Figure 3A . U2OS cells were transfected with siCTRL or with siDIS3 and 48 hours later were irradiated ( IR ) or left untreated ( NO IR ) . Cells were fixed 4 hours after IR and stained for RAD51 . Histograms show foci number per cell , analyzed with Cell Profiler . The average number of foci per cell was calculated from three independent experiments . At least 200 cells were counted for each point , error bars indicate s . e . m . ( n = 3 ) . * = p < 0 . 05 , two - way ANOVA , ns = not statistically significant , scale bar is 10 µD . Representative pictures of alkaline comet assays performed in U2OS silenced ( siDIS3 ) or not ( siCTRL ) for DIS3 , at sequential time points after exposure to 3 Gy irradiation . Relative comet tail moments were plotted for siCTRL ( black circles ) and siDIS3 ( red circles ) . Analysis was performed using Casplab software , error bars indicate s . e . m . Plots represent three biological replicates , with more than 150 cells for each siRNA . * * * * = p < 0 . 0001 , * * = p < 0 . 001 , ns = not statistically significant , two - way ANOVA , scale bar is 20 µm . F . Representative images of HPRT negative mutants in HT1080 cells infected with scramble shRNA ( shScr ) or shRNA against DIS3 ( shDIS3 ) . Histograms show the mutation frequency and the deletion length of DIS3 WT ( shScr ) and DIS3 depleted cells ( shDIS3 ) after I - SceI induced DSB . Error bars represent s . e . m . ( n = 3 ) . * * * = p < 0 . 001 , ns = not statistically significant Student 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 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 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

Figure 3A. U2OS cells were transfected with siCTRL or with siDIS3 and 48 hours later were irradiated (IR) or left untreated (NO IR). Cells were fixed 4 hours after IR and stained for RAD51. Histograms show foci number per cell, analyzed with Cell Profiler. The average number of foci per cell was calculated from three independent experiments. At least 200 cells were counted for each point, error bars indicate s.e.m. (n=3). * = p < 0.05, two-way ANOVA, ns= not statistically significant, scale bar is 10 µD. Representative pictures of alkaline comet assays performed in U2OS silenced (siDIS3) or not (siCTRL) for DIS3, at sequential time points after exposure to 3 Gy irradiation. Relative comet tail moments were plotted for siCTRL (black circles) and siDIS3 (red circles). Analysis was performed using Casplab software, error bars indicate s.e.m. Plots represent three biological replicates, with more than 150 cells for each siRNA. **** = p < 0.0001, ** = p < 0.001, ns= not statistically significant, two-way ANOVA, scale bar is 20 µm.F. Representative images of HPRT negative mutants in HT1080 cells infected with scramble shRNA (shScr) or shRNA against DIS3 (shDIS3). Histograms show the mutation frequency and the deletion length of DIS3 WT (shScr) and DIS3 depleted cells (shDIS3) after I-SceI induced DSB. Error bars represent s.e.m. (n=3). *** = p < 0.001, ns= not statistically significant Student t-test.


Figure 4A . U2OS cells were transfected with siCTRL or with siDIS3 and 48 hours later were 3 Gy irradiated ( IR ) or left untreated ( NO IR ) . Cells were fixed 4 hours after IR and stained for BRCA1 . Histograms show foci number per cell , analyzed with Cell Profiler . The average number of foci per cell was calculated from three independent experiments . At least 200 cells were counted for each point , error bars indicate s . e . m . ( n = 3 biological replicates ) . * * = p < 0 . 01 , ns = not statistically significant , two - way ANOVA , scale bar is 10 µm . C . Representative images of BRCA1 staining on U2OS cells transfected with siCTRL or with siDIS3 . 48 hours after transfection cells were irradiated and 4 hours later were treated with RNase H or left untreated ( UNTR ) . Histograms show the average foci number per cell from three independent experiments analyzed with Cell Profiler . At least 200 cells were counted for each point , error bars indicate s . e . m . ( n = 3 ) . * * = p < 0 . 01 , ns = not statistically significant , one - way ANOVA , scale bar is 10 µm . D . Representative pictures of comet assays performed under alkaline conditions in U2OS cells , silenced ( siDIS3 ) or not ( siCTRL ) for DIS3 untreated ( UNTR ) or treated with RNase H ( RNase H ) . Relative comet tail moments are plotted for siCTRL ( black circles ) and siDIS3 ( red circles ) . Analysis was performed using Casplab software , error bars indicate s . e . m . Plots represent three biological replicates , with 150 cells for each siRNA . * * * * = p < 0 . 0001 , one - way ANOVA , scale bar is 20 µm .

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 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 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 4A. U2OS cells were transfected with siCTRL or with siDIS3 and 48 hours later were 3 Gy irradiated (IR) or left untreated (NO IR). Cells were fixed 4 hours after IR and stained for BRCA1. Histograms show foci number per cell, analyzed with Cell Profiler. The average number of foci per cell was calculated from three independent experiments. At least 200 cells were counted for each point, error bars indicate s.e.m. (n=3 biological replicates). ** = p < 0.01, ns= not statistically significant, two-way ANOVA, scale bar is 10 µm.C. Representative images of BRCA1 staining on U2OS cells transfected with siCTRL or with siDIS3. 48 hours after transfection cells were irradiated and 4 hours later were treated with RNase H or left untreated (UNTR). Histograms show the average foci number per cell from three independent experiments analyzed with Cell Profiler. At least 200 cells were counted for each point, error bars indicate s.e.m. (n=3). ** = p < 0.01, ns= not statistically significant, one-way ANOVA, scale bar is 10 µm.D. Representative pictures of comet assays performed under alkaline conditions in U2OS cells, silenced (siDIS3) or not (siCTRL) for DIS3 untreated (UNTR) or treated with RNase H (RNase H). Relative comet tail moments are plotted for siCTRL (black circles) and siDIS3 (red circles). Analysis was performed using Casplab software, error bars indicate s.e.m. Plots represent three biological replicates, with 150 cells for each siRNA. **** = p < 0.0001, one-way ANOVA, scale bar is 20 µm.


Figure 5A . Representative confocal images upon irradiation with the 405nm laser . U2OS cells were presensitized with BrdU for 24 hours , micro - irradiated , fixed after 10 minutes , and stained with antibodies against γH2AX and DIS3 scale bar is 10 µm . B . Magnification image of the white box in panel A . C . Intensity profiles of γH2AX ( green ) and DIS3 signals through the yellow line in panel B . Quantification is performed with ImageJ software scale bar is 10 µm .

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

Figure 5A. Representative confocal images upon irradiation with the 405nm laser. U2OS cells were presensitized with BrdU for 24 hours, micro-irradiated, fixed after 10 minutes, and stained with antibodies against γH2AX and DIS3 scale bar is 10 µm. B. Magnification image of the white box in panel A. C. Intensity profiles of γH2AX (green) and DIS3 signals through the yellow line in panel B. Quantification is performed with ImageJ software scale bar is 10 µm.


Figure 6A . Representative pictures of comet assays performed under alkaline conditions in DIS3 WT MM cell lines MM . 1S and RPMI - 8226 ( RPMI ) , and in DIS3 mutated MM cell lines PCM6 and OPM2 , untreated ( UNTR ) or treated with RNase H ( RNase H ) . Tail moments analysis was performed using Casplab software , error bars indicate s . e . m . Plots represent three biological replicates , with 150 cells for each cell line . * * * * = p < 0 . 0001 ns = not statistically significant , two - way ANOVA , scale bar is 20 µm . B . Immunostaining with S9 . 6 ( green ) and DAPI ( blue ) in MM . 1S , RPMI - 8226 , PCM6 , and OPM2 cells , treated ( RNase H ) or not ( UNTR ) with RNase H for 1 hour . Histograms show S9 . 6 signal intensity per cell . More than 150 cells were counted , error bars indicate s . e . m . ( n = 3 biological replicates ) . * * * * = p < 0 . 0001 , one - way ANOVA , scale bar is 10 µm .

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 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

Figure 6A. Representative pictures of comet assays performed under alkaline conditions in DIS3 WT MM cell lines MM.1S and RPMI-8226 (RPMI), and in DIS3 mutated MM cell lines PCM6 and OPM2, untreated (UNTR) or treated with RNase H (RNase H). Tail moments analysis was performed using Casplab software, error bars indicate s.e.m. Plots represent three biological replicates, with 150 cells for each cell line. **** = p < 0.0001 ns= not statistically significant, two-way ANOVA, scale bar is 20 µm.B. Immunostaining with S9.6 (green) and DAPI (blue) in MM.1S, RPMI-8226, PCM6, and OPM2 cells, treated (RNase H) or not (UNTR) with RNase H for 1 hour. Histograms show S9.6 signal intensity per cell. More than 150 cells were counted, error bars indicate s.e.m. (n=3 biological replicates). **** = p < 0.0001, one-way ANOVA, scale bar is 10 µm.


Figure 7A . Relative mRNA expression level of interferon genes IFI6 , ISG15 , IFITM1 , IFIT3 , IFI27 , MCL1 , cGAS , and STING in OPM2 cells infected with a vector expressing DIS3 ( DIS3 ) or an empty vector ( EV ) . Values are relative to housekeeping gene expression , normalized on EV , and represent SE of three biological replicates . * * * = p < 0 . 001 , * * = p < 0 . 01 , multiple t - tests . Differentially expressed genes ( DEGs ) between mutant and WT samples ; p - values are from limma and false - discovery rate ( FDR ) is calculated using the Benjamini - Hochberg procedure ; FDR < 0 . 05 was used as cut off . Functional annotation analysis by IPA of the DEGs between DIS3 mutant and WT samples based on FDR cut - off . ( C ) Volcano plot of DEGs between mutant and WT samples ; labeled genes are the IFN - related genes with an FDR < 0 . 05 ( red horizontal line ) and absolute log 2 - fold change above or below 0 . 5 ( red vertical lines ) , respectively ; additional representative IFN - related genes are also labeled . ( F ) Violin plot of the number of non - synonymous ( NS ) mutation load comparison between DIS3 mutant versus DIS3 WT performed on the entire CoMMpass dataset , including 853 MM patients , 88 presenting DIS3 mutations ( all samples ) and on the CoMMpass dataset , stratified according to the intensity of the IFN responsiveness ( clusters C1 , C2 , C3 ) . Wilcoxon rank - sum test was used for comparison between DIS3 mutant vs WT . * * * * = p < , * * = p < 0 . 01 , * = p < 0 . 05 . Black color dots represent the median in each group .

