Figure 7Rab39a expression in splenocyte subsets was measured using an FDG assay . Shown are relative FDG fluorescence of Rab39a KO cells / WT background . Summary of two independent experiments . Splenic DCs were expanded in Rab39a KO and WT mice by injection of B16 - Flt3L tumor . 10 days post tumor implantation , DCIR2 + ( Cd8α - ) or XCR1 + ( Cd8α + ) dendritic cells were isolated from spleens via magnetic positive selection . 5 x 104 cells were plated in a 96 well plate and incubated with the indicated amounts of Ova - Fe and 1 : 1 of Reporter CD8 or CD4 T - cells overnight before measuring luciferase activity . Error bars indicate SD of 2 replicate wells . Shown are representative graphs of 3 independent experiments . * p < 0 . 05 using two - way ANOVA . CD11b + CD11c + dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection . Cells were fed with 3 µm biotin - ova - magnetic beads for 4 hours . Phagosomes were extracted , permeabilized and stained for the indicated proteins . For ROS measurements , phagocytosis was performed with 5 µM CellROX and the phagosomes were not permeabilized . For analysis , the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of target ( dox / no dox ) was calculated . Shown are three independent experiments combined . Error bars show SD between experiments . p values were calculated using two - tailed ratio - paired Student ' s t - test . CD11b + CD11c + dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection . Cells were fed with 3 µm magnetic beads conjugated to pHrodo . After 4 hours , cells were harvested for flow cytometry to measure fluorescence of eaten beads due to phagosomal pH . For analysis , the background fluorescence of unstained beads was subtracted from the resulting gMFI and the fold change ( dox / no dox ) was calculated . Shown are three independent experiments with error bars indicating the SD . p values were calculated using two - tailed ratio - paired Student ' s t - test . CD11b + CD11c + dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection . Cells were fed with 3 µm magnetic beads conjugated to Alexa fluor dye . After 4 hours , cells were harvested and resuspended in 1 mg / ml Trypan blue to quench fluorescence of uneaten beads . Cells were run for flow cytometry to measure phagocytosis . The ratio of KO cells with beads / WT cells with beads was then obtained . Graphs show three independent experiments and p values were calculated using two - tailed ratio - paired Student ' s t - test . Error bars show SD between experiments . Rab39a KO and WT mice were injected intravenously with 1 x 105 A20 - Ova cells . The next day , CFSE labeled OTI - splenocytes were injected i . v . . 3 days post OT - I transfer , spleens were harvested and assayed for OT - I proliferation . Three independent experiments ( WT vs KO ) were performed . To combine the data , the OT - I proliferation in each individual mouse was normalized to the average proliferation of the WT controls in that experiment . Error bars show SD between individual mice and p value was derived from two - tailed student ' s t - test . n = 14 ( WT ) or 9 ( KO ) .

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Figure 7Rab39a expression in splenocyte subsets was measured using an FDG assay. Shown are relative FDG fluorescence of Rab39a KO cells/WT background. Summary of two independent experiments.Splenic DCs were expanded in Rab39a KO and WT mice by injection of B16-Flt3L tumor. 10 days post tumor implantation, DCIR2+ (Cd8α-) or XCR1+ (Cd8α+) dendritic cells were isolated from spleens via magnetic positive selection. 5 x 104 cells were plated in a 96 well plate and incubated with the indicated amounts of Ova-Fe and 1:1 of Reporter CD8 or CD4 T-cells overnight before measuring luciferase activity. Error bars indicate SD of 2 replicate wells. Shown are representative graphs of 3 independent experiments. * p < 0.05 using two-way ANOVA.CD11b+ CD11c+ dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection. Cells were fed with 3 µm biotin-ova-magnetic beads for 4 hours. Phagosomes were extracted, permeabilized and stained for the indicated proteins. For ROS measurements, phagocytosis was performed with 5 µM CellROX and the phagosomes were not permeabilized. For analysis, the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of target (dox/no dox) was calculated. Shown are three independent experiments combined. Error bars show SD between experiments. p values were calculated using two-tailed ratio-paired Student's t-test.CD11b+ CD11c+ dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection. Cells were fed with 3 µm magnetic beads conjugated to pHrodo. After 4 hours, cells were harvested for flow cytometry to measure fluorescence of eaten beads due to phagosomal pH. For analysis, the background fluorescence of unstained beads was subtracted from the resulting gMFI and the fold change (dox/no dox) was calculated. Shown are three independent experiments with error bars indicating the SD. p values were calculated using two-tailed ratio-paired Student's t-test.CD11b+ CD11c+ dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection. Cells were fed with 3 µm magnetic beads conjugated to Alexa fluor dye. After 4 hours, cells were harvested and resuspended in 1 mg/ml Trypan blue to quench fluorescence of uneaten beads. Cells were run for flow cytometry to measure phagocytosis. The ratio of KO cells with beads / WT cells with beads was then obtained. Graphs show three independent experiments and p values were calculated using two-tailed ratio-paired Student's t-test. Error bars show SD between experiments.Rab39a KO and WT mice were injected intravenously with 1 x 105 A20-Ova cells. The next day, CFSE labeled OTI-splenocytes were injected i.v.. 3 days post OT-I transfer, spleens were harvested and assayed for OT-I proliferation. Three independent experiments (WT vs KO) were performed. To combine the data, the OT-I proliferation in each individual mouse was normalized to the average proliferation of the WT controls in that experiment. Error bars show SD between individual mice and p value was derived from two-tailed student's t-test. n = 14 (WT) or 9 (KO).


Figure 1C Co - crystal structure of gephyrin E - domain ( blue ) and bound GlyR ß - loop ( red - PDB code 2FTS ( Kim et al . , 2006 ) ) . The mutated gephyrin Gly375 residue is shown in orange .

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Figure 1C Co-crystal structure of gephyrin E-domain (blue) and bound GlyR ß-loop (red - PDB code 2FTS (Kim et al., 2006)). The mutated gephyrin Gly375 residue is shown in orange.


Figure 2A Representative images of GFP - gephyrin and GFP - G375D , expressed in hippocampal neurons at 9 + 4 days in vitro . Inset shows higher magnification . Scale bar = 20 µm . B Relative distribution of cluster density of gephyrin variants on dendrites from transfected neurons . Number of gephyrin clusters in each dendrite is normalized to 20 µm segments . C Representative segments of dendrites expressing GFP - gephyrin or GFP - G375D and immunostained with the presynaptic marker VGAT . Arrowheads show co - localization of gephyrin and VGAT clusters . Scale bar = 10 µm . D Quantification of co - localization between gephyrin and VGAT clusters ( gephyrin - VGAT 77 . 4 ± 2 . 9 % , G375D - VGAT 30 ± 4 . 4 % ) . 31 GFP - gephyrin and 30 GFP - G375D neurons from three independent cultures were used for quantifications . Results are expressed as mean ±

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Figure 2A Representative images of GFP-gephyrin and GFP-G375D, expressed in hippocampal neurons at 9 + 4 days in vitro. Inset shows higher magnification. Scale bar = 20 µm.B Relative distribution of cluster density of gephyrin variants on dendrites from transfected neurons. Number of gephyrin clusters in each dendrite is normalized to 20 µm segments.C Representative segments of dendrites expressing GFP-gephyrin or GFP-G375D and immunostained with the presynaptic marker VGAT. Arrowheads show co-localization of gephyrin and VGAT clusters. Scale bar = 10 µm.D Quantification of co-localization between gephyrin and VGAT clusters (gephyrin-VGAT 77.4±2.9 %, G375D-VGAT 30±4.4 %). 31 GFP-gephyrin and 30 GFP-G375D neurons from three independent cultures were used for quantifications. Results are expressed as mean ± SEM


Figure 3A Representative dendritic segment of a neuron expressing GFP - G375D and 3B11 - immunostained endogenous gephyrin ( red ) . B Quantification of density and ( C ) size of endogenous gephyrin clusters in neurons expressing GFP , GFP - G375D or non - transfected neighboring neurons . 20 GFP - G375 and non - transfected neurons and 15 GFP - expressing neurons from three independent cultures were used for quantifications . Results are expressed as mean ± SEM and data were analyzed by t - test .

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Figure 3A Representative dendritic segment of a neuron expressing GFP-G375D and 3B11-immunostained endogenous gephyrin (red).B Quantification of density and (C) size of endogenous gephyrin clusters in neurons expressing GFP, GFP-G375D or non-transfected neighboring neurons. 20 GFP-G375 and non-transfected neurons and 15 GFP-expressing neurons from three independent cultures were used for quantifications. Results are expressed as mean ± SEM and data were analyzed by t-test.


Figure 4A Representative neurons expressing myc - tagged gephyrin or G375D and pHluorin - tagged GABAAR γ2 . Surface - expressed γ2 - subunits were immunolabelled with GFP - antibodies in red , and the pHluorin - fluorescence ( total γ2 levels ) are shown in green . B Size and C fluorescence ( fl . ) intensity of surface - expressed γ2 clusters in myc - gephyrin and myc - G375D - expressing dendrites ( n = 20 myc - gephyrin and 21 myc - G375D neurons in B and 18 myc - gephyrin and myc - G375D neurons in C ) D Mean γ2 - pHluorin fluorescence in myc - gephyrin and myc - G375D - expressing dendrites ( n = 20 and 18 myc - gephyrin and myc - G375D expressing neurons ) . E Number of surface γ2 clusters on dendrites normalized to 20 µm segments ( n = 18 myc - gephyrin and myc - G375D expressing neurons ) . At least three independent cultures were used for each experiment . Results are expressed as mean ± SEM ; Data were analyzed by t - test . F Representative traces of miniature recordings on cultured hippocampal neurons transfected either with GFP - gephyrin or GFP - G375D . G mIPSC amplitudes from neurons expressing GFP - gephyrin or GFP - G375D . Numbers show analyzed cell - numbers . Data were analyzed by one - way ANOVA and post - hoc Tukey test . H Frequency of the mIPSCs from neurons expressing GFP - gephyrin or GFP - G375D . The horizontal line within the boxes indicates the median frequency for the represented condition ( bottom / top boundary indicates the 25th / 75th percentile ) . Top and bottom error bars indicate the 90th and 10th percentiles , respectively . Analysis by Kruskal - Wallis test followed by post - hoc Mann - Whitney test .

