scientific). S2 cells were harvested from culture plates by pipetting and centrifugation (5000 x g, 3 min), after which cells were homogenized and lysed in TRI reagent by pipetting up and down at least 10 times. Extraction from hemocytes was started by adding TRI reagent onto the frozen hemocyte pellet, and cells were homogenized and lysed by pipetting up and down at least 10 times. Thereafter the extraction was performed according to manufacturer's instructions. qRT-PCR was carried out from extracted RNAs (30-40 ng RNA/sample) using the iTaq Universal SYBR Green Onestep kit (Bio-Rad, Hercules, CA). The expression values obtained for selected genes were normalized to the expression of a gene encoding for ribosomal protein L32 (RpL32). The primers used are listed in Supplementary Table 1.

Statistics
Statistical analyses of luciferase assay measurements, qRT-PCR results and protein band intensity analyses were carried out using the two-tailed Student t-test for two arrays assuming equal variances. Statistical analyses of fly life span experiments were carried out with the log-rank (Mantel-Cox) test using Prism 6 (GraphPad) software. The difference was considered statistically significant if the p-value was < 0.05. Proportional data were analyzed using a Generalized linear model (GLM) with binomial distribution and combined with Tukey's post-hoc test for pairwise comparisons. The data on hemocyte numbers were analyzed using a negative binomial GLM followed by pairwise comparisons of groups using estimated marginal means. A Kruskal-Wallis rank sum test combined with Dunn's post hoc test was applied to GFP intensity comparisons in Figure 3D. R version 4.0.4 was used to perform GLM and Kruskal-Wallis rank sum tests.

Potential Interaction Partners of Eye Transformer (ET) Analyzed by Mass Spectrometry
In a previous study, we used a large-scale RNAi screen in Drosophila S2 cells to discover genes important for the Drosophila JAK/STAT pathway (31). In the screen we identified Eye transformer (ET) as a negative regulator of the JAK/STAT pathway (31). To screen for putative ET interactors, we created a Drosophila S2 cell-line stably overexpressing the ET protein with a V5 tag (ET-V5), and affinity-purified all the proteins in the ET complex with the V5 antibody. In Supplementary Figure 1A, it is shown that the ET-V5 signal can be detected with the V5-antibody (a-V5) in the lysates from ET-V5-expressing S2 cells. Since this control experiment showed that ET-V5 is successfully captured with this method, we next affinity purified proteins in complex with ET, separated them using SDS-PAGE and visualized the proteins by silver staining (Supplementary Figure 1B). Each line was cut, digested with trypsin and the protein composition was determined by mass spectrometry.
The potential ET interaction partners were studied in two situations, either with or without overexpression of the Dome receptor ligand upd1. When the ligand is present, the JAK/STAT pathway is activated, whereas without the ligand, the pathway stays inactive. In addition, untreated S2 cells and S2 cells transfected with an empty pMT vector were used as negative controls. After proteins that were also found in the negative controls were excluded, we identified in total 173 Drosophila melanogaster proteins in complex with ET under the conditions where the JAK/STAT pathway was inactive (ET-V5 alone) and 175 proteins when the ligand Upd1 was present (ET-V5 + Upd1myc). Out of these, 136 proteins were found in both situations (ET with and without Upd1 induction). The mass spectrometry raw data, including putative candidates for JAK/STAT pathway regulation, is shown in Supplementary Table 2.

The Proteasome Complex Components Negatively Regulate JAK/STAT Pathway Activation in S2 Cells
The mass spectrometry screen identified in total 212 unique putative interaction partners of ET (Supplementary Table 2). These genes/proteins were compared to the Kallio & coworker's screen for JAK/STAT components (31) as well as other literature, to search for links to the JAK/STAT pathway, including immunity, hematopoiesis or stress response (25). In total, nine genes were selected for further study: four gene products where the interaction was detected with ET alone, three gene products where the interaction with ET was detected upon Upd1 induction and two gene products where the interaction was detected with ET both with and without Upd1 induction. The selected genes are listed in Table 1, with a description of the gene function deduced from the www.flybase.org gene pages. To assess if the association of ET with these nine gene products signifies a regulatory effect on the JAK/STAT pathway, we carried out a JAK/STAT pathway reporter assay in S2 cells with dsRNAs targeting the genes ( Figure 1A). As shown in Figure 1A, induction with overexpression of either upd1 (gray bars) or hop Tum-l (black bars) causes an activation of the JAK/STAT pathway, as assessed by the production of the luciferase signal from the JAK/STAT target gene luciferase reporter construct Turandot M-luciferase (TotM-luc). GFP dsRNA was used as a negative control in both assays. Knockdown of ET was used as a positive control in measuring the Upd1-induced TotM-luc activity, but as ET is upstream of Hop, its knockdown does not have an increasing effect on Hop Tum-l -induced TotM-luc activity, but instead, a decreasing effect, as discussed e.g., in (31). Of the nine RNAi treatments tested, only Proteasome a6 subunit (Prosa6) causes hyperactivation of the JAK/STAT pathway upon hop Tum-l induction. Prosa6 is a component of the core particle of the proteasome complex [see below (43)]. The Upd1-induced TotMluc signal is enhanced when G protein a o subunit (Gao), Myosin light chain cytoplasmic (Mlc-c), Moesin (Moe), Prosa6 or Twins (tws) are knocked down by targeted RNAi.