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 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 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

Figure 7A. Relative mRNA expression level of interferon genes IFI6, ISG15, IFITM1, IFIT3, IFI27, MCL1, cGAS, and STING in OPM2 cells infected with a vector expressing DIS3 (DIS3) or an empty vector (EV). Values are relative to housekeeping gene expression, normalized on EV, and represent SE of three biological replicates. *** = p < 0.001, ** = p < 0.01, multiple t-tests.Differentially expressed genes (DEGs) between mutant and WT samples; p-values are from limma and false-discovery rate (FDR) is calculated using the Benjamini-Hochberg procedure; FDR < 0.05 was used as cut off. Functional annotation analysis by IPA of the DEGs between DIS3 mutant and WT samples based on FDR cut-off. (C) Volcano plot of DEGs between mutant and WT samples; labeled genes are the IFN-related genes with an FDR < 0.05 (red horizontal line) and absolute log 2-fold change above or below 0.5 (red vertical lines), respectively; additional representative IFN-related genes are also labeled.(F) Violin plot of the number of non-synonymous (NS) mutation load comparison between DIS3 mutant versus DIS3 WT performed on the entire CoMMpass dataset, including 853 MM patients, 88 presenting DIS3 mutations (all samples) and on the CoMMpass dataset, stratified according to the intensity of the IFN responsiveness (clusters C1, C2, C3). Wilcoxon rank-sum test was used for comparison between DIS3 mutant vs WT. **** = p <, ** = p < 0.01, * = p < 0.05. Black color dots represent the median in each group.


Figure 1A - E Chemokine - galectin interactions were detected by using a solid - phase immunoassay . For this , 46 human CC and CXC chemokines were adsorbed on nitrocellulose membranes and the stripes incubated in parallel with ( A ) TBS or ( B ) TBS containing biotinylated galectins ( the representative image shows a processed membrane tested with labeled Gal - 3 ) . Signals had been generated by using SA - HRP and chemiluminescence reagents . ( C - D ) The blots were subjected to densitometric analysis , and ( E ) all independent experiments were combined ( binding chemokines in light blue , Gal - 3 : n = 5 , Gal - 3 CRD : n = 5 , Gal - 1 n = 4 ) . For SPR - based experiments , ( F ) Gal - 3 ( density 650 RU ) , were immobilized and increasing concentrations of CXCL12 were passed over the flow cells . The red curve represents a single - site fit to the data . Insets are representative sensorgrams of CXCL12 testing on immobilized galectin . For SPR - based experiments , ( G ) Gal - 3 CRD ( density 1180 RU ) , were immobilized and increasing concentrations of CXCL12 were passed over the flow cells . The red curve represents a single - site fit to the data . Insets are representative sensorgrams of CXCL12 testing on immobilized galectin . For SPR - based experiments , ( H ) Gal - 1 ( density 130 RU ) were immobilized and increasing concentrations of CXCL12 were passed over the flow cells . The red curve represents a single - site fit to the data . Insets are representative sensorgrams of CXCL12 testing on immobilized galectin .

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 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 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

Figure 1A-E Chemokine-galectin interactions were detected by using a solid-phase immunoassay. For this, 46 human CC and CXC chemokines were adsorbed on nitrocellulose membranes and the stripes incubated in parallel with (A) TBS or (B) TBS containing biotinylated galectins (the representative image shows a processed membrane tested with labeled Gal-3). Signals had been generated by using SA-HRP and chemiluminescence reagents. (C-D) The blots were subjected to densitometric analysis, and (E) all independent experiments were combined (binding chemokines in light blue, Gal-3: n = 5, Gal-3 CRD: n = 5, Gal-1 n = 4).For SPR-based experiments, (F) Gal-3 (density 650 RU), were immobilized and increasing concentrations of CXCL12 were passed over the flow cells. The red curve represents a single-site fit to the data. Insets are representative sensorgrams of CXCL12 testing on immobilized galectin.For SPR-based experiments, (G) Gal-3 CRD (density 1180 RU), were immobilized and increasing concentrations of CXCL12 were passed over the flow cells. The red curve represents a single-site fit to the data. Insets are representative sensorgrams of CXCL12 testing on immobilized galectin.For SPR-based experiments, (H) Gal-1 (density 130 RU) were immobilized and increasing concentrations of CXCL12 were passed over the flow cells. The red curve represents a single-site fit to the data. Insets are representative sensorgrams of CXCL12 testing on immobilized galectin.


Figure 2a - b Expansions of 1H - 15N HSQC spectra are overlaid and Δδ values are plotted vs . the amino acid sequences , along with secondary structures : ( a ) 10 µM 15N - enriched CXCL12 alone ( black peaks ) and in the presence of 330 µM Gal - 3 CRD ( red peaks ; assignments reported by Murphy et al . [ 74 ] ) and ( b ) 30 µM 15N - enriched Gal - 3 CRD alone ( black peaks ) and in the presence of 500 µM CXCL12 ( red peaks ; assignments for Gal -

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

Figure 2a-b Expansions of 1H-15N HSQC spectra are overlaid and Δδ values are plotted vs. the amino acid sequences, along with secondary structures: (a) 10 µM 15N-enriched CXCL12 alone (black peaks) and in the presence of 330 µM Gal-3 CRD (red peaks; assignments reported by Murphy et al.[74]) and (b) 30 µM 15N-enriched Gal-3 CRD alone (black peaks) and in the presence of 500 µM CXCL12 (red peaks; assignments for Gal-3


Figure 3a - c Jurkat T cells migrated in the presence of 10 nM CXCL12 with increasing concentrations of ( a ) Gal - 3 , ( b ) Gal - 3 CRD , and ( c ) Gal - 1 ( A - C : n = 4 ) . D Human CD4 + T cells migrated in the presence of 10 nM CXCL12 alone and with 10 nM Gal - 3 ( dark green ) , Gal - 3 CRD ( light green ) , and Gal - 1 ( blue , n = 3 ) . E - F ( E ) Jurkat T cells migrated to increasing concentrations of CXCL12 alone ( black ) and CXCL12 in the presence of 0 . 1 nM of Gal - 3 CRD ( light green ) . ( F ) The inhibitory effect of Gal - 3 CRD is shown as percentage of the chemotactic effect of CXCL12 ( E - F : n = 3 ) . G Jurkat T cells were allowed to migrate in the presence of CXCL12 at increasing concentrations alone and with 0 . 1 nM Gal - 1 or 100 nM Gal - 1 ( n = 3 ) . H Jurkat T cells did not migrate in the sole presence of 1 nM to 1 µM Gal - 3 , Gal - 3 CRD ( both n = 5 ) , or Gal - 1 ( n = 4 ) . I The viability of Jurkat T cells was assessed after incubation with 1 µM CXCL12 alone and in the presence of Gal - 3 , Gal - 3 CRD and Gal - 1 ( n = 2 ) . ( J ) THP - 1 cells migrated in the presence of 10 nM CXCL12 alone and with 10 nM Gal - 3 , Gal - 3 CRD and Gal - 1 ( K ) neutrophils migrated in the presence of 10 nM CXCL12 alone and with 10 nM Gal - 3 , Gal - 3 CRD and Gal - 1L Human eosinophils migrated in the presence of 10 nM CCL26 alone and with 10 nM Gal - 3 , Gal - 3 CRD , and Gal - 1 ( n = 3 ) . M CD4 + T cells migrated in the presence of 1 nM CCL17 alone and with equimolar concentrations of Gal - 3 , Gal - 3 CRD , and Gal - 1 ( n = 4 ) .

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 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 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 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 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 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 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 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

Figure 3a-c Jurkat T cells migrated in the presence of 10 nM CXCL12 with increasing concentrations of (a) Gal-3, (b) Gal-3 CRD, and (c) Gal-1 (A-C: n = 4).D Human CD4+ T cells migrated in the presence of 10 nM CXCL12 alone and with 10 nM Gal-3 (dark green), Gal-3 CRD (light green), and Gal-1 (blue, n = 3).E-F (E) Jurkat T cells migrated to increasing concentrations of CXCL12 alone (black) and CXCL12 in the presence of 0.1 nM of Gal-3 CRD (light green). (F) The inhibitory effect of Gal-3 CRD is shown as percentage of the chemotactic effect of CXCL12 (E-F: n = 3).G Jurkat T cells were allowed to migrate in the presence of CXCL12 at increasing concentrations alone and with 0.1 nM Gal-1 or 100 nM Gal-1 (n=3).H Jurkat T cells did not migrate in the sole presence of 1 nM to 1 µM Gal-3, Gal-3 CRD (both n = 5), or Gal-1 (n = 4).I The viability of Jurkat T cells was assessed after incubation with 1 µM CXCL12 alone and in the presence of Gal-3, Gal-3 CRD and Gal-1 (n = 2).(J) THP-1 cells migrated in the presence of 10 nM CXCL12 alone and with 10 nM Gal-3, Gal-3 CRD and Gal-1(K) neutrophils migrated in the presence of 10 nM CXCL12 alone and with 10 nM Gal-3, Gal-3 CRD and Gal-1L Human eosinophils migrated in the presence of 10 nM CCL26 alone and with 10 nM Gal-3, Gal-3 CRD, and Gal-1 (n = 3).M CD4+ T cells migrated in the presence of 1 nM CCL17 alone and with equimolar concentrations of Gal-3, Gal-3 CRD, and Gal-1 (n = 4).


Figure 4A - B The peritoneal recruitment of ( A ) CD45 + / CD115 - / Ly6G + neutrophils and ( B ) CD45 + / CD115 + / Ly6Chi classical monocytes in C57BL / 6J mice was assessed 4 hours after intraperitoneal ( IP ) injection of 500 nM CXCL12 alone ( black ) and in the presence of 50 nM Gal - 3 ( dark green ; A - B : n = 7 mice ) . C The peritoneal recruitment of classical monocytes after IP injection of PBS ( n = 6 ) or TG in C57BL / 6J WT ( dark green , n = 10 mice ) and Gal - 3 - / - ( black , n = 5 mice ) mice was assessed after 18 h . Where indicated , the mice received an IP injection of CXCR4 antagonist AMD 3465 12 hours prior to the experiment . D The concentration of CXCL12 concentration was determined by ELISA on the peritoneal lavage normalized with levels from the mesenterium ( n = 4 mice ) . E CXCR4 expression levels on Ly6Chi monocytes of the blood and the peritoneal lavage after 18 h of TG stimulation were determined by flow cytometry and indicated as mean fluorescence intensity ( MFI ) ( n = 4 mice ) .

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 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 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 4A-B The peritoneal recruitment of (A) CD45+/CD115-/Ly6G+ neutrophils and (B) CD45+/CD115+/Ly6Chi classical monocytes in C57BL/6J mice was assessed 4 hours after intraperitoneal (IP) injection of 500 nM CXCL12 alone (black) and in the presence of 50 nM Gal-3 (dark green; A-B: n = 7 mice).C The peritoneal recruitment of classical monocytes after IP injection of PBS (n = 6) or TG in C57BL/6J WT (dark green, n = 10 mice) and Gal-3-/- (black, n = 5 mice) mice was assessed after 18 h. Where indicated, the mice received an IP injection of CXCR4 antagonist AMD 3465 12 hours prior to the experiment.D The concentration of CXCL12 concentration was determined by ELISA on the peritoneal lavage normalized with levels from the mesenterium (n = 4 mice).E CXCR4 expression levels on Ly6Chi monocytes of the blood and the peritoneal lavage after 18 h of TG stimulation were determined by flow cytometry and indicated as mean fluorescence intensity (MFI) (n = 4 mice).