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Figure 4A Representative neurons expressing myc-tagged gephyrin or G375D and pHluorin-tagged GABAAR γ2. Surface-expressed γ2-subunits were immunolabelled with GFP-antibodies in red, and the pHluorin-fluorescence (total γ2 levels) are shown in green.B Size and C fluorescence (fl.) intensity of surface-expressed γ2 clusters in myc-gephyrin and myc-G375D-expressing dendrites (n = 20 myc-gephyrin and 21 myc-G375D neurons in B and 18 myc-gephyrin and myc-G375D neurons in C)D Mean γ2-pHluorin fluorescence in myc-gephyrin and myc-G375D-expressing dendrites (n = 20 and 18 myc-gephyrin and myc-G375D expressing neurons).E Number of surface γ2 clusters on dendrites normalized to 20 µm segments (n = 18 myc-gephyrin and myc-G375D expressing neurons). At least three independent cultures were used for each experiment. Results are expressed as mean ± SEM; Data were analyzed by t-test.F Representative traces of miniature recordings on cultured hippocampal neurons transfected either with GFP-gephyrin or GFP-G375D.G mIPSC amplitudes from neurons expressing GFP-gephyrin or GFP-G375D. Numbers show analyzed cell-numbers. Data were analyzed by one-way ANOVA and post-hoc Tukey test.H Frequency of the mIPSCs from neurons expressing GFP-gephyrin or GFP-G375D. The horizontal line within the boxes indicates the median frequency for the represented condition (bottom/top boundary indicates the 25th /75th percentile). Top and bottom error bars indicate the 90th and 10th percentiles, respectively. Analysis by Kruskal-Wallis test followed by post-hoc Mann-Whitney test.


Figure 5A Western blot of HEK293 cells expressing GFP - tagged gephyrin or G375D using GFP - specific and polyclonal gephyrin antibodies . Note that both gephyrin variants are stably expressed and show no degradation in the cells . Asterisk in the poly - gephyrin blot shows endogenous gephyrin . B SDS - PAGE of E . coli expressed and purified 6His - gephyrins and CD spectra . Mean residue ellipticity of gephyrin and G375D show profiles with minima at 208 nm and 222 nm . C Size - exclusion chromatography of purified 6His - gephyrin and isolated E - domain variants . Full - length gephyrins and isolated E - domains were loaded onto a Superose 6 gel filtration column and elution of the protein was monitored by measuring absorbance at 280 nm ( mAU , milliabsorbance units ) . D Co - immunoprecipitation of HEK293 cell lysates expressing myc - tagged gephyrin together with GFP - tagged gephyrin or G375D using myc - specific antibodies . Control IgG were used to show specificity of the assay .

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Figure 5A Western blot of HEK293 cells expressing GFP-tagged gephyrin or G375D using GFP-specific and polyclonal gephyrin antibodies. Note that both gephyrin variants are stably expressed and show no degradation in the cells. Asterisk in the poly-gephyrin blot shows endogenous gephyrin.B SDS-PAGE of E. coli expressed and purified 6His-gephyrins and CD spectra. Mean residue ellipticity of gephyrin and G375D show profiles with minima at 208 nm and 222 nm.C Size-exclusion chromatography of purified 6His-gephyrin and isolated E-domain variants. Full-length gephyrins and isolated E-domains were loaded onto a Superose 6 gel filtration column and elution of the protein was monitored by measuring absorbance at 280 nm (mAU, milliabsorbance units).D Co-immunoprecipitation of HEK293 cell lysates expressing myc-tagged gephyrin together with GFP-tagged gephyrin or G375D using myc-specific antibodies. Control IgG were used to show specificity of the assay.


Figure 6B Representative pulldown - experiment and quantification of 6His - tagged gephyrin and G375D using immobilized α3 - peptide . 6His - tagged gephyrin variants were expressed in E . coli cells , purified to homogeneity and incubated with α3 - peptides immobilized to NeutrAvidin beads . n = 3 , normalized to 6His - gephyrin . 38 . 9 ± 1 . 5 % G375D were pulled down . C Representative SPR sensograms of 6His - gephyrin and 6His - G375D binding to immobilized GABAAR α3 - peptide . Response units are plotted against time . Injected gephyrin concentrations were 0 . 5 , 2 , 10 , 25 , 40 µM . D SPR binding isotherms of a representative experiment as in C . Calculated KD values of 3 . 2 and 10 µM for gephyrin and G375D , respectively , are indicated by vertical lines . The experiment was repeated three times with two independently purified protein batches and two sensor chips . E Representative ITC binding isotherms of the cytoplasmic loop of the GlyR ß - subunit ( GlyR - ßL , residue 378 - 426 ) and 6His - tagged gephyrin or G375D . The GlyR - ßL - gephyrin binding data ( black ) were fitted in a two - side model , whereas G375D binding could only be fitted to a single binding site . Dissociation constants KD and stoichiometry ( N = number of binding sites ) are given for each binding component . n = 5 from two independently purified protein batches . Results are expressed as mean ± SEM .

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Figure 6B Representative pulldown-experiment and quantification of 6His-tagged gephyrin and G375D using immobilized α3-peptide. 6His-tagged gephyrin variants were expressed in E. coli cells, purified to homogeneity and incubated with α3-peptides immobilized to NeutrAvidin beads. n = 3, normalized to 6His-gephyrin. 38.9±1.5% G375D were pulled down.C Representative SPR sensograms of 6His-gephyrin and 6His-G375D binding to immobilized GABAAR α3-peptide. Response units are plotted against time. Injected gephyrin concentrations were 0.5, 2, 10, 25, 40 µM.D SPR binding isotherms of a representative experiment as in C. Calculated KD values of 3.2 and 10 µM for gephyrin and G375D, respectively, are indicated by vertical lines. The experiment was repeated three times with two independently purified protein batches and two sensor chips.E Representative ITC binding isotherms of the cytoplasmic loop of the GlyR ß-subunit (GlyR-ßL, residue 378-426) and 6His-tagged gephyrin or G375D. The GlyR-ßL-gephyrin binding data (black) were fitted in a two-side model, whereas G375D binding could only be fitted to a single binding site. Dissociation constants KD and stoichiometry (N = number of binding sites) are given for each binding component. n = 5 from two independently purified protein batches. Results are expressed as mean ± SEM.


Figure 7A Biosynthesis pathway of Moco with all stable intermediates . Gephyrin G - and E - domain catalyze the last two steps as depicted . B In vitro Moco - synthesis assay using 150 pmol purified 6His - tagged gephyrins . D580A is an activity - deficient gephyrin mutation previously identified in Moco - deficient patients ( Reiss et al . , 2011 ) . Assays without the addition of gephyrin ( - gephyrin ) or molybdenum ( - Mo ) served as internal controls of the assay ( see also ( Belaidi Schwarz , 2013 ) ) . C Experiment as in B but with pre - incubation of 6His - gephyrin / G375D in a 1 : 1 ratio to simulate the heterozygous mutation . n = at least 3 with two independently purified protein batches . Results are expressed as mean ± SEM .

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Figure 7A Biosynthesis pathway of Moco with all stable intermediates. Gephyrin G- and E-domain catalyze the last two steps as depicted.B In vitro Moco-synthesis assay using 150 pmol purified 6His-tagged gephyrins. D580A is an activity-deficient gephyrin mutation previously identified in Moco-deficient patients (Reiss et al., 2011). Assays without the addition of gephyrin (-gephyrin) or molybdenum (-Mo) served as internal controls of the assay (see also (Belaidi Schwarz, 2013)).C Experiment as in B but with pre-incubation of 6His-gephyrin/G375D in a 1:1 ratio to simulate the heterozygous mutation. n = at least 3 with two independently purified protein batches. Results are expressed as mean ± SEM.


Figure 1 ( A ) Venn diagram of STK38 partners identified in both starvation and suspension conditions by proximity biotinylation assay coupled to mass spectrometry identification ( B ) Heatmap representation of common STK38 interactors identified in both starvation and suspension conditions according to their dynamic of association with STK38 ( with a minimal fold increase of 1 . 38 ± 0 . 06 ) . Unsupervised hierarchical clustering was generated based on their association fold using Pearson correlation . ( C ) Representation of the 10 most cellular component enriched terms of the two clusters ( assessed by using the Gene Ontology ( GO )

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Figure 1(A) Venn diagram of STK38 partners identified in both starvation and suspension conditions by proximity biotinylation assay coupled to mass spectrometry identification (B) Heatmap representation of common STK38 interactors identified in both starvation and suspension conditions according to their dynamic of association with STK38 (with a minimal fold increase of 1.38 ± 0.06). Unsupervised hierarchical clustering was generated based on their association fold using Pearson correlation. (C) Representation of the 10 most cellular component enriched terms of the two clusters (assessed by using the Gene Ontology (GO) database