The proteasome is a large protein complex consisting of a 20S core particle capped by 19S regulatory particles (43). Among the seven alpha rings of the proteasome core particle, RNAimediated silencing of Prosa1, Prosa6 and Prosa7 caused hyperactivation of the JAK/STAT pathway as did silencing of four of the seven beta ring sub-particles and one related gene, namely Prosb2, Prosb3, Prosb5, Prosb7 and Prosb2R1. Moreover, silencing of the Drosophila 19S regulatory particle genes Rpn1, Rpn2 and Rpn3, as well as proteasome-related chaperone protein Pomp, had similar effects (Table 2 and Figure 1B). In conclusion, RNAi-mediated silencing of the expression of proteasome genes causes hyperactivation of the JAK/STAT pathway in S2 cells [ Table 2 and Figure 1B    The total of twenty-five genes from the study of ET interaction partners ( Table 1 and Figure 1A) and from the Kallio & coworkers' RNAi screen for negative regulators of the JAK/ STAT pathway ( Table 2 and Figure 1B (31), were selected for further study. To investigate if these genes act in the regulation of other immune-related pathways besides JAK/STAT, we tested if their silencing affects the activation of the Imd or Toll pathways using reporter assays. To investigate effects on the Imd pathway, the pathway was induced by transfecting S2 cells with the Imd plasmid and relevant dsRNAs. GFP was used as a negative control and Relish dsRNA as a positive control. As shown in Figure 1C, most of the genes studied do not regulate the Imd pathway; however, knockdown of effete (eff) and Prosb2R1 leads to a slight decrease in pathway activation compared to GFP. Effete, the Drosophila homolog of the Ubc5 E2-ubiquitin conjugating enzyme, has previously been shown to be needed for Imd ubiquitination (45). Knockdown of Prosa6, Prosa7, heph and Fur1 leads to a slight elevation in the Imd pathway activation, whereas when Kap-a3, IntS2, and IntS6 are silenced, the pathway is markedly upregulated ( Figure 1C).
To investigate the effects of silencing of the selected 25 genes on the Toll pathway, the pathway was induced by overexpression of the Spz C106 plasmid. A MyD88 dsRNA treatment was used as a positive and GFP as a negative control of the pathway. As shown in Figure 1D, the silencing of several genes resulted in a reduction in Toll pathway activity, including mainly components of the proteasome: Rpn-genes, Pomp, Prosa-and Prosb-genes (but not Prosb2R1). A statistically significant reduction was seen also with Ced-6, Gao, Twins (tws) and heph. Three genes that negatively regulate the Toll pathway were identified: IntS2, IntS6 and eff. The effect of IntS2 and IntS6 on the Toll pathway has been previously shown (46), but the effect of eff on the Toll pathway has not been previously studied and remains to be investigated.
As a summary, the integrator complex members (IntS2 and IntS6) appear to negatively regulate all pathways tested indicating that their effect is not specific to the JAK/STAT pathway. Although Effete co-localizes with ET, it does not seem to regulate the TotMluc reporter gene activation mediated by the JAK/STAT pathway. Instead, Effete appears to negatively regulate the Toll pathway in S2 cells, which requires further exploration in the future. RNAi against the proteasome complex components does not have major effects on the Imd pathway, but silencing proteasome complex members decreases the activity of the Toll pathway indicating that the proteasome positively regulates the Toll pathway. Cactus, the Drosophila homologue of the mammalian Inhibitor of kB (IkB), is known to be degraded by the proteasome upon induction of the Toll pathway (47,48), so it is plausible that silencing components of the proteasome complex leads to accumulation of the Cactus protein and reduction in the activity of the Toll pathway also in this context. The effect of RNAi against the candidate genes identified in A and B on the Spz C106 -induced Toll pathway reporter (Drs-luc) activity. In all reporter assays, n=4 per dsRNA treatment, and luciferase reporter values were normalized to the values of the Act5C-bgal reporter activity. The relative reporter activity value of cells with an activated pathway treated with the negative dsRNA control (GFP) is set to 1. Statistical analyses were carried out using Student t test for two samples assuming equal variances. *p, 0.05, **p, 0.01, ***p, 0.001. n.s., not significant.