Figure 5A - B Jurkat T cells migrated in the presence of 10 nM CXCL12 with increasing concentrations of E185A ( orange , n = 4 ) and N222A ( red , n = 5 ) mutants of ( A ) Gal - 3 and ( B ) Gal - 3 CRD . C - D Jurkat T cells migrated in the presence of 10 nM CXCL12 with 10 nM ( C ) Gal - 3 and ( D ) Gal - 3 CRD mutants as indicated ( C - D : n = 3 ) . E Aggregation of Jurkat T cells in the presence of 1 nM to 1 µM Gal - 3 and Gal - 1 alone and with 70 mM lactose , and Gal - 3 CRD , was determined by flow cytometry ( n = 4 ) . Jurkat T cells migrated with 10 nM CXCL12 alone and in the presence of ( F ) 1 µM Gal - 3 , ( G ) Gal - 1 , ( H ) Gal - 3 CRD in the presence of 70 mM lactose or cellobiose or after treatment of cells with 150 µM DMJ . Jurkat T cells migrated with 10 nM CXCL12 alone and in the presence of ( I ) 0 . 1 nM Gal - 3 and ( J ) Gal - 3 CRD in the presence of 70 mM lactose or cellobiose or after treatment of cells with 150 µM DMJ .

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Figure 5A-B Jurkat T cells migrated in the presence of 10 nM CXCL12 with increasing concentrations of E185A (orange, n = 4) and N222A (red, n = 5) mutants of (A) Gal-3 and (B) Gal-3 CRD.C-D Jurkat T cells migrated in the presence of 10 nM CXCL12 with 10 nM (C) Gal-3 and (D) Gal-3 CRD mutants as indicated (C-D: n = 3).E Aggregation of Jurkat T cells in the presence of 1 nM to 1 µM Gal-3 and Gal-1 alone and with 70 mM lactose, and Gal-3 CRD, was determined by flow cytometry (n = 4).Jurkat T cells migrated with 10 nM CXCL12 alone and in the presence of (F) 1 µM Gal-3, (G) Gal-1, (H) Gal-3 CRD in the presence of 70 mM lactose or cellobiose or after treatment of cells with 150 µM DMJ.Jurkat T cells migrated with 10 nM CXCL12 alone and in the presence of (I) 0.1 nM Gal-3 and (J) Gal-3 CRD in the presence of 70 mM lactose or cellobiose or after treatment of cells with 150 µM DMJ.


Figure 6a HEK cells transfected with a luminescent cAMP sensor were incubated with 10 nM CXCL12 alone and in the presence of 10 nM Gal - 3 CRD followed by stimulation with forskolin , an activator of adenylate cyclase . Results are shown as luminescence relative to baseline ( RLU , example of n = 3 ) . b The effect of 10 nM Gal - 3 CRD upon stimulation with 10 nM CXCL12 prior to forskolin stimulation , was tested . Control experiments were performed as indicated ( n = 3 , four technical replicates ) . c HEK cells transfected with a RlucII - conjugated CXCR4 and an eYFP - β - arrestin 2 construct were stimulated with 10 nM CXCL12 alone and in the presence of 10 nM Gal - 3 CRD . Control experiments were performed as indicated . Results are given as the net BRET ratio ( i . e . ratio of emissions at 535 / 485 nm minus the ratio of mock cells , n = 5 , three technical replicates ) . D Internalization of CXCR4 with 10 nM CXCL12 alone and in the presence of 10 nM Gal - 3 CRD was assessed by incubation with an APC - conjugated anti - CXCR4 antibody ( baseline signal = 100 % , n = 8 , two technical replicates ) . E 100 nM CXCL12 and Gal - 3 were added to Jurkat T cells , and their co - localization was assessed on the cell surface by PLA . Control experiments were performed as indicated ( representative example of n = 3 ) . White scale bar : 10 µm . F - G Jurkat T cells were incubated with 100 nM of Gal - 3 CRD Alexa Fluor 555 alone , with 10 µM AMD 3100 , with 100 nM CXCL12 or with CXCL12 and AMD 3100 . Experiments were performed ( f ) without and ( g ) in the presence of 70 mM lactose ( F - G : n = 3 , two technical replicates ) . H Jurkat T cells were incubated with 100 nM CXCL12 FITC alone and with increasing concentrations of Gal - 3 CRD as indicated ( n = 4 , two technical replicates ) .

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Figure 6a HEK cells transfected with a luminescent cAMP sensor were incubated with 10 nM CXCL12 alone and in the presence of 10 nM Gal-3 CRD followed by stimulation with forskolin, an activator of adenylate cyclase. Results are shown as luminescence relative to baseline (RLU, example of n = 3).b The effect of 10 nM Gal-3 CRD upon stimulation with 10 nM CXCL12 prior to forskolin stimulation, was tested. Control experiments were performed as indicated (n = 3, four technical replicates).c HEK cells transfected with a RlucII-conjugated CXCR4 and an eYFP-β-arrestin 2 construct were stimulated with 10 nM CXCL12 alone and in the presence of 10 nM Gal-3 CRD. Control experiments were performed as indicated. Results are given as the net BRET ratio (i.e. ratio of emissions at 535/485 nm minus the ratio of mock cells, n = 5, three technical replicates).D Internalization of CXCR4 with 10 nM CXCL12 alone and in the presence of 10 nM Gal-3 CRD was assessed by incubation with an APC-conjugated anti-CXCR4 antibody (baseline signal = 100%, n = 8, two technical replicates).E 100 nM CXCL12 and Gal-3 were added to Jurkat T cells, and their co-localization was assessed on the cell surface by PLA. Control experiments were performed as indicated (representative example of n = 3). White scale bar: 10 µm.F-G Jurkat T cells were incubated with 100 nM of Gal-3 CRD Alexa Fluor 555 alone, with 10 µM AMD 3100, with 100 nM CXCL12 or with CXCL12 and AMD 3100. Experiments were performed (f) without and (g) in the presence of 70 mM lactose (F-G: n = 3, two technical replicates).H Jurkat T cells were incubated with 100 nM CXCL12 FITC alone and with increasing concentrations of Gal-3 CRD as indicated (n = 4, two technical replicates).


Figure 2ISCts midguts expressing YFP alone ( control ) , or expressing two non - overlapping EcR - RNAi , and quantification of the number of YFP - positive cells ( yellow ) . The graph also plots the number of GFP - positive cells in esgts midgut expressing GFP alone ( ctrl ) , or expressing EcR - RNAi # 1 , EcR - RNAi # 2 , and mcherry - RNAi as an additional negative control ( see EV2B ) . esgts midguts expressing GFP alone ( control ) , or expressing EcR - DN , and EcR - DN + pri . Samples were stained for GFP ( green ) . The graph shows quantification of the number of GFP - positive cells in the different genotypes . Posterior midguts containing control and Ubr3 null MARCM clones ( GFP , green ) . esgts midguts expressing GFP alone ( control ) , or expressing Ubr3 - RNAi , and Ubr3 - RNAi + OvoB . Samples were stained for GFP ( green ) . H ' quantification of GFP - positive cells from H .

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Figure 2ISCts midguts expressing YFP alone (control), or expressing two non-overlapping EcR-RNAi, and quantification of the number of YFP-positive cells (yellow). The graph also plots the number of GFP-positive cells in esgts midgut expressing GFP alone (ctrl), or expressing EcR-RNAi#1, EcR-RNAi#2, and mcherry-RNAi as an additional negative control (see EV2B).esgts midguts expressing GFP alone (control), or expressing EcR-DN, and EcR-DN + pri. Samples were stained for GFP (green). The graph shows quantification of the number of GFP-positive cells in the different genotypes.Posterior midguts containing control and Ubr3 null MARCM clones (GFP, green).esgts midguts expressing GFP alone (control), or expressing Ubr3-RNAi, and Ubr3-RNAi + OvoB. Samples were stained for GFP (green). H' quantification of GFP-positive cells from H.


Figure 3esgts midguts expressing GFP alone ( control ) or expressing SvbACT . Samples were stained for GFP ( green ) , and ß - catenin ( purple ) , DE - Cadherin ( white ) or Scribble ( yellow ) . Top and bottom pictures show separate channels for a same region . esgts midguts expressing GFP alone ( control ) , or expressing miR8 , OvoB , and mir8 + OvoB . Samples were stained for GFP ( green ) and ß - catenin ( purple ) . The graph shows quantification of GFP - positive cells for each genotype . The Y axis is drawn using a log ( 10 ) scale . esgts midguts expressing GFP alone ( control ) , or expressing Notch Intra Cellular Domain ( NICD ) , and NICD + OvoB . In closeups ( right ) , DAPI is shown in purple for improved contrast ; the arrow highlights a cell with intermediate phenotype .

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Figure 3esgts midguts expressing GFP alone (control) or expressing SvbACT. Samples were stained for GFP (green), and ß-catenin (purple), DE-Cadherin (white) or Scribble (yellow). Top and bottom pictures show separate channels for a same region.esgts midguts expressing GFP alone (control), or expressing miR8, OvoB, and mir8 + OvoB. Samples were stained for GFP (green) and ß-catenin (purple). The graph shows quantification of GFP-positive cells for each genotype. The Y axis is drawn using a log(10) scale.esgts midguts expressing GFP alone (control), or expressing Notch Intra Cellular Domain (NICD), and NICD + OvoB. In closeups (right), DAPI is shown in purple for improved contrast; the arrow highlights a cell with intermediate phenotype.


Figure 4esgtsF / O midguts expressing GFP alone ( control ) , or expressing RasV12 , and RasV12 + svb - RNAi ( top panels ) . Bottom panels show esgts midguts expressing GFP alone ( control ) , or expressing EGFR - DN , and EGFR - DN + OvoB . Samples were stained for GFP ( green ) . The graph shows quantification of the number of GFP - positive cells in esgts midguts expressing GFP alone ( ctrl ) , or expressing EGFR - DN , EGFR - DN + OvoB , and EGFR - DN + svb - RNAi . The Y axis is plotted as log ( 10 ) . esgts midguts expressing GFP alone ( control ) , or expressing ArmS10 , ArmS10 + svb - RNAi , ArmS10 + OvoB , TCF - DN , and TCF - DN + OvoB . Samples were stained for GFP ( green ) . The graph shows quantification of the number of GFP - positive cells in esgts midguts expressing GFP alone ( ctrl ) , or expressing TCF - DN , TCF - DN + OvoB , and TCF - DN + svb - RNAi . The Y axis is plotted as log ( 10 ) .