Figure 2 ( A ) STK38 interacts with XPO1 . HekRasV12 cells were transiently transfected with myc - STK38 ( wt ) together with either Flag - XPO1 ( wt ) plasmid , Flag - control ( ctrl = Sirt3 ) plasmid or without DNA . 24h later , cells were incubated with Okadaic Acid ( OA ) , ( final concentration = 1µM ) for 1 hour or with DMSO . Flag fusions were pulled - down and co - immunoprecipited proteins were analyzed by western blotting ( WB ) . Upper panel displays whole cell lysates ( WCL ) and lower panel represents immunoprecipitated proteins . STK38 accumulates in the cytoplasm upon nutrient starvation in a XPO1 - dependant manner . ( B ) HeLa cells were transfected with myc - STK38 ( wt ) plasmid . The next day , cells were incubated with DMEM or EBSS in the presence of XPO1 inhibitors KPT - 185 or KPT - 330 as indicated ( final concentration = 1 µM ) or DMSO for 4 hours . Cells were then fixed and stained for myc - tag . Representative images are shown and scale bars are 40 µm . STK38 accumulates in the cytoplasm upon nutrient starvation in a XPO1 - dependant manner . ( C ) Quantification of myc - STK38 ( wt ) nuclear / cytoplasmic staining ( n > 30 cells from 3 independent experiments , mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Mann - Whitney test ) . XPO1 activity is required for nutrient starvation - induced autophagy . ( D ) Immunoblotting with indicated antibodies of whole cell lysates of cells and its graphical representation ( n = 3 independent experiments , mean ± SEM ; * * * p < 0 . 001 , ns , not significant , Student ' s t test ) . As expected , p62 degradation in starvation conditions was inhibited in presence of XPO1 inhibitors . XPO1 activity is required for nutrient starvation - induced autophagy . ( E ) HeLa cells stably expressing the GFP - LC3 - RFP - LC3ΔG reporter autophagic probe [ 26 ] were incubated with DMEM or EBSS in the presence of XPO1 inhibitors KPT - 185 or KPT - 330 ( final concentration = 1 µM ) or DMSO for 4 hours . The GFP - LC3 ( degraded upon autophagy ) and RFP - LC3ΔG ( non - degraded upon autophagy ) signals were recorded by FACS analysis and then shown as a ratio recapitulating overall LC3 level ( n = 3 independent experiments , mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , ns , not significant , Mann - Whitney test ) . Here again , incubation with XPO1 inhibitors significantly impaired the autophagy

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) STK38 interacts with XPO1. HekRasV12 cells were transiently transfected with myc-STK38(wt) together with either Flag-XPO1(wt) plasmid, Flag-control (ctrl = Sirt3) plasmid or without DNA. 24h later, cells were incubated with Okadaic Acid (OA), (final concentration = 1µM) for 1 hour or with DMSO. Flag fusions were pulled-down and co-immunoprecipited proteins were analyzed by western blotting (WB). Upper panel displays whole cell lysates (WCL) and lower panel represents immunoprecipitated proteins.STK38 accumulates in the cytoplasm upon nutrient starvation in a XPO1-dependant manner. (B) HeLa cells were transfected with myc-STK38(wt) plasmid. The next day, cells were incubated with DMEM or EBSS in the presence of XPO1 inhibitors KPT-185 or KPT-330 as indicated (final concentration = 1 µM) or DMSO for 4 hours. Cells were then fixed and stained for myc-tag. Representative images are shown and scale bars are 40 µm.STK38 accumulates in the cytoplasm upon nutrient starvation in a XPO1-dependant manner. (C) Quantification of myc-STK38(wt) nuclear/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, Mann-Whitney test).XPO1 activity is required for nutrient starvation-induced autophagy. (D) Immunoblotting with indicated antibodies of whole cell lysates of cells and its graphical representation (n = 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Student's t test). As expected, p62 degradation in starvation conditions was inhibited in presence of XPO1 inhibitors.XPO1 activity is required for nutrient starvation-induced autophagy. (E) HeLa cells stably expressing the GFP-LC3-RFP-LC3ΔG reporter autophagic probe [26] were incubated with DMEM or EBSS in the presence of XPO1 inhibitors KPT-185 or KPT-330 (final concentration = 1 µM) or DMSO for 4 hours. The GFP-LC3 (degraded upon autophagy) and RFP-LC3ΔG (non-degraded upon autophagy) signals were recorded by FACS analysis and then shown as a ratio recapitulating overall LC3 level (n=3 independent experiments, mean ± SEM; *p<0.05, **p<0.01, ns, not significant, Mann-Whitney test). Here again, incubation with XPO1 inhibitors significantly impaired the autophagy process


Figure 3STK38 kinase activity is required and sufficient for its cytoplasmic accumulation upon nutrient starvation . ( A ) HeLa cells silenced for endogenous STK38 were transfected with myc - STK38 ( wt ) expressing plasmid , myc - STK38 ( K118R ) ( STK38 kinase - dead version ) plasmid or with HA - STK38 ( PIF ) ( STK38 constitutively active version ) plasmid . 24 hours later , cells were incubated with DMEM or EBSS for 4 hours , fixed and stained for myc - tag or HA - tag . Representative images are shown and scale bars are 40 µm . STK38 kinase activity is required and sufficient for its cytoplasmic accumulation upon nutrient starvation . ( B ) Graphical representation of tag - STK38 variants nuclear staining / cytoplasmic staining ( n > 30 cells from 3 independent experiments , mean ± SEM ; * * p < 0 . 01 , * * * p < 0 . 001 , ns , not significant , Mann - Whitney test ) . STK38 kinase activity is required and sufficient for nutrient - starvation induced autophagy . ( C ) HeLa cells stably expressing the GFP - LC3 - RFP - LC3ΔG autophagic probe [ 26 ] were transiently transfected with siRNA targeting the 3 ' UTR region of endogenous STK38 ( or with non - targeting siRNA ( siNT ) ) . The next day , cells were transiently transfected with the indicated STK38 mutants expressing plasmids 24 hours after plasmid transfection , cells were incubated with DMEM or EBSS for 4 hours . The GFP - LC3 ( degraded upon autophagy ) and RFP - LC3ΔG ( non - degraded upon autophagy ) signals were recorded by FACS analysis and then shown as a ratio recapitulating overall LC3 level ( n = 4 independent experiments , mean ± SEM ; * p < 0 . 05 , * * * p < 0 . 001 , ns , not significant , Mann - Whitney test ) . depleting STK38 prevents autophagy to take place . This effect was partially reversed by expressing the wt ORF as well as constitutively active STK38 whereas kinase - dead version failed to reproduce endogenous STK38 effect . Expression of constitutively active STK38 is sufficient to induce a substantial change in the autophagic flux upon nutrient rich conditionsSTK38 kinase activity is required and sufficient for nutrient - starvation induced autophagy . ( D ) HeLa cells were transiently transfected with siRNA targeting the 3 ' UTR region of endogenous STK38 ( or with non - targeting siRNA ( siNT ) ) and then transiently transfected with the indicated STK38 mutants expressing plasmids 48h after . The next day , the cells were incubated with DMEM or EBSS for 4 hours and overall p62 level was quantified for autophagy estimation ( n = 3 independent experiments , mean ± SEM ; * * * p < 0 . 001 , ns , not significant , Student ' s t test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3STK38 kinase activity is required and sufficient for its cytoplasmic accumulation upon nutrient starvation. (A) HeLa cells silenced for endogenous STK38 were transfected with myc-STK38(wt) expressing plasmid, myc-STK38(K118R) (STK38 kinase-dead version) plasmid or with HA-STK38(PIF) (STK38 constitutively active version) plasmid. 24 hours later, cells were incubated with DMEM or EBSS for 4 hours, fixed and stained for myc-tag or HA-tag. Representative images are shown and scale bars are 40 µm.STK38 kinase activity is required and sufficient for its cytoplasmic accumulation upon nutrient starvation. (B) Graphical representation of tag-STK38 variants nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; **p<0.01, ***p<0.001, ns, not significant, Mann-Whitney test).STK38 kinase activity is required and sufficient for nutrient-starvation induced autophagy. (C) HeLa cells stably expressing the GFP-LC3-RFP-LC3ΔG autophagic probe [26] were transiently transfected with siRNA targeting the 3'UTR region of endogenous STK38 (or with non-targeting siRNA (siNT)). The next day, cells were transiently transfected with the indicated STK38 mutants expressing plasmids 24 hours after plasmid transfection, cells were incubated with DMEM or EBSS for 4 hours. The GFP-LC3 (degraded upon autophagy) and RFP-LC3ΔG (non-degraded upon autophagy) signals were recorded by FACS analysis and then shown as a ratio recapitulating overall LC3 level (n=4 independent experiments, mean ± SEM; *p<0.05, ***p<0.001, ns, not significant, Mann-Whitney test). depleting STK38 prevents autophagy to take place. This effect was partially reversed by expressing the wt ORF as well as constitutively active STK38 whereas kinase-dead version failed to reproduce endogenous STK38 effect. Expression of constitutively active STK38 is sufficient to induce a substantial change in the autophagic flux upon nutrient rich conditionsSTK38 kinase activity is required and sufficient for nutrient-starvation induced autophagy. (D) HeLa cells were transiently transfected with siRNA targeting the 3'UTR region of endogenous STK38 (or with non-targeting siRNA (siNT)) and then transiently transfected with the indicated STK38 mutants expressing plasmids 48h after. The next day, the cells were incubated with DMEM or EBSS for 4 hours and overall p62 level was quantified for autophagy estimation (n = 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Student's t test).