Prosa6 Silencing Leads to Reduced Expression of ET and Reduced Amounts of the ET Protein
Because the proteasomal genes were identified in JAK/STAT pathway regulation in S2 cells ( Figures 1A, B), we selected Prosa6 to study in more detail. To study the interplay between ET and Prosa6 in the JAK/STAT pathway, we carried out a double knockdown experiment with ET and Prosa6. As shown in Figure 2A, the TotM-luc activity was induced with the pMThop Tum-l construct. As previously shown, ET knockdown reduces this hop Tum-l -induced TotM-luc activity [(31) and Figure 1A], while Prosa6 knockdown causes hyperactivation of this activity ( Figure 1A). When both ET and Prosa6 are knocked down simultaneously (Prosa6+ET), the result is additive, indicating that the effect of ET and Prosa6 proteins on the JAK/STAT pathway activation is partially independent.
Next, we investigated what happens to the expression of ET upon Prosa6 knockdown. S2 cells were treated with Prosa6 dsRNA and control dsRNAs and either transfected with pMT-Upd1-myc, which induces the JAK/STAT pathway, or transfected with the pMT-empty plasmid for control. ET expression values from RNAs extracted from the cells were measured and normalized with RpL32 expression. As shown in Figure 2B, Prosa6 knockdown causes reduction in the transcription of ET both in conditions where the JAK/STAT pathway is inactive, and in those where it is activated with overexpression of Upd1.
Further, we investigated the effect of Prosa6 knockdown on the amount of ET protein, by treating S2 cells with Prosa6 dsRNA and control dsRNAs and transfecting them with pMT-ET-V5 together with pMT-Upd1-myc, which induces the JAK/ STAT pathway. Treatment of cells with the pMT-empty vector and GFP dsRNA was used as a control. Cellular lysates were prepared and subjected to SDS-PAGE electrophoresis, Western blotting and antibody treatments and imaged. The ET-V5 protein band intensity was normalized to a-tubulin band intensity values in each sample. As shown in Figures 2C, D, the amount of ET-V5 protein is reduced in Prosa6 dsRNAtreated cell lysates compared to the negative control GFP dsRNA-treated lysates. Figure 2C shows an example of one experiment, and Figure 2D shows the quantification from three independent experiments including in total six or seven replicates per treatment. In ET dsRNA-treated cells, the ET-V5 band is not at all visible indicating that RNAi in S2 cells silences the protein production of the targeted gene very efficiently.
In conclusion, upon knockdown of Prosa6, ET transcription as well as the amount of the ET protein is decreased. As ET is the key negative regulator of the JAK/STAT pathway, these results in part explain the hyperactivation of the pathway by knockdown of Prosa6. Members of the 26S proteasome complex, components of the 19S regulatory particle

CG1519
Proteasome a7 subunit Prosa7 predicted endopeptidase activity PSMA3 (2 Members of the 26S proteasome complex, components of the 20S core particle, b subunits (inner rings)

CG3329
Proteasome b2 subunit Prosb2 predicted endopeptidase activity PSMB7 ( Prosa6 Silencing in Hemocytes Activates JAK/STAT Signaling Since our previous results indicated involvement of the proteasome complex in regulating JAK/STAT signaling in S2 cells, we next tested if this was also the case in vivo in Drosophila larval hemocytes. We used a STAT reporter 10xSTAT92E-GFP combined with a hemocyte driver He-GAL4 as a read-out of the JAK/STAT activity in hemocytes using flow cytometry. As a positive control we overexpressed a constitutively active Drosophila JAK/STAT pathway component hopscotch (hop) in hemocytes (10xStat92E-GFP;He-GAL4/UAS-hop Tum-l ), and as expected, detected an increase in the Stat92E-GFP signal compared to control hemocytes. We also detected a clear increase in the reporter expression when knocking down the Proteasome a6 subunit (10xStat92E-GFP;He-GAL4/UAS-Prosa6 GD ) compared to control hemocytes expressing only the reporter ( Figure 3A).