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Figure 4esgtsF/O midguts expressing GFP alone (control), or expressing RasV12, and RasV12 + svb-RNAi (top panels). Bottom panels show esgts midguts expressing GFP alone (control), or expressing EGFR-DN, and EGFR-DN + OvoB. Samples were stained for GFP (green). The graph shows quantification of the number of GFP-positive cells in esgts midguts expressing GFP alone (ctrl), or expressing EGFR-DN, EGFR-DN + OvoB, and EGFR-DN + svb-RNAi. The Y axis is plotted as log(10).esgts midguts expressing GFP alone (control), or expressing ArmS10, ArmS10 + svb-RNAi, ArmS10 + OvoB ,TCF-DN, and TCF-DN + OvoB. Samples were stained for GFP (green). The graph shows quantification of the number of GFP-positive cells in esgts midguts expressing GFP alone (ctrl), or expressing TCF-DN, TCF-DN + OvoB, and TCF-DN + svb-RNAi. The Y axis is plotted as log(10).


Figure 5esgts midguts expressing GFP alone ( control ) , or expressing SvbREP . Samples were stained for GFP ( green ) and Prospero ( red ) . Closeups correspond to boxed regions , with DAPI shown in purple and GFP - positive cells outlined in yellow . esgts - F / O midguts expressing GFP alone ( control ) , or expressing SvbREP . Samples were stained for GFP ( green ) and cleaved DCP1 ( red ) . E ' shows quantification of the number of GFP - positive cells in esgts guts expressing GFP alone ( ctrl ) , or DIAP , SvbREP , and SvbREP + DIAP .

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Figure 5esgts midguts expressing GFP alone (control), or expressing SvbREP. Samples were stained for GFP (green) and Prospero (red). Closeups correspond to boxed regions, with DAPI shown in purple and GFP-positive cells outlined in yellow.esgts-F/O midguts expressing GFP alone (control), or expressing SvbREP. Samples were stained for GFP (green) and cleaved DCP1 (red). E' shows quantification of the number of GFP-positive cells in esgts guts expressing GFP alone (ctrl), or DIAP, SvbREP, and SvbREP + DIAP.


Figure 6esgts midguts expressing GFP alone ( control ) , or expressing Notch - RNAi , Notch - RNAi + svb - RNAi , and Notch - RNAi + SvbREP . Samples were stained for GFP ( green ) and Prospero ( red ) . The graph shows quantification of the number of GFP - positive cells . esgts midguts expressing GFP alone ( control ) , or expressing STAT - RNAi , STAT - RNAi + svb - RNAi and STAT - RNAi + SvbREP . Samples were stained for GFP ( green ) and Prospero ( red ) . The graph shows quantification of the number of GFP - positive cells .

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Figure 6esgts midguts expressing GFP alone (control), or expressing Notch-RNAi, Notch-RNAi + svb-RNAi, and Notch-RNAi + SvbREP. Samples were stained for GFP (green) and Prospero (red). The graph shows quantification of the number of GFP-positive cells.esgts midguts expressing GFP alone (control), or expressing STAT-RNAi, STAT-RNAi + svb-RNAi and STAT-RNAi + SvbREP. Samples were stained for GFP (green) and Prospero (red). The graph shows quantification of the number of GFP-positive cells.


Figure 7Cross sections of control MyoIAts midguts ( expressing GFP and mCherry - RNAi ) , or expressing svb - RNAi , SvbACT , and Pri . Samples were stained for F - actin ( white ) , GFP ( green ) and Tsp2a ( yellow ) .

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Figure 7Cross sections of control MyoIAts midguts (expressing GFP and mCherry-RNAi), or expressing svb-RNAi, SvbACT, and Pri. Samples were stained for F-actin (white), GFP (green) and Tsp2a (yellow).


Figure 8MyoIAts midguts expressing GFP alone ( control ) , or expressing SvbACT . Samples were stained for GFP ( green ) , Scribble ( yellow ) , ß - catenin ( purple ) and DAPI ( Blue ) . A ' pictures display cross sections of the regions shown in A . MyoIAts midguts expressing GFP alone ( control ) , or expressing SvbACT . Samples were stained for GFP ( green ) and PH3 ( red ) . The graph plots number of mitotic PH3 - positive cells per midgut of MyoIAts guts expressing GFP alone ( ctrl ) , or expressing SvbACT , and SvbREP + pri .

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Figure 8MyoIAts midguts expressing GFP alone (control), or expressing SvbACT. Samples were stained for GFP (green), Scribble (yellow), ß-catenin (purple) and DAPI (Blue). A' pictures display cross sections of the regions shown in A.MyoIAts midguts expressing GFP alone (control), or expressing SvbACT. Samples were stained for GFP (green) and PH3 (red). The graph plots number of mitotic PH3-positive cells per midgut of MyoIAts guts expressing GFP alone (ctrl), or expressing SvbACT , and SvbREP + pri.


Figure 1 ( D ) Cells were treated with sodium arsenite ( 50 µM ) for 45 min , followed by recovery in drug - free medium . Kinetics of SG dissolution are reported . Images were taken over a time period of 4 hrs every 10 min . Dashed lines = 95 % confidence intervals . Number of cells counted : 159 ( siRNA control ) ; 166 ( siRNA Hsp90 α + β ) . ( E ) HeLa - Kyoto cells were treated with HS at 43 . 5 ˚ C for 1 hr . Cells were then allowed to recover at 37 ° C for 1 hr in drug - free medium ( control ) or in presence of VER ( 40 µM ) , GA ( 5 µM ) or 17AAG ( 5 µM ) . Cells were fixed , stained for the SG marker TIA - 1 and the percentage of cells with SGs was counted . Number of cells counted : 605 ( recovery control ) ; 510 ( recovery VER ) ; 1334 ( recovery GA ) ; 468 ( recovery 17AAG ) . n = 3 - 4 independent experiments , ± sem . p < 0 . 01 ( One - way ANOVA ) . ( F ) G3BP2 - GFP HeLa - Kyoto cells were treated with MG132 ( 20 µM ) for 3 hrs , followed by recovery for 2 hrs in drug - free medium ( control ) or in presence of VER ( 40 µM ) , GA ( 5 µM ) or 17AAG ( 5 µM ) . Cells were fixed and the percentage of cells with SGs was counted . Number of cells counted : 439 ( recovery control ) ; 649 ( recovery VER ) ; 637 ( recovery GA ) ; 649 ( recovery 17AAG ) . n = 3 independent experiments , ± sem . p < 0 . 0001 ( One - way ANOVA ) .

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Figure 1(D) Cells were treated with sodium arsenite (50 µM) for 45 min, followed by recovery in drug-free medium. Kinetics of SG dissolution are reported. Images were taken over a time period of 4 hrs every 10 min. Dashed lines = 95% confidence intervals. Number of cells counted: 159 (siRNA control); 166 (siRNA Hsp90 α+β).(E) HeLa-Kyoto cells were treated with HS at 43.5˚C for 1 hr. Cells were then allowed to recover at 37 °C for 1 hr in drug-free medium (control) or in presence of VER (40 µM), GA (5 µM) or 17AAG (5 µM). Cells were fixed, stained for the SG marker TIA-1 and the percentage of cells with SGs was counted. Number of cells counted: 605 (recovery control); 510 (recovery VER); 1334 (recovery GA); 468 (recovery 17AAG). n = 3-4 independent experiments, ± sem. p < 0.01 (One-way ANOVA). (F) G3BP2-GFP HeLa-Kyoto cells were treated with MG132 (20 µM) for 3 hrs, followed by recovery for 2 hrs in drug-free medium (control) or in presence of VER (40 µM), GA (5 µM) or 17AAG (5 µM). Cells were fixed and the percentage of cells with SGs was counted. Number of cells counted: 439 (recovery control); 649 (recovery VER); 637 (recovery GA); 649 (recovery 17AAG). n = 3 independent experiments, ± sem. p < 0.0001 (One-way ANOVA).


Figure 2 ( C ) HeLa cells were subjected to proximity ligation assay ( PLA ) using antibodies specific for endogenous Hsp90 and DYRK3 . PLA foci and nuclei were segmented , and PLA foci / cell were automatically quantified . The mean of PLA / foci in cells incubated with no antibodies ( - ) , only Hsp90 antibody , only DYRK3 antibody and both Hsp90 and DYRK3 antibodies is shown . Cells incubated with Hsp90 and DYRK3 antibodies and left untreated were used as control . n = 3 independent experiments , ± sem ; 368 - 504 cells counted / sample , p < 0 . 002 between the negative controls and cells incubated with Hsp90 and DYRK3 antibodies ; p = 10 - 6 between cells incubated with Hsp90 and DYRK3 antibodies and left untreated ( control ) or treated for 2 hrs with GA ( 5 µM ) ( One - way ANOVA ) . Scale bar is 10 µm . ( F ) HeLa cells were lipofected with a cDNA encoding for GFP or GFP - DYRK3 . 6 hrs post - transfection , cells were incubated in drug - free medium ( - ) or in presence of GA , 17AAG or VER ( concentrations are shown ) . Total proteins were extracted 16 hrs later . GFP and TUBA4A protein levels were analyzed by immunoblotting . Representative immunoblotting of 3 independent experiments . ( I ) HeLa cells were lipofected with a cDNA encoding for GFP - DYRK3 . 24 hrs post - transfection , cells were treated for 45 min with arsenite ( 50 µM ) alone ( control ) or with GA ( 5 µM ) or 17AAG ( 5 µM ) . Cell were then fixed and the percentage of cells expressing GFP - DYRK3 with SGs was counted . Total cells counted : 462 ( sodium arsenite ) ; 444 ( sodium arsenite and GA ) ; 561 ( sodium arsenite and 17AAG ) . n = 3 independent experiments , ± sem ; p < 0 . 001 ( One - way ANOVA ) .

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Figure 2(C) HeLa cells were subjected to proximity ligation assay (PLA) using antibodies specific for endogenous Hsp90 and DYRK3. PLA foci and nuclei were segmented, and PLA foci/cell were automatically quantified. The mean of PLA/foci in cells incubated with no antibodies (-), only Hsp90 antibody, only DYRK3 antibody and both Hsp90 and DYRK3 antibodies is shown. Cells incubated with Hsp90 and DYRK3 antibodies and left untreated were used as control. n = 3 independent experiments, ± sem; 368-504 cells counted/sample, p < 0.002 between the negative controls and cells incubated with Hsp90 and DYRK3 antibodies; p = 10-6 between cells incubated with Hsp90 and DYRK3 antibodies and left untreated (control) or treated for 2 hrs with GA (5 µM) (One-way ANOVA). Scale bar is 10 µm.(F) HeLa cells were lipofected with a cDNA encoding for GFP or GFP-DYRK3. 6 hrs post-transfection, cells were incubated in drug-free medium (-) or in presence of GA, 17AAG or VER (concentrations are shown). Total proteins were extracted 16 hrs later. GFP and TUBA4A protein levels were analyzed by immunoblotting. Representative immunoblotting of 3 independent experiments.(I) HeLa cells were lipofected with a cDNA encoding for GFP-DYRK3. 24 hrs post-transfection, cells were treated for 45 min with arsenite (50 µM) alone (control) or with GA (5 µM) or 17AAG (5 µM). Cell were then fixed and the percentage of cells expressing GFP-DYRK3 with SGs was counted. Total cells counted: 462 (sodium arsenite); 444 (sodium arsenite and GA); 561 (sodium arsenite and 17AAG). n = 3 independent experiments, ± sem; p < 0.001 (One-way ANOVA).