Figure 4 ( A - B ) STK38 is required for XPO1 _ S1055 phosphorylation . ( A ) HeLa cells were transiently transfected with the indicated siRNA and subjected to Flag - XPO1 ( wt ) transient transfection the following day . 48 hours later , cells were incubated with Okadaic Acid ( OA , final concentration = 1 µM ) for 1 hour or with DMSO . Immunoblotting was performed on whole cell lysates with indicated antibodies . ( B ) Graphical representation of the phospho S1055 - XPO1 signal on total XPO1 ( n = 3 independent experiments , mean ± SEM ; * p < 0 . 05 , ns , not significant , Mann - Whitney test ) . As expected , XPO1 is phosphorylated on its Ser1055 upon OA treatment but not when STK38 is silenced ( C ) STK38 kinase activity is required for XPO1 _ S1055 phosphorylation . HekRasV12 cells silenced ( or not ) for endogenous STK38 were transfected with Flag - XPO1 ( wt ) expression plasmid in addition to myc - STK38 ( wt ) or myc - STK38 ( K118R ) ( STK38 kinase - dead version ) plasmids . The next day , cells were treated with 1 µM OA or with vehicle ( DMSO ) for 1 hour . Flag fusions were immunoprecipited and pulled - down proteins were analyzed by western blotting . Upper panel displays immunoprecipited proteins and lower panel represents whole cell lysates .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A-B) STK38 is required for XPO1_S1055 phosphorylation. (A) HeLa cells were transiently transfected with the indicated siRNA and subjected to Flag-XPO1(wt) transient transfection the following day. 48 hours later, cells were incubated with Okadaic Acid (OA, final concentration = 1 µM) for 1 hour or with DMSO. Immunoblotting was performed on whole cell lysates with indicated antibodies. (B) Graphical representation of the phospho S1055-XPO1 signal on total XPO1 (n = 3 independent experiments, mean ± SEM; *p<0.05, ns, not significant, Mann-Whitney test). As expected, XPO1 is phosphorylated on its Ser1055 upon OA treatment but not when STK38 is silenced(C) STK38 kinase activity is required for XPO1_S1055 phosphorylation. HekRasV12 cells silenced (or not) for endogenous STK38 were transfected with Flag-XPO1(wt) expression plasmid in addition to myc-STK38(wt) or myc-STK38(K118R) (STK38 kinase-dead version) plasmids. The next day, cells were treated with 1 µM OA or with vehicle (DMSO) for 1 hour. Flag fusions were immunoprecipited and pulled-down proteins were analyzed by western blotting. Upper panel displays immunoprecipited proteins and lower panel represents whole cell lysates.


Figure 5 ( A ) Graphical representation of XPO1 protein . C528 to S amino acid mutation confers XPO1 resistance to both KPT - 185 and KPT - 330 chemical inhibitors [ 24 ] . S1055 amino acid correspond to the phosphorylation target of the STK38 . Finally , the ΔCter region is also highlighted corresponding to a XPO1 construct lacking the 39 C - terminal residues ( B - C ) XPO1 _ S1055 phosphorylation is required and sufficient for STK38 nuclear exit upon nutrient starvation . ( B ) HeLa cells were transiently transfected with myc - STK38 ( wt ) in addition to indicated Flag - XPO1 mutant plasmids . 24 hours later , cells were incubated with DMEM or EBSS for 4 hours both supplemented with KPT - 185 ( final concentration = 1 µM ) in order to inhibit endogenous XPO1 activity . Cells were then fixed and stained for Flag and myc tags . Only cells positives for both Flag - XPO1 and myc - STK38 ( wt ) were captured . Representative images are shown and scale bars are 40 µm . ( C ) Graphical representation of myc - STK38 ( wt ) nuclear staining / cytoplasmic staining ( n > 30 cells from 3 independent experiments , mean ± SEM ; * * * p < 0 . 001 , ns , not significant , Mann - Whitney test ) . Expression of wt XPO1 failed to induce cytoplasmic localization of STK38 upon nutrient starvation in presence of KPT - 185 where C528S mutant recapitulates results described in Figure 2B - C . Expression of phosphonegative XPO1 ( S1055A ) failed to induces STK38 cytoplasmic localization upon EBSS treatment while phosphomimetics XPO1 ( S1055D and S1055E ) were sufficient to promote STK38 cytoplasmic localization without autophagic stimuli . Finally , expression of XPO1 lacking in its 39 C - terminal residues mimicked phosphomimetics variants . ( D ) HAP1 cells carrying genomic XPO1 mutations of S1055 were subjected for IMDM ( nutrient rich medium ) or EBSS ( starvation ) incubation for 4 hours followed by western - blot analysis for p62 level measurement . This result indicates that p62 degradation in starvation conditions was inhibited in XPO1 _ S1055A HAP1 cells whereas p62 degradation was potentiated in both phosphomimetics variant ( S1055D & S1055E ) ( n = 4 independent experiments , mean ± SEM ; * p < 0 . 05 , ns , not significant , Mann - Whitney test ( E ) Hek - HT - iRFP - LC3 cells were transiently transfected with siRNA targeting endogenous STK38 ( or with non - targeting siRNA ( siNT ) ) . Two days after , cells were transiently transfected with indicated Flag - XPO1 mutant plasmids . 24 hours later , cells were incubated with DMEM or EBSS for 4 hours both supplemented with KPT - 185 ( final concentration = 1 µM ) in order to inhibit endogenous XPO1 activity and with chloroquine ( final concentration = 10 µM ) . Cells were then fixed and the number of iRFP - LC3 dots per cell was recorded only in cells positive for Flag - XPO1 mutants As expected , silencing of endogenous STK38 prevented the formation of LC3 dots upon starvation where introduction of phosphonegative XPO1 ( S1055A ) failed also to increase LC3 dots upon starvation . Interestingly , phosphomimetic XPO1 ( S1055D ) was sufficient to increase the number of LC3 dots even in complete and also STK38 - depleted conditions .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Graphical representation of XPO1 protein. C528 to S amino acid mutation confers XPO1 resistance to both KPT-185 and KPT-330 chemical inhibitors [24]. S1055 amino acid correspond to the phosphorylation target of the STK38. Finally, the ΔCter region is also highlighted corresponding to a XPO1 construct lacking the 39 C-terminal residues (B-C) XPO1_S1055 phosphorylation is required and sufficient for STK38 nuclear exit upon nutrient starvation. (B) HeLa cells were transiently transfected with myc-STK38(wt) in addition to indicated Flag-XPO1 mutant plasmids. 24 hours later, cells were incubated with DMEM or EBSS for 4 hours both supplemented with KPT-185 (final concentration = 1 µM) in order to inhibit endogenous XPO1 activity. Cells were then fixed and stained for Flag and myc tags. Only cells positives for both Flag-XPO1 and myc-STK38(wt) were captured. Representative images are shown and scale bars are 40 µm. (C) Graphical representation of myc-STK38(wt) nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Mann-Whitney test). Expression of wt XPO1 failed to induce cytoplasmic localization of STK38 upon nutrient starvation in presence of KPT-185 where C528S mutant recapitulates results described in Figure 2B-C. Expression of phosphonegative XPO1 (S1055A) failed to induces STK38 cytoplasmic localization upon EBSS treatment while phosphomimetics XPO1 (S1055D and S1055E) were sufficient to promote STK38 cytoplasmic localization without autophagic stimuli. Finally, expression of XPO1 lacking in its 39 C-terminal residues mimicked phosphomimetics variants.(D) HAP1 cells carrying genomic XPO1 mutations of S1055 were subjected for IMDM (nutrient rich medium) or EBSS (starvation) incubation for 4 hours followed by western-blot analysis for p62 level measurement. This result indicates that p62 degradation in starvation conditions was inhibited in XPO1_S1055A HAP1 cells whereas p62 degradation was potentiated in both phosphomimetics variant (S1055D & S1055E) (n = 4 independent experiments, mean ± SEM; *p<0.05, ns, not significant, Mann-Whitney test(E) Hek-HT-iRFP-LC3 cells were transiently transfected with siRNA targeting endogenous STK38 (or with non-targeting siRNA (siNT)). Two days after, cells were transiently transfected with indicated Flag-XPO1 mutant plasmids. 24 hours later, cells were incubated with DMEM or EBSS for 4 hours both supplemented with KPT-185 (final concentration = 1 µM) in order to inhibit endogenous XPO1 activity and with chloroquine (final concentration = 10 µM). Cells were then fixed and the number of iRFP-LC3 dots per cell was recorded only in cells positive for Flag-XPO1 mutants As expected, silencing of endogenous STK38 prevented the formation of LC3 dots upon starvation where introduction of phosphonegative XPO1 (S1055A) failed also to increase LC3 dots upon starvation. Interestingly, phosphomimetic XPO1 (S1055D) was sufficient to increase the number of LC3 dots even in complete and also STK38-depleted conditions.


Figure 6 ( A ) XPO1 and STK38 are required for Beclin1 cytoplasmic accumulation upon nutrient starvation - induced autophagy . HeLa cells were transiently transfected with the indicated siRNA ( control and KPT conditions = siNT ) . 72 hours later , cells were incubated with XPO1 inhibitors KPT - 185 or KPT - 330 as indicated ( final concentration = 1 µM ) or with DMSO for all other conditions for 2 hours prior incubation with DMEM or EBSS supplemented ( or not ) with inhibitors for 2 hours . Cells were fixed and stained for endogenous Beclin1 . Graphical representation of endogenous Beclin1 nuclear staining / cytoplasmic staining ( n > 30 cells from 3 independent experiments , mean ± SEM ; * p < 0 . 05 , * * * p < 0 . 001 , ns , not significant , Mann - Whitney test ) . XPO1 activity inhibition by both KPT - 185 and KPT - 330 as well as STK38 silencing failed to induce Beclin1 cytoplasmic accumulation upon nutrient starvation ( B ) XPO1 _ S1055 phosphorylation is required and sufficient for Beclin1 nuclear exit upon nutrient starvation . Genome edited XPO1 mutant HAP1 cells were incubated with IMDM ( complete ) or EBSS ( starvation ) for 2 hours . Cells were then fixed and stained for endogenous Beclin1 . Graphical representation of endogenous Beclin1 nuclear staining / cytoplasmic staining ( n > 30 cells from 3 independent experiments , mean ± SEM ; * * * p < 0 . 001 , ns , not significant , Mann - Whitney test ) . Here , phosphonegative XPO1 failed to induce Beclin1 cytoplasmic accumulation upon nutrient starvation whereas phosphomimetics variant ( S1055D & S1055E ) potentiated Beclin1 cytoplasmic accumulation ( C ) Genome edited XPO1 mutant HAP1 cells were transiently transfected with siRNA targeting endogenous STK38 ( siSTK38 # 206 or with non - targeting siRNA ( siNT ) ) . 72 hours later , cells were incubated with IMDM ( complete ) or EBSS ( starvation ) for 2 hours . Cells were then fixed and stained for endogenous Beclin1 . Graphical representation of endogenous Beclin1 nuclear staining / cytoplasmic staining ( n = 30 cells from 2 independent experiments ) . Here , phosphomimetic XPO1 ( S1055D ) potentiated Beclin1 cytoplasmic localization in both complete and starvation condition even in the absence of STK38 as compared to cells carrying wt