The FSC-area (FSC-A) vs. FSC-height (FSC-H) plot is often used to exclude cell doublets from further flow cytometry analysis taking advantage of the fact that round particles will appear as a population at a 45°angle, while doublets have larger area than height. We observed that while the control animals had mainly round hemocytes (plasmatocytes) (49), appearing at a 45°a ngle in the FSC-A vs. FSC-H plot, Prosa6 silencing resulted in cells detected outside of this area, similar to when hop Tum-l was overexpressed in hemocytes ( Figure 3B). Overexpression of hop Tum-l is known to cause hematopoietic neoplasia leading to the formation of melanotic nodules and a specific hemocyte type, the lamellocyte (6,42). Furthermore, Avet-Rochex and coworkers (50) identified Prosa6 (then called Pros35) as a suppressor of melanotic nodules in Drosophila larval hemocytes. Therefore, we reasoned that at least a fraction of these hemocytes outside the 45°area might be lamellocytes, which, due to their flat and discoidal morphology (49), fall outside the area where the round plasmatocytes are detected in the control animals. Since lamellocytes can be easily separated from plasmatocytes by morphology, we stained hemocytes from 10xStat92E-GFP;He-GAL4/UAS-Prosa6 GD and 10xStat92E-GFP; He-GAL4/w GD 3 rd instar larvae with the F-actin stain Phalloidin to visualize hemocyte morphology. We found that hemocytes from the Prosa6 knockdown larvae had more variation in their morphology when compared to the control hemocytes and observed the presence of hemocytes with a lamellocyte morphology ( Figure 3C).
Next, we analyzed the GFP signal intensity of the Stat92E-GFP reporter and the proportion of fluorescent hemocytes separately in all hemocytes and in the plasmatocyte fraction  for (A, B). (C) Fluorescence microscopy of hemocytes from the control and Prosa6 knockdown larvae expressing the 10xSTAT-GFP and stained with the nuclear stain DAPI and the F-actin stain Phalloidin. Hemocytes with lamellocyte morphology (marked with arrowheads) were found in Prosa6 knockdown larvae but not in controls. Of note, DAPI staining appears dimmer in the Prosa6 knockdown sample, but this was likely due to slide-to-slide variation. Scale bars 10 µm. (D) Quantification of 10xSTAT-GFP signal intensity and percentage of fluorescent hemocytes in controls and in the Prosa6 knockdown animals. All hc, the whole hemocyte population; pc, hemocytes inside the gate shown in (B). Hemocytes were analyzed from two replicates of w GD (nine animals each) and three replicates of Prosa6 GD (10 animals each), comprising of an order of 10 4 cells in each replicate. Error bars show mean and lower and upper confidence limits (cl). Grey dots represent individual animals. ***p < 0.001. Intensity values were analyzed using a Kruskal-Wallis rank sum test combined with Dunn's post hoc test. The proportions of fluorescent cells were analyzed using a GLM with binomial distribution combined with Tukey's post-hoc test.
( Figure 3B). In both cases, the GFP signal was significantly higher in the hemocytes where Prosa6 was silenced compared to controls ( Figure 3D, i). Also, the fraction of GFP-positive hemocytes increased significantly in both populations after Prosa6 knockdown ( Figure 3D, ii). These data indicate that silencing Prosa6 leads to activation of JAK/STAT signaling also in vivo.
Silencing Prosa6 in Hemocytes Leads to Activation of Hemocytes and Differentiation of an Infection-Induced Hemocyte Type, the Lamellocyte Prompted by the observation of the appearance of lamellocytes and JAK/STAT reporter activation after Prosa6 silencing in hemocytes, we studied the hemocyte phenotype further. To investigate if silencing of Prosa6 in hemocytes causes hemocyte differentiation, we utilized hemocyte reporter constructs eaterGFP (expressed in plasmatocytes) and msnCherry (expressed in lamellocytes) combined with the Hml D -GAL4 and He-GAL4 to silence Prosa6 in hemocytes and to quantify hemocyte types using a flow cytometer. Flow cytometry analysis of hemocytes showed that while in a wildtype larva, the majority of the hemocyte pool consists of eaterGFP-positive plasmatocytes, the silencing of Prosa6 caused aberrant lamellocyte differentiation and hemocyte activation ( Figure 4A, i-iii). The hemocyte response was stronger with the UAS-Prosa6 GD construct, where the numbers of all identified hemocyte types were increased, including lamelloblasts, the putative lamellocyte precursors, and prelamellocytes ( Figure 4B, i-vi). A similar, but milder, response was seen also with the UAS-Prosa6 KK construct. Furthermore, around 25% of Hml D -GAL4;He-GAL4/UAS-Prosa6 GD larvae exhibited melanotic nodules, while in Hml D -GAL4;He-GAL4/UAS-Prosa6 KK larvae this phenotype was again milder ( Figure 4C, i-ii).