Figure 3 ( A , HeLa cells were left untreated ( control ) or treated with GA ( 5 µM ) , 17AAG ( 5 µM ) or VER ( 10 µM ) for 8 hrs . Cells were then fixed and immunostained for the nuclear speckle marker SC35 and DYRK3 . Automatic segmentation of nuclear speckles is based on SC35 . An automated imaging assay was used to quantify the percentage of nuclear speckles highly enriched for DYRK3 ( using a fluorescent ratio 1 . 5 as threshold ) . Scale bar is 10 µm . D ) Representative confocal microscopy images of cells with SC35 - positive bodies ( D ) Scale bar is 10 µm . ( F ) HeLa cells were lipofected with a cDNA encoding for GFP - DYRK3 - dN . 24 hrs post - transfection GFP - DYRK3 - dN cells were either left untreated or exposed to GA ( 5 µM ) for 4 hrs . In untreated cells ( control ) , GFP - DYRK3 - dN is diffusely distributed in the cytosol and in the nucleus . Upon GA treatment , GFP - DYRK3 - dN forms perinuclear ( PN ) aggregates . Representative confocal images are shown . Scale bar is 10 µm . ( I ) mCherry - G3BP1 expressing HeLa cells were lipofected with cDNAs encoding for GFP - DYRK3 - dN or Sup35 - NM - GFP - DYRK3 - dN . 24 hrs post - transfection cells were treated for 45 min with sodium arsenite to induce SGs , followed by recovery for 4 hrs in presence of GA ( 5 µM ; rec . GA ) . Representative confocal microscopy images are shown . Nucleic acid was stained with DAPI . Scale bar is 10 µm .

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Figure 3(A, HeLa cells were left untreated (control) or treated with GA (5 µM), 17AAG (5 µM) or VER (10 µM) for 8 hrs. Cells were then fixed and immunostained for the nuclear speckle marker SC35 and DYRK3. Automatic segmentation of nuclear speckles is based on SC35. An automated imaging assay was used to quantify the percentage of nuclear speckles highly enriched for DYRK3 (using a fluorescent ratio 1.5 as threshold). Scale bar is 10 µm.D) Representative confocal microscopy images of cells with SC35-positive bodies (D) Scale bar is 10 µm.(F) HeLa cells were lipofected with a cDNA encoding for GFP-DYRK3-dN. 24 hrs post-transfection GFP-DYRK3-dN cells were either left untreated or exposed to GA (5 µM) for 4 hrs. In untreated cells (control), GFP-DYRK3-dN is diffusely distributed in the cytosol and in the nucleus. Upon GA treatment, GFP-DYRK3-dN forms perinuclear (PN) aggregates. Representative confocal images are shown. Scale bar is 10 µm.(I) mCherry-G3BP1 expressing HeLa cells were lipofected with cDNAs encoding for GFP-DYRK3-dN or Sup35-NM-GFP-DYRK3-dN. 24 hrs post-transfection cells were treated for 45 min with sodium arsenite to induce SGs, followed by recovery for 4 hrs in presence of GA (5 µM; rec. GA). Representative confocal microscopy images are shown. Nucleic acid was stained with DAPI. Scale bar is 10 µm.


Figure 4 ( C ) Quantitation of the fluorescence intensity recovery after bleach of GFP - DYRK3 in G3BP1 - mCherry HeLa - Kyoto cells 24 h after transfection . Cells were treated with sodium arsenite ( 50 µM ) for 45 min to induce SGs , which were visualized with G3BP1 - mCherry ; then , sodium arsenite was removed and the cells were incubated in drug - free medium or in presence of GA ( 5 µM ) or of GSK ( 5 µM ) . FRAP was performed during the stress recovery period . A representative image of cells treated with arsenite followed by recovery in drug - free medium is shown . Arrowheads and dotted circle indicate the ROI . Scale bar is 5 µm . The mean of 13 FRAP curves ( recovery control ) , 12 FRAP curves ( recovery GA ) and 14 FRAP curves ( recovery GSK ) is shown in red ; the sem is shown in gray .

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

Figure 4(C) Quantitation of the fluorescence intensity recovery after bleach of GFP-DYRK3 in G3BP1-mCherry HeLa-Kyoto cells 24 h after transfection. Cells were treated with sodium arsenite (50 µM) for 45 min to induce SGs, which were visualized with G3BP1-mCherry; then, sodium arsenite was removed and the cells were incubated in drug-free medium or in presence of GA (5 µM) or of GSK (5 µM). FRAP was performed during the stress recovery period. A representative image of cells treated with arsenite followed by recovery in drug-free medium is shown. Arrowheads and dotted circle indicate the ROI. Scale bar is 5 µm. The mean of 13 FRAP curves (recovery control), 12 FRAP curves (recovery GA) and 14 FRAP curves (recovery GSK) is shown in red; the sem is shown in gray.


Figure 5 ( B ) Confocal microscopy images of P525L FUS eGFP MNs treated with sodium arsenite for 2 hrs and showing colocalization of FUS with TIAR - positive stress granules . Scale bar is 10 µm . ( C ) P525L FUS eGFP MNs were treated with sodium arsenite for 2 hrs , followed by recovery in drug - free medium ( + rec . control ) or in presence of 17AAG ( 30 μM ; + rec . 17AAG ) or of GSK ( 5 μM ; + rec . GSK ) for 6 hrs . Quantitation of the number of SGs per cell in P525L FUS eGFP MNs . Number of P525L cells counted and n / condition : 2488 , n = 13 ( arsenite ) ; 1045 , n = 12 ( arsenite + rec . control ) ; 646 , n = 6 ( arsenite + rec . 17AAG ) ; 924 , n = 3 ( arsenite + rec . GSK ) , ± sem ( One - way ANOVA ) . ( I ) Double immunofluorescence labeling using antibodies against DYRK3 and FUS in lumbar spinal cord α - MNs of healthy subjects ( control ) and fALS patients carrying the p . 521C mutation in the FUS gene . Control ( upper panel ) : α - MNs showing uniform cytoplasmic ( white arrows ) and speckled pattern of strong nuclear immunoreactivity ( white arrowhead ) of DYRK3 , as well as diffuse nuclear FUS immunoreactivity . fALS ( p . R521C - FUS ; lower panel ) : surviving α - MNs harboring large FUS aggregates ( yellow arrows ) showed markedly reduced DYRK3 immunoreactivity both in the cytoplasm as well as in the nucleus ( red arrowheads ) . Instead , α - MNs devoid of FUS aggregates ( white arrows ) showed normal DYRK3 immunoreactivity similar to the one of α - MNs from normal controls . Asterix ( * ) represents non - specific lipofuscin granules in one of the α - MN in FUS - ALS . Paraffin sections ; scale bars is 50 µm . ( J ) Quantification of α - MNs showing low or absent DYRK3 staining ( upper graphic ) and FUS aggregates ( lower graphic ) in lumbar spinal cord from five healthy subjects ( control ) and five fALS patients carrying the p . 521C mutation in the FUS gene . Total number of α - MNs analyzed : 403 ( control ) ; 132 ( fALS ) . n = 5 , ± sem ( Student ' s t - test ) .

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 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

Figure 5(B) Confocal microscopy images of P525L FUS eGFP MNs treated with sodium arsenite for 2 hrs and showing colocalization of FUS with TIAR-positive stress granules. Scale bar is 10 µm. (C) P525L FUS eGFP MNs were treated with sodium arsenite for 2 hrs, followed by recovery in drug-free medium (+ rec. control) or in presence of 17AAG (30 μM; + rec. 17AAG) or of GSK (5 μM; +rec. GSK) for 6 hrs. Quantitation of the number of SGs per cell in P525L FUS eGFP MNs. Number of P525L cells counted and n/condition: 2488, n = 13 (arsenite); 1045, n = 12 (arsenite + rec. control); 646, n = 6 (arsenite + rec. 17AAG); 924, n = 3 (arsenite + rec. GSK), ± sem (One-way ANOVA). (I) Double immunofluorescence labeling using antibodies against DYRK3 and FUS in lumbar spinal cord α-MNs of healthy subjects (control) and fALS patients carrying the p.521C mutation in the FUS gene. Control (upper panel): α-MNs showing uniform cytoplasmic (white arrows) and speckled pattern of strong nuclear immunoreactivity (white arrowhead) of DYRK3, as well as diffuse nuclear FUS immunoreactivity. fALS (p.R521C-FUS; lower panel): surviving α-MNs harboring large FUS aggregates (yellow arrows) showed markedly reduced DYRK3 immunoreactivity both in the cytoplasm as well as in the nucleus (red arrowheads). Instead, α-MNs devoid of FUS aggregates (white arrows) showed normal DYRK3 immunoreactivity similar to the one of α-MNs from normal controls. Asterix (*) represents non-specific lipofuscin granules in one of the α-MN in FUS-ALS. Paraffin sections; scale bars is 50 µm. (J) Quantification of α-MNs showing low or absent DYRK3 staining (upper graphic) and FUS aggregates (lower graphic) in lumbar spinal cord from five healthy subjects (control) and five fALS patients carrying the p.521C mutation in the FUS gene. Total number of α-MNs analyzed: 403 (control); 132 (fALS). n = 5, ± sem (Student's t-test).


Figure 1A HeLa cells were transfected with control ( Ctr ) or three different GCN2 - targeting siRNA - s and progression from prophase to anaphase was observed by live - cell imaging . Averages ( lines ) and SEM ( bands ) from five ( siCtr ) or four ( each siGCN2 ) independent experiments are shown ; n = 379 for siCtr , 241 for siGCN2 # 1 , 170 for siGCN2 # 2 , 239 for siGCN2 # 3 . * * * * p < 0 . 0001 for each of the siRNA - s ( Kolmogorov - Smirnov test ) . t = 0 denotes entry into prophase as judged by the first frame showing chromatin condensation . E , Chromosome congression is affected by the absence of GCN2 . E Immunofluorescence images of HeLa cells stained for a kinetochore marker , CREST ( grey ) and DAPI for DNA ( blue ) . Arrowheads indicate misaligned chromosomes . Scale bars represent 5 µm . G Increased number of mitotic cells with tripolar spindles after GCN2 knock - down . HeLa cells were transfected with control ( Ctr ) or GCN2 - targeting siRNA and fixed for immunofluorescence . The number of spindle poles was determined by using a pericentrin antibody . Mean and SEM are shown from five independent experiments . n = 61 , 27 , 19 , 24 and 28 for siCtr ; n = 49 , 29 , 22 , 26 and 26 for siGCN2 ; p = 0 . 0125 , paired two - tailed t - test .