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Figure 6(A) XPO1 and STK38 are required for Beclin1 cytoplasmic accumulation upon nutrient starvation-induced autophagy. HeLa cells were transiently transfected with the indicated siRNA (control and KPT conditions = siNT). 72 hours later, cells were incubated with XPO1 inhibitors KPT-185 or KPT-330 as indicated (final concentration = 1 µM) or with DMSO for all other conditions for 2 hours prior incubation with DMEM or EBSS supplemented (or not) with inhibitors for 2 hours. Cells were fixed and stained for endogenous Beclin1. Graphical representation of endogenous Beclin1 nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; *p<0.05, ***p<0.001, ns, not significant, Mann-Whitney test). XPO1 activity inhibition by both KPT-185 and KPT-330 as well as STK38 silencing failed to induce Beclin1 cytoplasmic accumulation upon nutrient starvation(B) XPO1_S1055 phosphorylation is required and sufficient for Beclin1 nuclear exit upon nutrient starvation. Genome edited XPO1 mutant HAP1 cells were incubated with IMDM (complete) or EBSS (starvation) for 2 hours. Cells were then fixed and stained for endogenous Beclin1. Graphical representation of endogenous Beclin1 nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Mann-Whitney test). Here, phosphonegative XPO1 failed to induce Beclin1 cytoplasmic accumulation upon nutrient starvation whereas phosphomimetics variant (S1055D & S1055E) potentiated Beclin1 cytoplasmic accumulation(C) Genome edited XPO1 mutant HAP1 cells were transiently transfected with siRNA targeting endogenous STK38 (siSTK38#206 or with non-targeting siRNA (siNT)). 72 hours later, cells were incubated with IMDM (complete) or EBSS (starvation) for 2 hours. Cells were then fixed and stained for endogenous Beclin1. Graphical representation of endogenous Beclin1 nuclear staining/cytoplasmic staining (n = 30 cells from 2 independent experiments). Here, phosphomimetic XPO1 (S1055D) potentiated Beclin1 cytoplasmic localization in both complete and starvation condition even in the absence of STK38 as compared to cells carrying wt XPO1


Figure 7 ( A ) XPO1 and STK38 are required for YAP1 nuclear export at high confluence . A549 cells were transiently transfected with the indicated siRNA ( control and KPT conditions = siNT ) at low or high confluency . 48 hours later , cells were incubated overnight in the presence of XPO1 inhibitors KPT - 185 and KPT - 330 ( final concentration = 1 µM ) or with DMSO for all other conditions . The next day , cells were fixed and stained for endogenous YAP1 . Quantitative representation of YAP1 nuclear fluorescence intensity ( n > 300 cells from 3 independent experiments , mean ± SEM ; * * * p < 0 . 001 , Mann - Whitney test ) . XPO1 activity inhibition by both KPT - 185 and KPT - 330 as well as STK38 silencing failed to induce YAP1 nuclear exit at high confluence ( B ) XPO1 _ S1055 phosphorylation is required and sufficient for YAP nuclear exit . Genome edited XPO1 HAP1 cells were cultured for two days at low versus high confluency . Cells were then fixed and stained for endogenous YAP1 . Quantitative representation of YAP1 nuclear fluorescence intensity ( n > 300 cells from 3 independent experiments , mean ± SEM ; * * * p < 0 . 001 , Mann - Whitney test ) . Here , phosphonegative XPO1 failed to induce YAP nuclear exit at high confluence whereas phosphomimetics variant ( S1055D & S1055E ) potentiated YAP1 nuclear exit ( C ) Genome edited XPO1 mutant HAP1 cells were transiently transfected with siRNA targeting endogenous STK38 ( siSTK38 # 206 or with non - targeting siRNA ( siNT ) ) at low or high confluence . 72 hours later , cells were fixed and stained for endogenous YAP1 . Quantitative representation of YAP1 nuclear fluorescence intensity ( n = 150 cells from 2 independent experiments ) . Here , phosphomimetic XPO1 ( S1055D ) potentiated YAP1 nuclear exit at both low and high confluences even in the absence of STK38 as compared to cells carrying wt

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Figure 7(A) XPO1 and STK38 are required for YAP1 nuclear export at high confluence. A549 cells were transiently transfected with the indicated siRNA (control and KPT conditions = siNT) at low or high confluency. 48 hours later, cells were incubated overnight in the presence of XPO1 inhibitors KPT-185 and KPT-330 (final concentration = 1 µM) or with DMSO for all other conditions. The next day, cells were fixed and stained for endogenous YAP1. Quantitative representation of YAP1 nuclear fluorescence intensity (n > 300 cells from 3 independent experiments, mean ± SEM; ***p<0.001, Mann-Whitney test). XPO1 activity inhibition by both KPT-185 and KPT-330 as well as STK38 silencing failed to induce YAP1 nuclear exit at high confluence(B) XPO1_S1055 phosphorylation is required and sufficient for YAP nuclear exit. Genome edited XPO1 HAP1 cells were cultured for two days at low versus high confluency. Cells were then fixed and stained for endogenous YAP1. Quantitative representation of YAP1 nuclear fluorescence intensity (n > 300 cells from 3 independent experiments, mean ± SEM; ***p<0.001, Mann-Whitney test). Here, phosphonegative XPO1 failed to induce YAP nuclear exit at high confluence whereas phosphomimetics variant (S1055D & S1055E) potentiated YAP1 nuclear exit(C) Genome edited XPO1 mutant HAP1 cells were transiently transfected with siRNA targeting endogenous STK38 (siSTK38#206 or with non-targeting siRNA (siNT)) at low or high confluence. 72 hours later, cells were fixed and stained for endogenous YAP1. Quantitative representation of YAP1 nuclear fluorescence intensity (n = 150 cells from 2 independent experiments). Here, phosphomimetic XPO1 (S1055D) potentiated YAP1 nuclear exit at both low and high confluences even in the absence of STK38 as compared to cells carrying wt XPO1


Figure 2 ( A ) Heatmap showing viability measurements for the 192 samples ( rows ) subject to each of 17 microenvironmental stimuli ( columns ) . The data are shown on a robust z - score scale , i . e . , the logarithm of the relative viability measurements were scaled by the median absolute deviation within each row . Limits were applied to scaling factor for optimal visualisation . Red indicates increased viability , blue indicates decreased viability . Samples are annotated for genetic features , sex and pre - treatment status .

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Figure 2(A) Heatmap showing viability measurements for the 192 samples (rows) subject to each of 17 microenvironmental stimuli (columns). The data are shown on a robust z-score scale, i.e., the logarithm of the relative viability measurements were scaled by the median absolute deviation within each row. Limits were applied to scaling factor for optimal visualisation. Red indicates increased viability, blue indicates decreased viability. Samples are annotated for genetic features, sex and pre-treatment status.


Figure 4Logarithm of the relative viability after treatment with Resiquimod ( A , n = 169 ) , IL4 ( B , n = 174 ) stratified by trisomy 12 , and in A , also by IGHV status . P - values from Student ' s t - test ( two - sided , non - paired ) . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively . Logarithm of the relative viability after treatment with TGFβ ( C , n = 174 ) stratified by trisomy 12 , P - values from Student ' s t - test ( two - sided , non - paired ) . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively .

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Figure 4Logarithm of the relative viability after treatment with Resiquimod (A, n=169), IL4 (B, n=174) stratified by trisomy 12, and in A, also by IGHV status. P-values from Student's t-test (two-sided, non-paired). The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.Logarithm of the relative viability after treatment with TGFβ (C, n=174) stratified by trisomy 12, P-values from Student's t-test (two-sided, non-paired). The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.


Figure 5 ( A ) Graphical representation of the four drug - stimulus interaction categories . Categories are defined according to the nature of the interaction ( synergistic or antagonistic ) , and whether the viability is increased or decreased by the stimulus ( positive or negative ) . x - axis shows treatment type , y - axis shows viability with each treatment . Red and blue points and lines depict a representative treatment response pattern for given interaction type . Blue horizontal lines represent the expected viability for combinatorial treatment in the absence of an interaction between drug - stimulus ( i . e . additive effects ) , black horizontal lines represent the measured viability after combinatorial treatment . The difference between the black and blue lines indicates the impact of the interaction on expected viability . ( D - I ) Examples of drug - stimulus interactions , for each category . Plots show the natural logarithm of the relative viability of 192 CLL samples after treatment with the respective drug and stimulus . Each line represents one patient sample linked across treatments . Black horizontal lines in each single treatment indicate the expected viability based on linear modelling . Black and blue horizontal lines in the combinatorial treatment indicate the expected viability based on the additive effect of the drug and stimulus ( blue ) , and the expected viability accounting for the additive effect and the interaction ( black ) . ( D + E ) Ibrutinib , a clinically used BTK inhibitor , is blocked by IL4 and IFNγ . ( F ) The JAK inhibitor pyridone - 6 inhibits the pro - survival effect of sCD40L + IL4 stimulation . ( G ) The p38 inhibitor ralimetinib and IFNγ show a synergistic pro - survival effect not observed in either single treatment . ( H ) TLR agonists , including CpG ODN ( shown ) increase sensitivity to BTK inhibition by ibrutinib , despite increasing viability as single treatments . ( I ) Soluble anti - IgM sensitises CLL samples to HSP90 inhibition by luminespib .