Since silencing Prosa6 in hemocytes led to hemocyte activation and melanotic nodule formation at the larval stage, we further studied the effects of Prosa6 silencing on egg-to-adult development and the lifespan of the flies. We found that while egg-to-pupal development of the animals with Prosa6 silencing in hemocytes was comparable to the controls ( Supplementary  Figure 2A), the pupal eclosion rate was lower in animals expressing UAS-Prosa6 GD in hemocytes, whereas animals expressing UAS-Prosa6 K K eclosed at a normal rate (Supplementary Figure 2A). Inspection of the pupal cases revealed melanotic nodules in a fraction of animals with Prosa6 silencing (1% in msnCherry,eaterGFP;Hml D -GAL4;He-GAL4//UAS-Prosa6 KK and 20% in msnCherry,eaterGFP;Hml D -GAL4;He-GAL4//UAS-Prosa6 GD ; Supplementary Figure 2B), similar to the levels observed in larvae ( Figure 4C). The lifespan of the msnCherry,eaterGFP;Hml D -GAL4;He-GAL4/ UAS-Prosa6 GD flies, both males and females, was shorter than that of the control flies, whereas the lifespan of msnCherry, eaterGFP;Hml D -GAL4;He-GAL4/UAS-Prosa6 KK flies was not affected (Supplementary Figures 2C, D). When the expression of Prosa6 normalized to RpL32 was measured from hemocytes by qRT-PCR, it was shown that in the Hml D -GAL4;He-GAL4/ UAS-Prosa6 GD hemocytes, Prosa6 expression was 43 ± 14% from the respective control (w/Prosa6 GD ), whereas in the Hml D -GAL4;He-GAL4/UAS-Prosa6 KK hemocytes, Prosa6 expression was 52 ± 17% from the control (w/UAS-Prosa6 KK ) hemocytes (Supplementary Figure 2E).
To conclude, knocking down Prosa6 in hemocytes causes immune activation characterized by the differentiation and activation of hemocytes and the formation of melanotic nodules, but also decreases the viability of the flies. It is possible that there is a threshold level of silencing needed for the full phenotypic effects, as with the KK line, only the mild activation of hemocytes, but not the other phenotypes, is seen.

JAK/STAT and JNK Signaling Are Needed for the Full Hemocyte Activation Caused by Prosa6 Silencing
To further verify that Prosa6 silencing induces hemocyte activation via JAK/STAT signaling, we opted for a genetic rescue experiment. We knocked down Prosa6 using the GD construct simultaneously with Stat92E and checked the effect on the hemocyte phenotype. Since also the JNK pathway has been shown to be important in lamellocyte formation (6), we also tested whether it has a role in hemocyte activation in the Prosa6 silencing background. To this end, we knocked down Prosa6 simultaneously with hemipterous (hep), a Drosophila JNKpathway component and checked the hemocyte composition. Knocking down Stat92E alone in hemocytes resulted in a mild activating effect; a small number of lamelloblasts and lamellocytes were detected in an otherwise wild type background ( Figure 5A). Knocking down hep led to a reduction in total hemocyte numbers compared to the control ( Figure 5B). Neither Stat92E nor hep knockdown in the Prosa6silenced background led to the rescue of total hemocytes, or of plasmatocytes, lamelloblasts or activated plasmatocytes, back to the levels detected in the control animals ( Figures 5A, B, i-iv). Both, however, reduced the formation of prelamellocytes and lamellocytes induced by Prosa6 silencing (Figures 5A, B, v-vi). Even though knocking down neither Stat92E nor hep in the Prosa6 background rescued the hemocyte phenotype completely to the wild-type levels, the number of lamellocytes produced by the Prosa6 knockdown (600 on average) was reduced approximately seven-fold with Stat92E knock-down (51) and four-fold with hep knockdown (148). Taken together, these results suggest that Prosa6 silencing-induced hemocyte activation requires the function of the JAK/STAT pathway, and that the JNK pathway functions in parallel or downstream of JAK/STAT eliciting hemocyte activation.