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Figure 1A HeLa cells were transfected with control (Ctr) or three different GCN2-targeting siRNA-s and progression from prophase to anaphase was observed by live-cell imaging. Averages (lines) and SEM (bands) from five (siCtr) or four (each siGCN2) independent experiments are shown; n= 379 for siCtr, 241 for siGCN2 #1, 170 for siGCN2 #2, 239 for siGCN2 #3. ****p<0.0001 for each of the siRNA-s (Kolmogorov-Smirnov test). t=0 denotes entry into prophase as judged by the first frame showing chromatin condensation.E, Chromosome congression is affected by the absence of GCN2. E Immunofluorescence images of HeLa cells stained for a kinetochore marker, CREST (grey) and DAPI for DNA (blue). Arrowheads indicate misaligned chromosomes. Scale bars represent 5 µm.G Increased number of mitotic cells with tripolar spindles after GCN2 knock-down. HeLa cells were transfected with control (Ctr) or GCN2-targeting siRNA and fixed for immunofluorescence. The number of spindle poles was determined by using a pericentrin antibody. Mean and SEM are shown from five independent experiments. n= 61, 27, 19, 24 and 28 for siCtr; n=49, 29, 22, 26 and 26 for siGCN2; p=0.0125, paired two-tailed t-test.


Figure 2A , B There is no significant difference in global translation rates in mitosis after GCN2 knock - down . A HeLa cells were pulse labeled with OPP and fixed with 70 % ethanol . OPP was detected by Click - IT ( AF 647 ) and mitotic cells were identified by staining for phospho - histone H3 . 2D flow cytometry plots are shown , where AF647 intensity corresponds to OPP incorporation . Translation levels normalized to siCtr G1 . The graph shows averages and SEM from three independent experiments . Paired two - tailed t - tests . D , Mitotic translation of selected proteins . Hela cells were incubated with 5 µM Eg5i overnight and mitotic cells were shaken off . MG132 was added to 50 µM ( t = 0 ) , then after 10 minutes the cells were split in two ( t = 10 ) . GCN2i ( Gcn2 - IN - 1 , MedChem Express HY - 100877 ) was added to 2 µM to one of the cell suspensions and samples were taken for immunoblotting every 10 minutes ( D ) Representative immunoblots of three biological replicates are shown . E Mitotic translation of selected proteins . Hela cells were incubated with 5 µM Eg5i overnight and mitotic cells were shaken off . MG132 was added to 50 µM ( t = 0 ) , then after 10 minutes the cells were split in two ( t = 10 ) . GCN2i ( Gcn2 - IN - 1 , MedChem Express HY - 100877 ) was added to 2 µM to one of the cell suspensions and samples were taken for immunoblotting every 10 minutes Representative ( E ) quantifications of three biological replicates ; averages and SEM are shown . Values are normalized to t = 0 .

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Figure 2A, B There is no significant difference in global translation rates in mitosis after GCN2 knock-down. A HeLa cells were pulse labeled with OPP and fixed with 70% ethanol. OPP was detected by Click-IT (AF 647) and mitotic cells were identified by staining for phospho-histone H3. 2D flow cytometry plots are shown, where AF647 intensity corresponds to OPP incorporation. Translation levels normalized to siCtr G1. The graph shows averages and SEM from three independent experiments. Paired two-tailed t-tests.D, Mitotic translation of selected proteins. Hela cells were incubated with 5 µM Eg5i overnight and mitotic cells were shaken off. MG132 was added to 50 µM (t=0), then after 10 minutes the cells were split in two (t=10). GCN2i (Gcn2-IN-1, MedChem Express HY-100877) was added to 2 µM to one of the cell suspensions and samples were taken for immunoblotting every 10 minutes (D) Representative immunoblots of three biological replicates are shown.E Mitotic translation of selected proteins. Hela cells were incubated with 5 µM Eg5i overnight and mitotic cells were shaken off. MG132 was added to 50 µM (t=0), then after 10 minutes the cells were split in two (t=10). GCN2i (Gcn2-IN-1, MedChem Express HY-100877) was added to 2 µM to one of the cell suspensions and samples were taken for immunoblotting every 10 minutes Representative (E) quantifications of three biological replicates; averages and SEM are shown. Values are normalized to t=0.


Figure 3A Verification of the mutants . HeLa cells transduced with the indicated GCN2 - siR constructs were transfected with control ( Ctr ) or GCN2 - targeting siRNA . Halofuginone ( HFG ) was added to 40 nM for 4 hours , and GCN2 autophosphorylation , ATF4 induction and eIF2α phosphorylation were assessed by immunoblotting . γ - tubulin is shown as loading control . The line in the GCN2 blot indicates that a longer exposure of the same blot is shown on the left than on the right . C , GCN2 kinase activity is regulated in the cell cycle . HeLa cells were arrested at the G1 / S transition using thymidine and released from the cell cycle at time 0 h . Samples were taken at the indicated time - points for immunoblotting Aurora A and Aurora A - P are shown to indicate the timing of mitosis , GCN2 - P is shown to assess GCN2 activity . Representative immunoblot of GCN2 phosphorylation normalized to GCN2 ( total ) , three biological repeats , three technical repeats each .

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Figure 3A Verification of the mutants. HeLa cells transduced with the indicated GCN2-siR constructs were transfected with control (Ctr) or GCN2-targeting siRNA. Halofuginone (HFG) was added to 40 nM for 4 hours, and GCN2 autophosphorylation, ATF4 induction and eIF2α phosphorylation were assessed by immunoblotting. γ-tubulin is shown as loading control. The line in the GCN2 blot indicates that a longer exposure of the same blot is shown on the left than on the right.C, GCN2 kinase activity is regulated in the cell cycle. HeLa cells were arrested at the G1/S transition using thymidine and released from the cell cycle at time 0 h. Samples were taken at the indicated time-points for immunoblotting Aurora A and Aurora A-P are shown to indicate the timing of mitosis, GCN2-P is shown to assess GCN2 activity. Representative immunoblot of GCN2 phosphorylation normalized to GCN2 (total), three biological repeats, three technical repeats each.


Figure 4B GCN2 coimmunoprecipitates with PP1α and γ . HeLa cells stably transfected with GFP - PP1α or GFP - PP1γ were transduced to express either GCN2 - siR ( wild - type ) or GCN2 - siR - AAXA . Cells were arrested in prometaphase with Eg5i , GFP - PP1α and GFP - PP1γ were immunoprecipitated using GFP - trap and the immunoprecipitates were immunoblotted for GCN2 . The vertical line in the PP1α and PP1γ blots indicates that different exposures of the same blot are shown for the inputs and the IP - s . D GCN2 can phosphorylate PP1α and γ in vitro . HeLa cells carrying GFP - PP1α or GFP - PP1γ were arrested in prometaphase with an Eg5 inhibitor ( Lane 3 ) , then GFP - tagged PP1α or PP1γ were immunoprecipitated using GFP - trap ( input , Lane 2 ) . The immunoprecipitates were incubated in kinase buffer and ATP with constitutively active , recombinant GCN2 . The samples were run on phos - tag gels and immunoblotted with PP1γ or PP1α antibody . F PP1α and PP1γ bandshifts are diminished in cells depleted for GCN2 and arrested in mitosis . HeLa cells were transfected with control or GCN2 - targeting siRNA and arrested in prometaphase using a PLK1 inhibitor . Representative quantifications of the ratios of the phospho - PP1 / PP1 signal ( average and SEM ) from four independent experiments are shown . * p = 0 . 0217 ( PP1α ) and * * p = 0 . 0069 ( PP1γ ) ( paired two - tailed t - tests ) .

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Figure 4B GCN2 coimmunoprecipitates with PP1α and γ. HeLa cells stably transfected with GFP-PP1α or GFP-PP1γ were transduced to express either GCN2-siR (wild-type) or GCN2-siR-AAXA. Cells were arrested in prometaphase with Eg5i, GFP-PP1α and GFP-PP1γ were immunoprecipitated using GFP-trap and the immunoprecipitates were immunoblotted for GCN2. The vertical line in the PP1α and PP1γ blots indicates that different exposures of the same blot are shown for the inputs and the IP-s.D GCN2 can phosphorylate PP1α and γ in vitro. HeLa cells carrying GFP-PP1α or GFP-PP1γ were arrested in prometaphase with an Eg5 inhibitor (Lane 3), then GFP-tagged PP1α or PP1γ were immunoprecipitated using GFP-trap (input, Lane 2). The immunoprecipitates were incubated in kinase buffer and ATP with constitutively active, recombinant GCN2. The samples were run on phos-tag gels and immunoblotted with PP1γ or PP1α antibody.F PP1α and PP1γ bandshifts are diminished in cells depleted for GCN2 and arrested in mitosis. HeLa cells were transfected with control or GCN2-targeting siRNA and arrested in prometaphase using a PLK1 inhibitor. Representative quantifications of the ratios of the phospho-PP1/PP1 signal (average and SEM) from four independent experiments are shown. *p=0.0217 (PP1α) and **p=0.0069 (PP1γ) (paired two-tailed t-tests).


Figure 5HeLa cells were transfected with control or GCN2 - targeting siRNA and fixed for immunofluorescence . Representative image of mitotic cells stained for A ChTOG ( magenta ) , tubulin ( green ) and DNA ( blue ) , Scale bars represent 5 µm . Percentage of cells displaying the aberrant localizations Averages and SEM from three independent experiments are shown . For chTOG n = 29 , 37 , 44 for siCtr ; n = 14 , 41 , 28 for siGCN2 ; * * p = 0 . 0063 ( paired two - tailed t - tests ) . HeLa cells were transfected with control or GCN2 - targeting siRNA and fixed for immunofluorescence . Representative image of mitotic cells stained for B CENPE ( magenta ) , P - Aurora B ( yellow ) , tubulin ( green ) and DNA ( blue ) are shown . Scale bars represent 5 µm . D Percentage of cells displaying the aberrant localizations Averages and SEM from three independent experiments are shown . For CENPE n = 15 , 35 , 43 for siCtr ; n = 11 , 21 , 20 for siGCN2 ; * * p = 0 . 001 , for P - Aurora B n = 29 , 37 , 44 for siCtr ; n = 14 , 41 , 28 for siGCN2 ; * * p = 0 . 0053 ( paired two - tailed t - tests ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5HeLa cells were transfected with control or GCN2-targeting siRNA and fixed for immunofluorescence. Representative image of mitotic cells stained for A ChTOG (magenta), tubulin (green) and DNA (blue), Scale bars represent 5 µm. Percentage of cells displaying the aberrant localizations Averages and SEM from three independent experiments are shown. For chTOG n= 29, 37, 44 for siCtr; n= 14, 41, 28 for siGCN2; **p=0.0063 (paired two-tailed t-tests).HeLa cells were transfected with control or GCN2-targeting siRNA and fixed for immunofluorescence. Representative image of mitotic cells stained for B CENPE (magenta), P-Aurora B (yellow), tubulin (green) and DNA (blue) are shown. Scale bars represent 5 µm. D Percentage of cells displaying the aberrant localizations Averages and SEM from three independent experiments are shown. For CENPE n= 15, 35, 43 for siCtr; n= 11, 21, 20 for siGCN2; **p=0.001, for P-Aurora B n=29, 37, 44 for siCtr; n= 14, 41, 28 for siGCN2; **p=0.0053 (paired two-tailed t-tests).