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Figure 5(A) Graphical representation of the four drug-stimulus interaction categories. Categories are defined according to the nature of the interaction (synergistic or antagonistic), and whether the viability is increased or decreased by the stimulus (positive or negative). x-axis shows treatment type, y-axis shows viability with each treatment. Red and blue points and lines depict a representative treatment response pattern for given interaction type. Blue horizontal lines represent the expected viability for combinatorial treatment in the absence of an interaction between drug-stimulus (i.e. additive effects), black horizontal lines represent the measured viability after combinatorial treatment. The difference between the black and blue lines indicates the impact of the interaction on expected viability.(D - I) Examples of drug-stimulus interactions, for each category. Plots show the natural logarithm of the relative viability of 192 CLL samples after treatment with the respective drug and stimulus. Each line represents one patient sample linked across treatments. Black horizontal lines in each single treatment indicate the expected viability based on linear modelling. Black and blue horizontal lines in the combinatorial treatment indicate the expected viability based on the additive effect of the drug and stimulus (blue), and the expected viability accounting for the additive effect and the interaction (black). (D+E) Ibrutinib, a clinically used BTK inhibitor, is blocked by IL4 and IFNγ. (F) The JAK inhibitor pyridone-6 inhibits the pro-survival effect of sCD40L + IL4 stimulation. (G) The p38 inhibitor ralimetinib and IFNγ show a synergistic pro-survival effect not observed in either single treatment. (H) TLR agonists, including CpG ODN (shown) increase sensitivity to BTK inhibition by ibrutinib, despite increasing viability as single treatments. (I) Soluble anti-IgM sensitises CLL samples to HSP90 inhibition by luminespib.


Figure 6Beeswarm - boxplots of the natural logarithm of the relative viability of 169 CLL samples , for ( C ) fludarabine + CpG ODN and combinatorial treatments , faceted by IGHV status and trisomy 12 status . P - values from paired Student ' s t - test . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively . Beeswarm - boxplots of the natural logarithm of the relative viability of 169 CLL samples , for ( D ) ibrutinib + IL4 single and combinatorial treatments , faceted by IGHV status and trisomy 12 status . P - values from paired Student ' s t - test . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively .

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Figure 6Beeswarm-boxplots of the natural logarithm of the relative viability of 169 CLL samples, for (C) fludarabine + CpG ODN and combinatorial treatments, faceted by IGHV status and trisomy 12 status. P-values from paired Student's t-test . The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.Beeswarm-boxplots of the natural logarithm of the relative viability of 169 CLL samples, for (D) ibrutinib + IL4 single and combinatorial treatments, faceted by IGHV status and trisomy 12 status. P-values from paired Student's t-test . The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.


Figure 7 ( A Mean ( A ) pSTAT6 staining intensity in CLL - infiltrated ( n = 100 for pSTAT and tumour - free lymph node biopsies ( n = 98 for pSTAT6 after background subtraction ( y axis ) , p - values from Student ' s t - test . Each dot represents the mean of all cells in the tissue microarray cores per patient sample . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively . B ) Mean ( B ) pIRAK4 staining intensity in CLL - infiltrated ( n = 100 for pIRAK4 ) and tumour - free lymph node biopsies n = 100 for pIRAK4 ) after background subtraction ( y axis ) , p - values from Student ' s t - test . Each dot represents the mean of all cells in the tissue microarray cores per patient sample . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively . Example images of immunohistochemistry sections : ( C + D ) pSTAT6 levels in ( C ) CLL - infiltrated and ( D ) tumour - free samples . Scale bar defines 20µm in all images . Example images of immunohistochemistry sections : ( E + F ) pIRAK4 levels in ( E ) CLL - infiltrated and ( F ) tumour - free samples . Scale bar defines 20µm in all images .

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Figure 7(A Mean (A) pSTAT6 staining intensity in CLL-infiltrated (n=100 for pSTAT and tumour-free lymph node biopsies (n=98 for pSTAT6 after background subtraction (y axis), p-values from Student's t-test. Each dot represents the mean of all cells in the tissue microarray cores per patient sample. The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.B) Mean (B) pIRAK4 staining intensity in CLL-infiltrated (n=100 for pIRAK4) and tumour-free lymph node biopsies n=100 for pIRAK4) after background subtraction (y axis), p-values from Student's t-test. Each dot represents the mean of all cells in the tissue microarray cores per patient sample. The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.Example images of immunohistochemistry sections: (C + D) pSTAT6 levels in (C) CLL-infiltrated and (D) tumour-free samples. Scale bar defines 20µm in all images.Example images of immunohistochemistry sections: (E + F) pIRAK4 levels in (E) CLL-infiltrated and (F) tumour-free samples. Scale bar defines 20µm in all images.


Figure 1ISRE - luciferase reporter assay in 293T cells infected with BTV - 8 or mock - infected ( Control ) and treated with 1000 U / ml of universal - IFN Luminescence fold induction was measured at different time points p . i . Mean luciferase activity fold inductions in control or BTV - infected in three experiments are presented . * GAS - luciferase reporter assay in Vero cells infected with BTV - 8 or mock - infected ( Control ) and treated wit 5 ng / ml of IFN - γ . Luminescence fold induction was measured at different time points p . i . Mean luciferase activity fold inductions in control or BTV - infected in three experiments are presentedqRT - PCR analysis of ISGs induction in mock - or BTV - 8 - infected 293T cells after type I IFN treatment for 8 hr . Mean mRNA fold inductions for 3 experiments are presented . qRT - PCR analysis of ISGs induction in mock - or BTV - 8 - infected Vero cells after type II IFN treatment for 8 hr . Mean mRNA fold inductions for 3 experiments are presented .

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Figure 1ISRE-luciferase reporter assay in 293T cells infected with BTV-8 or mock-infected (Control) and treated with 1000 U/ml of universal-IFN Luminescence fold induction was measured at different time points p.i. Mean luciferase activity fold inductions in control or BTV-infected in three experiments are presented. *GAS-luciferase reporter assay in Vero cells infected with BTV-8 or mock-infected (Control) and treated wit 5 ng/ml of IFN-γ. Luminescence fold induction was measured at different time points p.i. Mean luciferase activity fold inductions in control or BTV-infected in three experiments are presentedqRT-PCR analysis of ISGs induction in mock- or BTV-8-infected 293T cells after type I IFN treatment for 8 hr. Mean mRNA fold inductions for 3 experiments are presented.qRT-PCR analysis of ISGs induction in mock- or BTV-8-infected Vero cells after type II IFN treatment for 8 hr. Mean mRNA fold inductions for 3 experiments are presented.


Figure 2Immunoblot of Vero cells infected with BTV - 8 for 16 h , and probed for total and phosphorylated STAT1 / 2 levels . Vero cells infected with BTV - 8 were fixed at 16hpi , and stained with anti - BTV - VP7 antibody . STAT1 phosphorylation in IFN - I - treated Vero cells infected with BTV - 8 detected by ( C ) flow cytometry and ( D ) confocal microscopy ( Scale bar = 30 µm ) . Means ± SD of P - STAT1 mean fluorescence intensity ( MFI ) in 3 experiments are shown . STAT2 expression in BTV - 8 - infected Vero cells detected by ( E ) flow cytometry and ( F ) confocal microscopy ( Scale bar = 30 µm ) . Means ± SD of STAT2 MFI in 3 experiments are shown .

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Figure 2Immunoblot of Vero cells infected with BTV-8 for 16 h, and probed for total and phosphorylated STAT1/2 levels.Vero cells infected with BTV-8 were fixed at 16hpi, and stained with anti-BTV-VP7 antibody.STAT1 phosphorylation in IFN-I-treated Vero cells infected with BTV-8 detected by (C) flow cytometry and (D) confocal microscopy (Scale bar = 30 µm). Means ± SD of P-STAT1 mean fluorescence intensity (MFI) in 3 experiments are shown.STAT2 expression in BTV-8-infected Vero cells detected by (E) flow cytometry and (F) confocal microscopy (Scale bar = 30 µm). Means ± SD of STAT2 MFI in 3 experiments are shown.


Figure 3Vero cells were infected at MOI 1 ( or mock infected ) and treated with proteasome inhibitors MG132 ( MG 20 µM ) , lactacystin ( LC 20 µM ) , chloroquine ( CQ 50 µM ) , 3 - MA ( 5 mM ) , or bafilomycin - A1 ( BAF - A1 0 . 2 µM ) for the final 6h . Representative flow cytometry histogram of BTV - VP7 expression in inhibitor - treated cells . Vero cells were infected at MOI 1 ( or mock infected ) and treated with proteasome inhibitors MG132 ( MG 20 µM ) , lactacystin ( LC 20 µM ) , chloroquine ( CQ 50 µM ) , 3 - MA ( 5 mM ) , for the final 6h . Immunoblots probed for STAT1 , STAT2 , LC3 and GAPDH expression . Vero cells were infected at MOI 1 ( or mock infected ) and treated with bafilomycin - A1 ( BAF - A1 0 . 2 µM ) for the final 6h . Immunoblots probed for STAT1 , STAT2 , LC3 and GAPDH expression . Immunofluorescence of BTV - 8 or mock - infected Vero cells ( treated for the last 6h with BAF - A1 ) stained for BTV proteins , STAT2 and p62 . Inset shows detail of STAT2 and p62 colocalization . Nuclei were counterstained with DAPI . Arrowheads in inset indicate p62 and STAT2 colocalization . Scale bar = 20 μm . Inset scale bar = 3 μm .

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Figure 3Vero cells were infected at MOI 1 (or mock infected) and treated with proteasome inhibitors MG132 (MG 20 µM), lactacystin (LC 20 µM), chloroquine (CQ 50 µM), 3-MA (5 mM), or bafilomycin-A1 (BAF-A1 0.2 µM) for the final 6h. Representative flow cytometry histogram of BTV-VP7 expression in inhibitor-treated cells.Vero cells were infected at MOI 1 (or mock infected) and treated with proteasome inhibitors MG132 (MG 20 µM), lactacystin (LC 20 µM), chloroquine (CQ 50 µM), 3-MA (5 mM), for the final 6h. Immunoblots probed for STAT1, STAT2, LC3 and GAPDH expression.Vero cells were infected at MOI 1 (or mock infected) and treated with bafilomycin-A1 (BAF-A1 0.2 µM) for the final 6h. Immunoblots probed for STAT1, STAT2, LC3 and GAPDH expression.Immunofluorescence of BTV-8 or mock-infected Vero cells (treated for the last 6h with BAF-A1) stained for BTV proteins, STAT2 and p62. Inset shows detail of STAT2 and p62 colocalization. Nuclei were counterstained with DAPI. Arrowheads in inset indicate p62 and STAT2 colocalization. Scale bar = 20 μm. Inset scale bar = 3 μm.