DISCUSSION
In this study we have investigated the potential interaction partners of ET, and other putative regulators of the JAK/STAT pathway using our previously unpublished RNAi screen data. Among the 25 genes that were selected for further study, 12 have a described function in the proteasome pathway. These were Rpn1, Rpn2, Rpn3, Prosa1, Prosa6, Prosa7, Prosb2, Prosb3, Prosb5, Prosb7, Prosb2R1 and Pomp, all of which caused elevation of TotM-luc activity upon activation of the JAK/ STAT pathway by hop Tum-l in S2 cells. The 26S proteasome is a large complex composed of many subunits and under normal conditions, it degrades most proteins in the cell (52). It is known that the half-life of a protein varies from minutes to months, but at some point, every protein is marked and brought to degradation. In eukaryotes, unwanted proteins are marked for proteasomal degradation mainly with K48-linked polyubiquitin chains (53), which are recognized by the proteasome 19S regulatory particle. The regulatory particle functions in binding the ubiquitinated target proteins and deubiquitinating, unfolding and translocating them to the 20S proteasome core particle for cleavage (43). The core particle is a barrel shaped structure containing two outer rings each formed by seven a-subunits, and two inner rings formed by seven b-subunits. The b-subunits create a chamber where the proteolytically active sites are located. The N-termini of a-subunits form a gate that lets the substrates (target proteins) in through the central a-ring channel (52).
The ubiquitin-proteasome pathway has been implicated in the regulation of the JAK/STAT pathway in mammals: treatment with proteasome inhibitors prolongs the activation of the JAK/ STAT pathway in response of many cytokine stimuli, e.g (54- He-GAL4/w GD and msnCherry,eaterGFP;Hml D -GAL4; He-GAL4/w KK ) larvae and from larvae with Prosa6 silencing in hemocytes (msnCherry,eaterGFP;Hml D -GAL4; He-GAL4/UAS-Prosa6 GD and msnCherry,eaterGFP;Hml D -GAL4; He-GAL4/UAS-Prosa6 KK ). Each genotype was replicated three times, 10 animals in each replicate. Error bars show mean and lower and upper 95% confidence limits (cl). Grey dots represent individual animals. Data on hemocytes were analyzed using a negative binomial GLM. Stars indicate a significant difference when compared to the control sample. The two control backgrounds were also compared to each other. n.s., not significant; ***p < 0.001; pc, plasmatocyte; act pc, activated plasmatocyte; lb, lamelloblast; pre lc, prelamellocyte; lc, lamellocyte. (C), i) Examples of larvae without and with melanotic hemocyte aggregates of varying sizes, some of which are marked with arrowheads. For nodule quantification, each genotype was replicated three times, with 100 animals in each replicate (ii). Data were analyzed as a proportion of animals bearing nodules, using a GLM with binomial distribution combined with Tukey's post-hoc test. ***p < 0.001; **p < 0.01. 56). The mechanisms of proteosome-related regulation of the JAK/STAT pathway are complex: treatment with proteasome inhibitors causes stabilization of tyrosine-phosphorylated forms of STAT1 (56), STAT4, STAT5, STAT6 (57) and Jak2 (58), and thereby prolonged JAK/STAT activation. Furthermore, treatment with proteasome inhibitors also causes stabilization of the complex of the Jak2 protein with SOCS-1 (Suppressor Of Cytokine Signaling-1); SOCS-1 expression would normally enhance the proteasomal degradation of Jak2, but when the proteasome function is blocked, the Jak2/SOCS-1 complexes accumulate (58). So in mammals, the SOCS proteins (eight proteins, SOCS-1-7 and CIS) (59) bring another layer to the regulation of the JAK/STAT pathway. The Drosophila JAK/ STAT pathway has similar core pathway components as mammals, but there is less redundancy. For instance, in Drosophila, three members of the SOCS family have been identified, out of which Socs36E appears to be the main negative regulator of the pathway (25,60). Similarities have been found also in the regulation of the Drosophila and mammalian JAK/STAT pathways, such as between the Drosophila non-signaling receptor/inhibitor ET and the mammalian gp130, the soluble form of which can inhibit signaling (25,31,61).
We show that the knockdown of the Proteasome a6 subunit results in reduction in ET transcription, and reduction in the amount of the ET protein in Drosophila S2 cells (31). It appears that Prosa6 expression is needed for full ET expression to prevent aberrant activation of the JAK/STAT pathway. This regulation may be very complex: for example, Prosa6 knockdown, i.e., inhibition of the normal proteasome function, may cause an accumulation of ii-vi) Differential hemocyte counts. "-/+" indicate the presence of Prosa6 and hep knockdowns. Stars refer to the statistical difference compared to the wild-type control larvae (msnCherry,eaterGFP;Hml D -GAL4;He-GAL4/w GD ), the first sample in each plot. Underlined stars refer to the statistical difference of the simultaneous knockdown of Prosa6 and hep (msnCherry, eaterGFP;Hml D -GAL4;He-GAL4/UAS-Prosa6 GD ;UAS-hep GD ) to the single knockdowns of Prosa6 (short line) and hep (longer line). Each genotype was replicated three times, except for the msnCherry,eaterGFP;Hml D -GAL4;He-GAL4/w GD control, which was replicated 6 times, 10 animals in each replicate. Note that the control and Prosa6 hemocyte data is the same in (A, B) and has been plotted separately for clarity. Statistical analyses have been conducted on the whole dataset and pvalues adjusted according to multiple comparisons. Data was analyzed using a negative binomial GLM. n.s., not significant; *p < 0.05; ***p < 0.001; pc, plasmatocyte; act pc, activated plasmatocyte; lb, lamelloblast; pre lc, prelamellocyte; lc, lamellocyte.