Figure 6HeLa cells were transfected with control or GCN2 - targeting siRNA , synchronized in the cell cycle by thymidine block - and - release and samples were collected for flow cytometry at the indicated time points . A Cell - cycle progression in the cells synchronized by thymidine block - and - release , three independent experiments , averages and SEM are shown . HeLa cells were transfected with control or GCN2 - targeting siRNA , synchronized in the cell cycle by thymidine block - and - release and samples were collected for immunoblotting at the indicated time points . B Representative immunoblots of TACC3 - S558 phosphorylation and Aurora A - T288 phosphorylationF Dephosphorylation of PP1 substrates after Aurora A inhibition in the presence and absence of GCN2 activity . HeLa cells were treated with Aurora Ai ( MLN8237 , MedChemTronica HY - 10971 ) , and with both Aurora Ai and GCN2i . Cell suspensions were incubated at 37oC and at the indicated times samples were removed , mixed with Laemmli sample buffer and boiled . F The immunoblots were probed with the indicated antibodies .

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 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 O 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 6HeLa cells were transfected with control or GCN2-targeting siRNA, synchronized in the cell cycle by thymidine block-and-release and samples were collected for flow cytometry at the indicated time points. A Cell-cycle progression in the cells synchronized by thymidine block-and-release, three independent experiments, averages and SEM are shown.HeLa cells were transfected with control or GCN2-targeting siRNA, synchronized in the cell cycle by thymidine block-and-release and samples were collected for immunoblotting at the indicated time points. B Representative immunoblots of TACC3-S558 phosphorylation and Aurora A-T288 phosphorylationF Dephosphorylation of PP1 substrates after Aurora A inhibition in the presence and absence of GCN2 activity. HeLa cells were treated with Aurora Ai (MLN8237, MedChemTronica HY-10971), and with both Aurora Ai and GCN2i. Cell suspensions were incubated at 37oC and at the indicated times samples were removed, mixed with Laemmli sample buffer and boiled. F The immunoblots were probed with the indicated antibodies.


Figure 7B HeLa cells were transfected with control or GCN2 - targeting siRNA or treated with the indicated concentrations of GCN2 inhibitor , cells were fixed after 72 h incubation , and the number of cells containing micronuclei was determined by DAPI staining . Four independent experiments , averages with SD are shown . n = 187 , 72 , 162 , 531 for siCtr ; n = 159 , 112 , 200 , 656 for siGCN2 ; p = 0 . 009 ; n = 721 , 821 , 682 for 0 µM ; n = 607 , 585 , 635 for 1 µM , n = 414 , 510 , 723 for 2 µM ; n = 123 , 147 , 280 for 3 µM ; p = 0 . 004 for 1 µM , * * p = 0 . 005 for 2 µM , * * p = 0 . 005 for 3 µM ( paired two - tailed t - tests ) . C Progression from prophase to anaphase in the presence of the GCN2i at the indicated concentrations was observed by live - cell imaging in C hTert - RPE1 cells . Averages ( lines ) and SEM ( bands ) from three ( hTert - RPE1 ) independent experiments are shown t = 0 denotes entry into prophase . for hTert - RPE1 cells n = 276 , 284 , 231 and 228 for 0 , 1 , 2 , and 3 µM GCN2i , respectively . * p = 0 . 0168 ; * * * * < 0 . 0001 ( Kolmogorov - Smirnov test ) . E PP1α and PP1γ bandshifts are diminished in cells arrested in mitosis upon pharmacological inhibition of GCN2 . HeLa or hTert - RPE1 cells were arrested in mitosis using a PLK1 inhibitor and GCN2 - IN1 was added to 2 µM for 3 h before harvesting the cells . Representative E quantifications of the ratios of the phospho - PP1 / PP1 signal ( average and SEM ) from four independent experiments are shown . HeLa : * * * p = 0 . 0007 ( PP1α ) ; * * p = 0 . 0023 ( PP1γ ) ; htert - RPE1 : * p = 0 . 0111 ( PP1α ) and * * * p = 0 . 0007 ( PP1γ ) , paired two - tailed t - tests .

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 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 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

Figure 7B HeLa cells were transfected with control or GCN2-targeting siRNA or treated with the indicated concentrations of GCN2 inhibitor, cells were fixed after 72 h incubation, and the number of cells containing micronuclei was determined by DAPI staining. Four independent experiments, averages with SD are shown. n= 187, 72, 162, 531 for siCtr; n= 159, 112, 200, 656 for siGCN2; p= 0.009; n=721, 821, 682 for 0 µM; n= 607, 585, 635 for 1 µM, n= 414, 510, 723 for 2 µM; n= 123, 147, 280 for 3 µM; p=0.004 for 1 µM, **p=0.005 for 2 µM, **p=0.005 for 3 µM (paired two-tailed t-tests).C Progression from prophase to anaphase in the presence of the GCN2i at the indicated concentrations was observed by live-cell imaging in C hTert-RPE1 cells. Averages (lines) and SEM (bands) from three (hTert-RPE1) independent experiments are shown t=0 denotes entry into prophase. for hTert-RPE1 cells n= 276, 284, 231 and 228 for 0, 1, 2, and 3 µM GCN2i, respectively. *p=0.0168; ****<0.0001 (Kolmogorov-Smirnov test).E PP1α and PP1γ bandshifts are diminished in cells arrested in mitosis upon pharmacological inhibition of GCN2. HeLa or hTert-RPE1 cells were arrested in mitosis using a PLK1 inhibitor and GCN2-IN1 was added to 2 µM for 3 h before harvesting the cells. Representative E quantifications of the ratios of the phospho-PP1/PP1 signal (average and SEM) from four independent experiments are shown. HeLa: ***p=0.0007 (PP1α); **p=0.0023 (PP1γ); htert-RPE1: *p=0.0111 (PP1α) and ***p=0.0007(PP1γ), paired two-tailed t-tests.


Figure 8Progression from prophase to anaphase in the presence of the Aurora A and GCN2 inhibitors was observed by live - cell imaging in HeLa and hTert - RPE1 cells . GCN2 inhibitor ( " G " ) and Aurora A inhibitor ( " A " ) was added C , D Representative images from experiments shown in A and B , respectively . E , F Percentages of cells with micronuclei and mitotic slippage in the experiments shown in A and B . Averages and SEM from three independent experiments are shown . HeLa mitotic slippage * p = 0 . 0137 , HeLa micronculei p = 0 . 0774 , hTert - RPE1 mitotic slippage * * p = 0 . 0011 , hTert - RPE1 micronuclei p = 0 . 0640 ; ( two - tailed t - tests ) .

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 O O O

Figure 8Progression from prophase to anaphase in the presence of the Aurora A and GCN2 inhibitors was observed by live-cell imaging in HeLa and hTert-RPE1 cells. GCN2 inhibitor ("G") and Aurora A inhibitor ("A") was added C, D Representative images from experiments shown in A and B, respectively.E, F Percentages of cells with micronuclei and mitotic slippage in the experiments shown in A and B. Averages and SEM from three independent experiments are shown. HeLa mitotic slippage *p=0.0137, HeLa micronculei p=0.0774, hTert-RPE1 mitotic slippage **p=0.0011, hTert-RPE1 micronuclei p=0.0640; (two-tailed t-tests).


Figure 9B HeLa and Tert - RPE1 cells stably expressing the cell - cycle markers CDT1 - Ruby and GMNN - Neongreen were treated with the Aurora A and GCN2 inhibitors and cell - cycle progression was followed by live - cell imaging . Representative examples of HeLa and hTert - RPE1 cells are shown from the supplementary videos 4 and 5 . Scale bars represent 5 µM . C HeLa and hTert - RPE1 cells were seeded in triplicate and incubated in the presence of the GCN2 and Aurora A inhibitors for 7 ( HeLa ) or 10 ( RPE1 ) days , then fixed and colonies were counted . Shown are averages and SEM from three biological replicates . For HeLa cells * p = 0 . 0267 for A versus G2A ; * p = 0 . 0432 A versus G3A ( paired two - tailed t - tests ) . For hTert - RPE1 cells * p = 0 . 0391 A versus G2A ; * * p = 0 . 021 A versus G3A ( paired two - tailed t - tests ) . D HeLa and Tert - RPE1 cells were treated with the indicated inhibitors for 72 h , stained with Hoechst and analyzed by flow cytometry . The graph shows the percentage of Hoechst - positive ( dead ) cells in four independent experiments . Averages and SEM are shown . For HeLa cells * * p = 0 . 0087 A versus AG ; * * p = 0 . 0027 Ctr versus AG ; * p = 0 . 0237 Ctr versus G ; p = 0 . 0809 Ctr versus A ( paired two - tailed t - test ) . For hTert - RPE1 cells * * * p = 0 . 0005 A versus AG ; * * p = 0 . 0036 Ctr versus AG ; * p = 0 . 0210 Ctr versus G ; p = 0 . 29184 Ctr versus A ( paired two - tailed t - tests ) . E , F HeLa and Tert - RPE1 cells were treated with the indicated inhibitors for 72 h , stained with the CellEvent ™ Caspase - 3 / 7 green detection reagent and Hoechst and analyzed by flow cytometry . Representative 2D plots of Alexa - Fluor 488 ( a measure of Caspase - 3 / 7 activity ) and Hoechst intensity are shown for E HeLa and F RPE1 cells . All single cells are plotted in magenta , Hoechst - negative cells are overlaid in grey . Note that all Hoechst - positive ( dead ) cells are also stained with the Caspase - 3 / 7 green reagent , indicating apoptosis .