Figure 4ISRE - luciferase reporter assays in 293T cells stimulated with IFN - U co - transfected with BTV - VP6 , - VP7 , - NS1 , - NS2 , - NS3 , or - NS4 - expressing plasmids or empty expression plasmid ( Control ) . Representative mean fold inductions of luciferase activity of three independent experiments are shown . GAS - luciferase reporter assays in Vero cells stimulated with IFN - γ co - transfected with BTV - VP6 , - VP7 , - NS1 , - NS2 , - NS3 , or - NS4 - expressing plasmids or empty expression plasmid ( Control ) . Representative mean fold inductions of luciferase activity of three independent experiments are shown . Immunoblot of 293T cells transfected with BTV - NS2 , - NS3 , - NS4 ( FLAG ) plasmids and NiV - V ( HA ) and WNV - NS5 ( HA ) used as control for STAT1 phosphorylation inhibition and DENV - NS5 ( HA ) used as control for STAT2 degradation . Three independent immunoblot experiments as in ( C ) were quantified for STAT1 , P - STAT1 , STAT2 and P - STAT2 . Mean ± SD normalized to GAPDH and non - transfected cells . Immunoprecipitation ( IP ) for STAT1 / 2 binding using 293T transfected with BTV - NS2 , - NS3 , or - NS4 plasmids . NiV - V used as positive control for STAT1 and STAT2 binding , DENV - NS5 used as positive control for STAT2 binding . Cells were treated ( or left untreated ) with 1000 U / ml universal - IFN for the final 16 h . Immunoblots of WCE and IP were probed ( IB ) for STAT1 , STAT2 , HA ( NiV V , DENV NS5 ) , FLAG ( NS2 , NS3 , NS4 ) and GAPDH . STAT1 phosphorylation and ( G ) STAT2 nuclear translocation in BTV - NS3 - transfected Vero cells detected by immunofluorescence and confocal microscopy after 30 min IFN treatment . ( Scale bar = 20 µm ) . Arrowheads in merge images indicate examples of transfected cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4ISRE-luciferase reporter assays in 293T cells stimulated with IFN-U co-transfected with BTV -VP6, -VP7, -NS1, -NS2, -NS3, or -NS4-expressing plasmids or empty expression plasmid (Control). Representative mean fold inductions of luciferase activity of three independent experiments are shown.GAS-luciferase reporter assays in Vero cells stimulated with IFN-γ co-transfected with BTV -VP6, -VP7, -NS1, -NS2, -NS3, or -NS4-expressing plasmids or empty expression plasmid (Control). Representative mean fold inductions of luciferase activity of three independent experiments are shown.Immunoblot of 293T cells transfected with BTV -NS2, -NS3, -NS4 (FLAG) plasmids and NiV-V (HA) and WNV-NS5 (HA) used as control for STAT1 phosphorylation inhibition and DENV-NS5 (HA) used as control for STAT2 degradation.Three independent immunoblot experiments as in (C) were quantified for STAT1, P-STAT1, STAT2 and P-STAT2. Mean ± SD normalized to GAPDH and non-transfected cells.Immunoprecipitation (IP) for STAT1/2 binding using 293T transfected with BTV -NS2, -NS3, or -NS4 plasmids. NiV-V used as positive control for STAT1 and STAT2 binding, DENV-NS5 used as positive control for STAT2 binding. Cells were treated (or left untreated) with 1000 U/ml universal-IFN for the final 16 h. Immunoblots of WCE and IP were probed (IB) for STAT1, STAT2, HA (NiV V, DENV NS5), FLAG (NS2, NS3, NS4) and GAPDH.STAT1 phosphorylation and (G) STAT2 nuclear translocation in BTV-NS3-transfected Vero cells detected by immunofluorescence and confocal microscopy after 30 min IFN treatment. (Scale bar = 20 µm). Arrowheads in merge images indicate examples of transfected cells.


Figure 5Immunoblots of BTV - NS3 - transfected 293T cells treated with proteasome inhibitors MG or LC . Membranes were probed for STAT1 , STAT2 , BTV - NS3 ( FLAG ) and GAPDH . ( B - D ) Immunoblots of BTV - NS3 - transfected 293T cells treated with increasing concentration of lysosome acidification inhibitors ( B ) CQ or ( C ) NH4Cl or with increasing concentration of the ( D ) autophagosome formation inhibitor 3 - MA , and probed ( IB ) for STAT1 , STAT2 , BTV - NS3 ( FLAG ) and GAPDH expression . 293T cells were co - transfected with NS3 expression plasmid ( or empty plasmid as control ) and siRNA for Atg7 Lysates were obtained from FACS - sorted transfected cells . Immunoblots of sorted cells were probed for STAT1 ; STAT2 ; Atg7 FLAG - tagged NS3 ; LC3 and GAPDH . 293T cells were co - transfected with NS3 expression plasmid ( or empty plasmid as control ) and siRNA for Beclin - 1 ( or siRNA control or no RNA as controls ) . Lysates were obtained from FACS - sorted transfected cells . Immunoblots of sorted cells were probed for STAT1 ; STAT2 ; Beclin - 1 FLAG - tagged NS3 ; LC3 and GAPDH . Representative flow cytometry dot plots of Vero cells infected with rgBTV - 8 or BTV - 8 ΔNS3 and stained for BTV - VP7 . Gating on VP7 + cells was used for sorting to obtain lysates from infected cells . Immunoblot of FACS - sorted Vero cells infected with rgBTV - 8 or BTV - 8 ΔNS3 and treated with IFN - U for 30min prior to fixation , staining and sorting . Membranes were probed for STAT1 , P - STAT1 , STAT2 and GAPDH . Immunofluorescence confocal images of Vero cells infected with rgBTV - 8 , BTV - 8 ΔNS3 or mock - infected and treated with IFN - U for 30min prior to fixation and staining . Cells were stained for BTV - VP7 and ( I ) P - STAT1 or ( J ) STAT2 . Nuclei were counterstained with DAPI . ( Scale bar = 30 μm ) . ( I ) Arrowhead in merge image indicate partial inhibition of STAT1 phosphorylation in BTV - 8 ΔNS3 infected cells . ( J ) Arrowhead in merge image shows STAT2 translocation to nucleus in BTV - 8 ΔNS3 - infected cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Immunoblots of BTV-NS3-transfected 293T cells treated with proteasome inhibitors MG or LC. Membranes were probed for STAT1, STAT2, BTV-NS3 (FLAG) and GAPDH. (B-D) Immunoblots of BTV-NS3-transfected 293T cells treated with increasing concentration of lysosome acidification inhibitors (B) CQ or (C) NH4Cl or with increasing concentration of the (D) autophagosome formation inhibitor 3-MA, and probed (IB) for STAT1, STAT2, BTV-NS3 (FLAG) and GAPDH expression.293T cells were co-transfected with NS3 expression plasmid (or empty plasmid as control) and siRNA for Atg7 Lysates were obtained from FACS-sorted transfected cells. Immunoblots of sorted cells were probed for STAT1; STAT2; Atg7 FLAG-tagged NS3; LC3 and GAPDH.293T cells were co-transfected with NS3 expression plasmid (or empty plasmid as control) and siRNA for Beclin-1 (or siRNA control or no RNA as controls). Lysates were obtained from FACS-sorted transfected cells. Immunoblots of sorted cells were probed for STAT1; STAT2; Beclin-1 FLAG-tagged NS3; LC3 and GAPDH.Representative flow cytometry dot plots of Vero cells infected with rgBTV-8 or BTV-8 ΔNS3 and stained for BTV-VP7. Gating on VP7+ cells was used for sorting to obtain lysates from infected cells.Immunoblot of FACS-sorted Vero cells infected with rgBTV-8 or BTV-8 ΔNS3 and treated with IFN-U for 30min prior to fixation, staining and sorting. Membranes were probed for STAT1, P-STAT1, STAT2 and GAPDH.Immunofluorescence confocal images of Vero cells infected with rgBTV-8, BTV-8 ΔNS3 or mock-infected and treated with IFN-U for 30min prior to fixation and staining. Cells were stained for BTV-VP7 and (I) P-STAT1 or (J) STAT2. Nuclei were counterstained with DAPI. (Scale bar = 30 μm). (I) Arrowhead in merge image indicate partial inhibition of STAT1 phosphorylation in BTV-8 ΔNS3 infected cells. (J) Arrowhead in merge image shows STAT2 translocation to nucleus in BTV-8 ΔNS3-infected cells.