other regulatory protein(s) causing a reduction in ET expression.
The reduction mechanism appears to inhibit ET production already at the transcriptional level, and this effect is seen in both basal conditions and in conditions, where the JAK/STAT pathway is activated.
Moreover, we show that silencing Prosa6 in Drosophila hemocytes in vivo and thereby blocking the proteasomal degradation/turn-over step leads to overactivation of JAK/ STAT signaling, aberrant activation of immune cells and the formation of clusters of activated immune cells, known as melanotic nodules or pseudotumors. Our findings are in line with an earlier in vivo RNAi screen conducted by Avet-Rochex & coworkers (50), who looked for genes involved in blood cell homeostasis in Drosophila. In the screen, they identified Prosa6 (Pros35) as a suppressor of melanotic nodules in hemocytes. Moreover, we show that there is partial rescue of the Prosa6 knockdown-induced hemocyte activation phenotype not only with knockdown of STAT92E, but also hemipterous (hep), the Jun-kinase in Drosophila. These results may indicate that the pathways act synergistically, both being needed for full hemocyte activation. For example, the JNK and JAK/STAT pathways act in cooperation in the wing imaginal disc resulting in the loss of cell fate specification as a response to damage (62). Alternatively, it has been shown that the JAK/STAT ligands upd1-3 are transcriptional targets of the JNK pathway (63). Therefore, it is possible that the silencing of the JNK pathway leads to reduced upd expression, and hence has a dampening effect on the overall activity of the JAK/STAT signaling in hemocytes. So, in addition to the JAK/STAT pathway activation, the JNK pathway seems to be needed for full blown hemocyte activation brought upon by Prosa6 silencing, but the exact mechanism of this interaction remains to be elucidated. The proteasome has also been shown to be involved in the turnover and regulation of several key proteins in immune signaling. On the one hand, proteasomal degradation of inhibitory molecules such as the Inhibitor of kB (IkB) is essential for pathway activation (64) but on the other hand, shutting down un-needed immune activation is equally important, and proteasomal degradation of key factors is one such regulatory mechanism (58,65). In Drosophila, it has been shown that the proteasome represses the Imd pathway, probably by causing the degradation of Relish (NF-kB factor in the Drosophila Imd pathway) (66). Here we show that the proteasome is needed for proper Toll pathway activity, likely because Cactus (Drosophila IkB in the Toll pathway) has to be degraded upon pathway activation (47,48).
In addition to proteasome-related genes, we identified additional genes that interact with ET and/or affect the JAK/STAT activity (Gao, Gai, Mlc-c, Moe, tws, eff, Fur, heph, Kap-a3, IntS2 and IntS6). Importantly, the effect of a specific knockdown was dependent on the means of the pathway activation. Silencing of two out of eight genes studied with both inducers (Upd1 and Hop Tum-l ) resulted in a change on the TotM-luc reporter expression with both inducers: Mlc-c (to the same direction) and tws (to opposite directions). Mlc-c forms an essential light chain of Non-muscle myosin II, which is involved in shaping the actin cytoskeleton of cells in e.g., development (67). Since JAK/STAT functions in the control of myogenic differentiation (68), it might be that in this case the activation of the signaling pathway is in response to reduced levels of myosin II. Drosophila tws encodes a regulatory B/PR55 subunit of protein phosphatase 2A (PP2A), and it has been shown to play a role in e.g., cell division, tissue patterning and multiple signaling pathways (69,70). In human T-cell lines, inhibition of PPA2 has been shown to attenuate at least STAT3, STAT5 and STAT6 function (71)(72)(73), and there is some indication that it may act similarly in Drosophila neuroblasts (74).