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 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 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

Figure 9B HeLa and Tert-RPE1 cells stably expressing the cell-cycle markers CDT1-Ruby and GMNN-Neongreen were treated with the Aurora A and GCN2 inhibitors and cell-cycle progression was followed by live-cell imaging. Representative examples of HeLa and hTert-RPE1 cells are shown from the supplementary videos 4 and 5. Scale bars represent 5 µM.C HeLa and hTert-RPE1 cells were seeded in triplicate and incubated in the presence of the GCN2 and Aurora A inhibitors for 7 (HeLa) or 10 (RPE1) days, then fixed and colonies were counted. Shown are averages and SEM from three biological replicates. For HeLa cells *p=0.0267 for A versus G2A; *p= 0.0432 A versus G3A (paired two-tailed t-tests). For hTert-RPE1 cells *p=0.0391 A versus G2A; **p=0.021 A versus G3A (paired two-tailed t-tests). D HeLa and Tert-RPE1 cells were treated with the indicated inhibitors for 72 h, stained with Hoechst and analyzed by flow cytometry. The graph shows the percentage of Hoechst-positive (dead) cells in four independent experiments. Averages and SEM are shown. For HeLa cells **p=0.0087 A versus AG; **p=0.0027 Ctr versus AG; *p= 0.0237 Ctr versus G; p= 0.0809 Ctr versus A (paired two-tailed t-test). For hTert-RPE1 cells ***p=0.0005 A versus AG; **p=0.0036 Ctr versus AG; *p=0.0210 Ctr versus G; p= 0.29184 Ctr versus A (paired two-tailed t-tests).E, F HeLa and Tert-RPE1 cells were treated with the indicated inhibitors for 72 h, stained with the CellEvent™ Caspase-3/7 green detection reagent and Hoechst and analyzed by flow cytometry. Representative 2D plots of Alexa-Fluor 488 (a measure of Caspase-3/7 activity) and Hoechst intensity are shown for E HeLa and F RPE1 cells. All single cells are plotted in magenta, Hoechst-negative cells are overlaid in grey. Note that all Hoechst-positive (dead) cells are also stained with the Caspase-3/7 green reagent, indicating apoptosis.


Figure 1E and F , Naïve WT CD4 + CD62Lhi T cells were cultured under Th17 polarizing conditions . On day 3 , cells were transfected with control or siRNA targeting Batf , rested overnight , used for total RNA isolation to assess gene expression ( E ) , or restimulated with anti - CD3 for 24 h to measure cytokine production by ELISA ( F ) . Data information : , mean ± s . e . m . of 3 - 5 independent experiments ( * p < 0 . 05 ; two - sided t - test or one - way ANOVA followed by Tukey ' s test ) . RQ , relative quantification . Naïve CD4 + CD62Lhi T cells from IL - 17a reporter mice ( Il17aeGFP ) were cultured under Th17 polarizing conditions . On day 5 , eGFP + / IL - 17a + cells were sorted and cultured for two additional rounds in the presence of anti - CD3 alone ( TCR ( R3 ) ) or Th17 polarizing cytokines ( Th17 ( R3 ) ) ( G ) Data information : Data are representative of 3 - 5 independent experimentsWT and Batf KO mice were orally infected by gavage with 2x109 cfu / ml of Citrobacter rodentium ( Cr ) expressing GFP quantification of the signal ( O ) at the indicated time points . Data information : mean ± s . e . m . of n = 5 - 6 mice ( O ) ( * p < 0 . 05 ; two - sided t - test or one - way ANOVA followed by Tukey ' s test ) . WT and Batf KO mice were orally infected by gavage with 2x109 cfu / ml of Citrobacter rodentium ( Cr ) expressing GFP Cr infected WT and Batf KO were sacrificed on day 4 and day 10 . Cells were isolated from the colon , re - stimulated with PMA and ionomycin , and stained intracellularly for flow cytometric analysis of cytokine production and Foxp3 expression presented as density plots ( P ) . Data are gated on viable CD4 + TCRβ + . Data information : Data are representative of two independent

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 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 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 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

Figure 1E and F, Naïve WT CD4+CD62Lhi T cells were cultured under Th17 polarizing conditions. On day 3, cells were transfected with control or siRNA targeting Batf, rested overnight, used for total RNA isolation to assess gene expression (E), or restimulated with anti-CD3 for 24 h to measure cytokine production by ELISA (F). Data information: , mean ± s.e.m. of 3-5 independent experiments (*p<0.05; two-sided t-test or one-way ANOVA followed by Tukey's test). RQ, relative quantification.Naïve CD4+CD62Lhi T cells from IL-17a reporter mice (Il17aeGFP) were cultured under Th17 polarizing conditions. On day 5, eGFP+/IL-17a+ cells were sorted and cultured for two additional rounds in the presence of anti-CD3 alone (TCR(R3)) or Th17 polarizing cytokines (Th17(R3)) (G) Data information: Data are representative of 3-5 independent experimentsWT and Batf KO mice were orally infected by gavage with 2x109 cfu/ml of Citrobacter rodentium (Cr) expressing GFP quantification of the signal (O) at the indicated time points. Data information: mean ± s.e.m. of n=5-6 mice (O) (*p<0.05; two-sided t-test or one-way ANOVA followed by Tukey's test).WT and Batf KO mice were orally infected by gavage with 2x109 cfu/ml of Citrobacter rodentium (Cr) expressing GFP Cr infected WT and Batf KO were sacrificed on day 4 and day 10. Cells were isolated from the colon, re-stimulated with PMA and ionomycin, and stained intracellularly for flow cytometric analysis of cytokine production and Foxp3 expression presented as density plots (P). Data are gated on viable CD4+TCRβ+. Data information: Data are representative of two independent experiments


Figure 2Naïve WT and Batf KO CD4 + CD62Lhi T cells were cultured under Th17 polarizing conditions for 5 days . ChIP - seq for transcription factor ( Batf ) and histone modifications ( H3K4me3 , H3K27me3 , and H3K27ac ) was performed using day 5 WT and Batf KO Th17 cells ( A Data information : Data are representative of two independent experiments with similar results ( A , J , Naïve WT - 2BiT and Batf KO - 2BiT CD4 + CD62Lhi T cells were cultured under Th17 polarizing conditions . IL - 2 - Thy1 . 1 production comparing WT - 2BiT and Batf KO - 2BiT cells was measured by flow cytometry at the indicated time points and frequencies of positive cells determined . Data information : Data are representative of or mean ± s . e . m . of 3 - 5 independent experiments ( * p < 0 . 05 ; two - sided t - test ) . L and M , ELISA was used to quantify IL - 2 production in supernatants derived from cells in Figures 1G ( L ) and 1J ( M ) after 24 h of anti - CD3 stimulation . Data information : Data are representative of or mean ± s . e . m . of 3 - 5 independent experiments L - M ) ( * p < 0 . 05 ; two - sided 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 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 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

Figure 2Naïve WT and Batf KO CD4+CD62Lhi T cells were cultured under Th17 polarizing conditions for 5 days. ChIP-seq for transcription factor (Batf) and histone modifications (H3K4me3, H3K27me3, and H3K27ac) was performed using day 5 WT and Batf KO Th17 cells (A Data information: Data are representative of two independent experiments with similar results (A,J, Naïve WT-2BiT and Batf KO-2BiT CD4+CD62Lhi T cells were cultured under Th17 polarizing conditions. IL-2-Thy1.1 production comparing WT-2BiT and Batf KO-2BiT cells was measured by flow cytometry at the indicated time points and frequencies of positive cells determined. Data information: Data are representative of or mean ± s.e.m. of 3-5 independent experiments (*p<0.05; two-sided t-test).L and M, ELISA was used to quantify IL-2 production in supernatants derived from cells in Figures 1G (L) and 1J (M) after 24 h of anti-CD3 stimulation. Data information: Data are representative of or mean ± s.e.m. of 3-5 independent experiments L-M) (*p<0.05; two-sided t-test).


Figure 3ChIP - seq for Stat3 and Stat5 was performed after 1 h of IL - 6 and IL - 2 stimulation , respectively , using WT and Batf KO cells that were cultured under Th17 polarizing conditions for 5 days . Histograms show Batf , Stat3 , and Stat5 binding occupied around ± 1 kb of peak centers of co - bound Stat3 - Stat5 sites that were annotated near Th1 , Th17 , or Treg - specific genes ( C ) . Data information : Data are mean ± s . e . m . representative of two independent experiments with similar results ( c ( * p < 0 . 05 ; two - sided t - test ) . D , ChIP - seq for Stat3 and Stat5 was performed after 1 h of IL - 6 and IL - 2 stimulation , respectively , using WT and Batf KO cells that were cultured under Th17 polarizing conditions for 5 days . Tracks show Batf , Stat5 , and Stat3 ChIP - seq data compared between WT and Batf KO Th17 in the indicated gene loci ( WT , grey ; Batf KO , red ) . Red arrows indicate differential Stat5 binding comparing WT and Batf KO cells ( D ) . Data information : Data are mean ± s . e . m . representative of two independent experiments with similar results d ) , or ( * p < 0 . 05 ; two - sided 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 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

Figure 3ChIP-seq for Stat3 and Stat5 was performed after 1 h of IL-6 and IL-2 stimulation, respectively, using WT and Batf KO cells that were cultured under Th17 polarizing conditions for 5 days. Histograms show Batf, Stat3, and Stat5 binding occupied around ±1 kb of peak centers of co-bound Stat3-Stat5 sites that were annotated near Th1, Th17, or Treg-specific genes (C). Data information: Data are mean ± s.e.m. representative of two independent experiments with similar results (c (*p<0.05; two-sided t-test).D, ChIP-seq for Stat3 and Stat5 was performed after 1 h of IL-6 and IL-2 stimulation, respectively, using WT and Batf KO cells that were cultured under Th17 polarizing conditions for 5 days. Tracks show Batf, Stat5, and Stat3 ChIP-seq data compared between WT and Batf KO Th17 in the indicated gene loci (WT, grey; Batf KO, red). Red arrows indicate differential Stat5 binding comparing WT and Batf KO cells (D). Data information: Data are mean ± s.e.m. representative of two independent experiments with similar results d), or (*p<0.05; two-sided t-test).


Figure 4B , Naïve WT and Batf KO CD4 + CD62Lhi T cells were cultured under Th17 polarizing conditions for 5 days . The kinetics of Ets1 and Runx1 gene expression was assessed by RT - PCR in the indicated time points ( data normalized to naïve WT cells ) . Data information : Data are mean ± s . e . m . of 3 independent experiments ( B ) ( * p < 0 . 05 ; two - sided t - test ) . RQ , relative quantification .

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

Figure 4B, Naïve WT and Batf KO CD4+CD62Lhi T cells were cultured under Th17 polarizing conditions for 5 days. The kinetics of Ets1 and Runx1 gene expression was assessed by RT-PCR in the indicated time points (data normalized to naïve WT cells). Data information: Data are mean ± s.e.m. of 3 independent experiments (B) (*p<0.05; two-sided t-test). RQ, relative quantification.