Figure 6WCE and FLAG - tag NS3 IP assay immunoblots of 293T transfected with BTV - NS3 - FLAG plasmids and increasing amounts of Ubiquitin - HA plasmids , and probed ( IB ) for ubiquitin ( HA ) , BTV - NS3 ( FLAG ) or GAPDH expression . Immunofluorescence staining of Vero cells co - transfected with BTV - NS3 and ubiquitin and acquired with Airyscan super - resolution . Scale bar = 10 μm . Inset shows detailed area and arrowheads indicate NS3 and ubiquitin signal co - incidence ( scale bar = 2 μm ) . Fluorescence intensity profile of cell section indicated by the white line ( blue : DAPI , green : NS3 ; red : Ub ) . Arrowheads on image and arrows on intensity profile indicate matching fluorescence signal for NS3 and Ub . FLAG - tag IP assay of BTV - NS3 and WCE immunoblot of 293T co - transfected with BTV FLAG - NS3 - , - NS3 - K13R , - NS3 - K15R , - NS3 - K13 / 15R or - NS3 - PPRY / AARH plasmid and Ubiquitin - HA plasmid and probed ( IB ) for ubiquitin ( HA ) , BTV - NS3 ( FLAG ) , STAT2 and GAPDH expression . WCE and HA - tag Ubiquitin IP assay immunoblots of 293T transfected with Ubiquitin - HA plasmids and increasing amounts of BTV - FLAG - NS3 - K13 / 15R , and probed ( IB ) for ubiquitin ( HA ) , BTV - NS3 - K13 / 15R ( FLAG ) or GAPDH expression . ISRE - driven luciferase reporter assay in 293T cells transfected with empty expression plasmid ( Control ) , BTV - NS2 ( 300 ng ) , - NS3 ( 50 , 300 and 600 ng ) , or - NS3 - K13 / 15R ( 50 , 300 and 600 ng ) - expressing plasmids . Mean % inductions of luciferase activity of three independent experiments are shown . FLAG - tag IP assay of BTV - NS3 and WCE immunoblot of 293T co - transfected FLAG - BTV - NS3 plasmid and HA - UB - WT or HA - UB lysine ( K to R ) mutants . IP were probed ( IB ) for ubiquitin ( HA ) and BTV - NS3 ( FLAG ) expression . WCE were probed for STAT1 , STAT2 , ubiquitin ( HA ) , BTV - NS3 ( FLAG ) , and GAPDH expression .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6WCE and FLAG-tag NS3 IP assay immunoblots of 293T transfected with BTV-NS3-FLAG plasmids and increasing amounts of Ubiquitin-HA plasmids, and probed (IB) for ubiquitin (HA), BTV-NS3 (FLAG) or GAPDH expression.Immunofluorescence staining of Vero cells co-transfected with BTV-NS3 and ubiquitin and acquired with Airyscan super-resolution. Scale bar = 10 μm. Inset shows detailed area and arrowheads indicate NS3 and ubiquitin signal co-incidence (scale bar = 2 μm).Fluorescence intensity profile of cell section indicated by the white line (blue: DAPI, green: NS3; red: Ub). Arrowheads on image and arrows on intensity profile indicate matching fluorescence signal for NS3 and Ub.FLAG-tag IP assay of BTV-NS3 and WCE immunoblot of 293T co-transfected with BTV FLAG-NS3-, -NS3-K13R, -NS3-K15R, -NS3-K13/15R or -NS3-PPRY/AARH plasmid and Ubiquitin-HA plasmid and probed (IB) for ubiquitin (HA), BTV-NS3 (FLAG), STAT2 and GAPDH expression.WCE and HA-tag Ubiquitin IP assay immunoblots of 293T transfected with Ubiquitin-HA plasmids and increasing amounts of BTV-FLAG-NS3-K13/15R, and probed (IB) for ubiquitin (HA), BTV-NS3-K13/15R (FLAG) or GAPDH expression.ISRE-driven luciferase reporter assay in 293T cells transfected with empty expression plasmid (Control), BTV -NS2 (300 ng), -NS3 (50, 300 and 600 ng), or -NS3-K13/15R (50, 300 and 600 ng)-expressing plasmids. Mean % inductions of luciferase activity of three independent experiments are shown.FLAG-tag IP assay of BTV-NS3 and WCE immunoblot of 293T co-transfected FLAG-BTV-NS3 plasmid and HA-UB-WT or HA-UB lysine (K to R) mutants. IP were probed (IB) for ubiquitin (HA) and BTV-NS3 (FLAG) expression. WCE were probed for STAT1, STAT2, ubiquitin (HA), BTV-NS3 (FLAG), and GAPDH expression.


Figure 1 ( C - D ) RNA - Seq data from GTEx of the normalized expression of human CSAG1 ( C ) and CSAG2 ( D ) in the indicated tissues . Number of biological replicates are as follows : testis 361 , brain 255 , small intestine 187 , white adipose 663 , muscle 803 , skin 604 , esophagus 555 , stomach 359 , liver 226 , heart 429 . blood vessel 663 , lung 578 , colon 373 , nerve 619 , pituitary 283 , blood 755 , adrenal gland 258 , kidney 85 , prostate 245 , salivary gland 162 , ovary 180 , breast 459 , pancreas 328 , vagina 156 , uterus 142 , spleen 241 , fallopian tube 9 , bladder 21 , cervix 10 . Central band indicates median , boxes define 25 and 75 percentiles , and whiskers define 5 and 95 percentiles . ( E - F ) Expression of CSAG1 ( E ) and CSAG2 ( F ) in normal adjacent ( blue ) or tumor tissue ( red ) from TCGA RNA - Seq data . N . D . represents normal adjacent is not available . Data visualized by firebrowse . Number of biological replicates are as follows : ACC 41 , BLCA 307 , BRCA 524 , CESC 175 , CHOL 11 , COAD 153 , COADREAD 232 , DLBC 36 , ESCA 121 , HNSC 416 , KICH 46 , KIPAN 515 , KIRC 411 , KIRP 58 , LAML 7 , LGG 528 , LUAD 301 , LUSC 400 , MESO 31 , OV 183 , PCPG 70 , PRAD 204 , READ 79 , SARC 156 , SKCM 422 , STAD 288 , STES 409 , TGCT 126 , THCA 408 , THYM 75 , UCEC 306 , and UCS 37 . Central band indicates median , boxes define 25 and 75 percentiles , and whiskers define 5 and 95 percentiles . ( G ) Xq28 MAGE and CSAG genes are frequently co - expressed in lung squamous carcinoma tumors ( n = 184 ) . RNA - Seq gene expression data analyzed from TCGA . Statistics shown were calculated by linear regression analysis . ( H - K ) Expression of CSAG1 ( H - I ) or CSAG2 ( J - K ) in patients from indicated cancer types correlates with poor overall survival . Data is from TCGA and visualized by kmplotter .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(C-D) RNA-Seq data from GTEx of the normalized expression of human CSAG1 (C) and CSAG2 (D) in the indicated tissues. Number of biological replicates are as follows: testis 361, brain 255, small intestine 187, white adipose 663, muscle 803, skin 604, esophagus 555, stomach 359, liver 226, heart 429. blood vessel 663, lung 578, colon 373, nerve 619, pituitary 283, blood 755, adrenal gland 258, kidney 85, prostate 245, salivary gland 162, ovary 180, breast 459, pancreas 328, vagina 156, uterus 142, spleen 241, fallopian tube 9, bladder 21, cervix 10. Central band indicates median, boxes define 25 and 75 percentiles, and whiskers define 5 and 95 percentiles.(E-F) Expression of CSAG1 (E) and CSAG2 (F) in normal adjacent (blue) or tumor tissue (red) from TCGA RNA-Seq data. N.D. represents normal adjacent is not available. Data visualized by firebrowse. Number of biological replicates are as follows: ACC 41, BLCA 307, BRCA 524, CESC 175, CHOL 11, COAD 153, COADREAD 232, DLBC 36, ESCA 121, HNSC 416, KICH 46, KIPAN 515, KIRC 411, KIRP 58, LAML 7, LGG 528, LUAD 301, LUSC 400, MESO 31, OV 183, PCPG 70, PRAD 204, READ 79, SARC 156, SKCM 422, STAD 288, STES 409, TGCT 126, THCA 408, THYM 75, UCEC 306, and UCS 37. Central band indicates median, boxes define 25 and 75 percentiles, and whiskers define 5 and 95 percentiles.(G) Xq28 MAGE and CSAG genes are frequently co-expressed in lung squamous carcinoma tumors (n=184). RNA-Seq gene expression data analyzed from TCGA. Statistics shown were calculated by linear regression analysis.(H-K) Expression of CSAG1 (H-I) or CSAG2 (J-K) in patients from indicated cancer types correlates with poor overall survival. Data is from TCGA and visualized by kmplotter.


Figure 2 ( A - B ) The effect of stable knockdown CSAG2 in HCT116 ( A ) or A375 ( B ) were assayed for clonogenic growth on plastic for 7 days . The number of colonies was quantified and is shown . Data are represented as the mean ± SD , n = 3 biological replicates . ( C - D ) The effect of stable knockdown CSAG2 in HCT116 ( C ) or A375 ( D ) were determined for anchorage - independent growth in soft agar colony formation assays for 14 days . The number of colonies was quantified and is shown . Data are represented as the mean ± SD , n = 3 biological replicates . ( E - F ) Knockdown of CSAG2 in A375 ( F ) or HCT116 ( E ) cells decreases xenograft tumor growth in mice . Cells with doxycycline - inducible shRNAs were implanted into NOD SCID gamma mice . Doxycycline ( 2 mg / mL ) was administered via drinking water . Tumor growth was monitored over time . Data are represented as the mean ± SD , n = 6 mice for each group . ( G - H ) Stable expression of CSAG2 in HCT116 ( G ) and CSAG2 - negative H460 ( H ) cells increases anchorage - independent growth in soft agar colony formation assays , n = 3 biological replicates . Data are represented as the mean ± SD .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A-B) The effect of stable knockdown CSAG2 in HCT116 (A) or A375 (B) were assayed for clonogenic growth on plastic for 7 days. The number of colonies was quantified and is shown. Data are represented as the mean ± SD, n=3 biological replicates.(C-D) The effect of stable knockdown CSAG2 in HCT116 (C) or A375 (D) were determined for anchorage-independent growth in soft agar colony formation assays for 14 days. The number of colonies was quantified and is shown. Data are represented as the mean ± SD, n=3 biological replicates.(E-F) Knockdown of CSAG2 in A375 (F) or HCT116 (E) cells decreases xenograft tumor growth in mice. Cells with doxycycline-inducible shRNAs were implanted into NOD SCID gamma mice. Doxycycline (2 mg/mL) was administered via drinking water. Tumor growth was monitored over time. Data are represented as the mean ± SD, n=6 mice for each group.(G-H) Stable expression of CSAG2 in HCT116 (G) and CSAG2-negative H460 (H) cells increases anchorage-independent growth in soft agar colony formation assays, n=3 biological replicates. Data are represented as the mean ± SD.