Silencing of all the rest of the genes appeared to affect the JAK/ STAT signaling activity only in a specific induction context. It is also possible that in some cases, the lack of significant effect may be due to insufficient silencing of the corresponding genes, since in this screen for putative interactors, we only tested one dsRNA per gene. Gao and Gai belong to the conserved family of the heterotrimeric G protein a subunits, which act as effector molecules of G-protein coupled receptors (GPCRs) (75). Both Gao and Gai were found to activate STAT3 in a murine fibroblast cell line (76,77), whereas we found that knockdown of Gao enhanced the Upd1-induced JAK/ STAT pathway activity and knock-down of Gai reduced the Hop Tum-l -induced JAK/STAT. Moesin is a member of the FERM domain (band 4.1, Ezrin, Radixin and Moesin), which is also one of the protein domains at the N-terminus of Jaks enabling the adaptor and scaffolding interactions (78,79). Similarly in Drosophila, moesin is a member of the FERM protein domain, and is an important factor in processes including cell adhesion, cell movement and membrane trafficking (51).
The ubiquitin-conjugating enzymes function in mediating a variety of ubiquitin modifications, including the K48-linked polyubiquitination that directs the ubiquitinated proteins for degradation by the 26S proteasome (80) as well as K63-linked activating polyubiquitination events (45,81). Eff is an E2 ubiquitin-conjugating enzyme, and it has previously been shown to be needed for the K63-linked polyubiquitination of the cleaved Imd molecule for functional Imd signaling (45). Similarly to the Imd pathway, eff knockdown has a positive regulatory effect on the Hop Tum-l -induced JAK/STAT pathway in S2 cells. On the other hand, our results indicate that Eff negatively regulates the Toll pathway in S2 cells, but it appears not to be needed in the fat body mediated defense in vivo. Furins are a family of evolutionarily conserved serine endoprotease enzymes that cleave precursor proteins into their mature forms (82). In accordance with our results, silencing of Drosophila Fur1 in the fat body has previously been shown to elevate the transcription of stress response genes including the JAK/STAT pathway target gene TotM (83).
The Notch signaling pathway is well conserved from flies to humans and has been shown to be essential for blood cell development and lineage speciation (11,84). Kap-a3 (also called importin-a3) has been shown to be required for regulating Notch signaling (85) and Heph has been shown to be an important protein regulating Notch signaling in wing development (86). Both Kap-a3 and Heph negatively regulate the Hop Tum-l -induced JAK/STAT pathway in S2 cells indicating interplay between the Notch and JAK/STAT signaling pathways in this context. The integrator complex genes, in turn, have been suggested to have multiple functions: the main function of the complex is to mediate the 3' processing of small nuclear RNAs (87). In addition, silencing of the zebrafish Integrator 5 or 11 has been shown to result in defects in hematopoiesis; this was suggested to be due to misprocessing of snRNA, which leads to splicing defects in the mRNA of genes required for hematopoiesis (88). We show that silencing of IntS2 and IntS6, members of the Drosophila Integrator complex, results in an increase in the activity of the JAK/STAT pathway. The integrator complex has previously also been shown to negatively regulate the Toll pathway (46).
We also identified proteins that did interact with ET but did not affect JAK/STAT activity in our S2 assay (Rala and ced-6). Rala, which belongs to the family of Ras-like (Ral) small GTPases homologous to Ga-proteins, is shown to be required cellautonomously in regulating polar-cell specific markers, including the JAK/STAT pathway ligand Upd, in the developing oocyte (89). Although associating in the complex with ET, it appears that in the S2-cell context, Rala knockdown affects neither the Upd1 nor the Hop Tum-l -induced activity of the JAK/STAT pathway. Ced-6 has been identified as an essential adaptor protein for apoptotic clearance of unneeded cells by hemocytes in the developing embryo (90) as well as phagocytosis of S. aureus gram-positive bacteria in Drosophila adults upon infection (91). It has not previously been implicated in the regulation of JAK/STAT signaling to our knowledge.
The JAK/STAT signaling pathway, like many other signaling pathways, has core and tissue/cell type specific signaling outcomes (92,93). Accordingly, also the regulatory events outside the core pathway components might vary among the cell types. Our data revealed several putative JAK/STAT regulators in S2 cells, whose mode of action was often dependent on the way the pathway was activated. Importantly, silencing of the proteasome complex member Prosa6 consistently enhanced the JAK/STAT pathway activity. Moreover, knockdown of Prosa6 is sufficient to induce the activation of Drosophila blood cells, the hemocytes, in vivo, via a JAK/STAT pathway-dependent mechanism. The interplay between the Drosophila JAK/STAT pathway components and negative regulators including ET and the proteasome appears complex and remains to be further explored in detail.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.