A . FRAP images of CBX4 bodies to indicate the internal diffusion and diffusion across boundary properties . GFP - tagged CBX4 was overexpressed in HEK293T cells . These live cells were proceeded to time series imaging 24 hours post transfection . Six GFP - CBX4 bodies were circled and marked as region of interest 1 ( ROI 1 ) , ROI 2 , ROI 3 , ROI 4 , ROI 5 and ROI 6 . Three CBX4 bodies ( ROI 4 , ROI 5 and ROI 6 ) were bleached with strong 488 nm laser pulse . Another three unbleached CBX4 bodies ( ROI 1 , ROI 2 and ROI 3 ) were set as control . Images were captured every 4 s . The right histogram showed relative fluorescence intensities of unbleached and bleached CBX4 bodies in each time point . Data information : Data represented mean ± SEM in biological triplicate in ( A ) . Scale bars in ( A ) represented 5 μm . All the samples were imaged to obtain at least three sets of images .

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A. FRAP images of CBX4 bodies to indicate the internal diffusion and diffusion across boundary properties. GFP-tagged CBX4 was overexpressed in HEK293T cells. These live cells were proceeded to time series imaging 24 hours post transfection. Six GFP-CBX4 bodies were circled and marked as region of interest 1 (ROI 1), ROI 2, ROI 3, ROI 4, ROI 5 and ROI 6. Three CBX4 bodies (ROI 4, ROI 5 and ROI 6) were bleached with strong 488 nm laser pulse. Another three unbleached CBX4 bodies (ROI 1, ROI 2 and ROI 3) were set as control. Images were captured every 4 s. The right histogram showed relative fluorescence intensities of unbleached and bleached CBX4 bodies in each time point. Data information: Data represented mean ± SEM in biological triplicate in (A). Scale bars in (A) represented 5 μm. All the samples were imaged to obtain at least three sets of images.


P . RFP - tagged EZH2 was co - overexpressed with GFP - tagged CBX4 mutant named CBX4mut which was CBX4 - LTVTmut . SIM images were captured . The line scan profile was shown on the right . The red arrow within merged image indicated the line scan position . Data information represented 5 μm . All the samples were imaged to obtain at least three sets of images .

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P. RFP-tagged EZH2 was co-overexpressed with GFP-tagged CBX4 mutant named CBX4mut which was CBX4-LTVTmut. SIM images were captured. The line scan profile was shown on the right. The red arrow within merged image indicated the line scan position. Data information represented 5 μm. All the samples were imaged to obtain at least three sets of images.


G . HA - tagged EZH2 was co - overexpressed with Flag - tagged SUMO1 , Flag - tagged UBC9 or Flag - tagged CBX4 , and siNC . In the last group , HA - tagged EZH2 was co - overexpressed with Flag - tagged SUMO1 , Flag - tagged UBC9 and siCBX4 . EZH2 was IP with anti - HA beads . Both total and IP samples were IB with antibodies against HA , Flag and GAPDH . The expression ratios of SUMO1 - EZH2 within each group were showed below the figure . H . HA - tagged EZH2 was co - overexpressed with Flag - tagged SUMO4 , Flag - tagged UBC9 or Flag - tagged CBX4 , and siNC . In the last group , HA - tagged EZH2 was co - overexpressed with Flag - tagged SUMO4 , Flag - tagged UBC9 and siCBX4 . EZH2 was IP with anti - HA beads . Both total and IP samples were IB with antibodies against HA , Flag and GAPDH . The expression ratios of SUMO4 - EZH2 within each group were showed below the figure .

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G. HA-tagged EZH2 was co-overexpressed with Flag-tagged SUMO1, Flag-tagged UBC9 or Flag-tagged CBX4, and siNC. In the last group, HA-tagged EZH2 was co-overexpressed with Flag-tagged SUMO1, Flag-tagged UBC9 and siCBX4. EZH2 was IP with anti-HA beads. Both total and IP samples were IB with antibodies against HA, Flag and GAPDH. The expression ratios of SUMO1-EZH2 within each group were showed below the figure. H. HA-tagged EZH2 was co-overexpressed with Flag-tagged SUMO4, Flag-tagged UBC9 or Flag-tagged CBX4, and siNC. In the last group, HA-tagged EZH2 was co-overexpressed with Flag-tagged SUMO4, Flag-tagged UBC9 and siCBX4. EZH2 was IP with anti-HA beads. Both total and IP samples were IB with antibodies against HA, Flag and GAPDH. The expression ratios of SUMO4-EZH2 within each group were showed below the figure.


B . Two micrograms ( 2 μg ) of HA - tagged EZH2 were co - overexpressed with 4 μg of Flag - tagged SUMO4 , 250 ng of Flag - tagged UBC9 , 250 ng of Flag - tagged CBX4 or CBX4mut within 6 cm dishes . The CBX4mut was the LLPS - deficient CBX4 . EZH2 was IP with anti - HA beads . Both total and IP samples were IB with antibodies against HA , Flag and GAPDH . The expression ratios of SUMO4 - EZH2 were marked below the panel .

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B. Two micrograms (2 μg) of HA-tagged EZH2 were co-overexpressed with 4 μg of Flag-tagged SUMO4, 250 ng of Flag-tagged UBC9, 250 ng of Flag-tagged CBX4 or CBX4mut within 6 cm dishes. The CBX4mut was the LLPS-deficient CBX4. EZH2 was IP with anti-HA beads. Both total and IP samples were IB with antibodies against HA, Flag and GAPDH. The expression ratios of SUMO4-EZH2 were marked below the panel.


D . The endogenous EZH2 in TZM - bl cells within 6 cm dishes was knocked down by siRNAs targeting 3 ' UTR of EZH2 mRNA , followed by the overexpression of 2 μg of EZH2 , 4 μg of Flag - tagged SUMO4 , 250 ng of Flag - tagged UBC9 or 250 ng of Flag - tagged CBX4 . EZH2 was IP with anti - EZH2 antibodies . IP samples were IB with anti - EZH2 antibodies . Total samples were IB with anti - EZH2 , anti - H3K27me3 , anti - Histone H3 and anti - GAPDH antibodies . The expression ratios of SUMO4 - EZH2 and H3K27me3 were marked below the panel .

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D. The endogenous EZH2 in TZM-bl cells within 6 cm dishes was knocked down by siRNAs targeting 3'UTR of EZH2 mRNA, followed by the overexpression of 2 μg of EZH2, 4 μg of Flag-tagged SUMO4, 250 ng of Flag-tagged UBC9 or 250 ng of Flag-tagged CBX4. EZH2 was IP with anti-EZH2 antibodies. IP samples were IB with anti-EZH2 antibodies. Total samples were IB with anti-EZH2, anti-H3K27me3, anti-Histone H3 and anti-GAPDH antibodies. The expression ratios of SUMO4-EZH2 and H3K27me3 were marked below the panel.


E . In the first group , HEK293T cells within 6 cm dishes were transfected with empty vectors . In the second group , 3 μg of EZH2 was overexpressed . In the third group , 3 μg of EZH2 was co - overexpressed with 2 μg of EED and 2 μg of SUZ12 . In the fourth and sixth group , 3 μg of EZH2 was co - overexpressed with 2 μg of EED , 2 μg of SUZ12 , 2 μg of SUMO4 , 500 ng of UBC9 and 2 μg of CBX4 . In the fifth group , 3 μg of EZH2mut ( Y731D ) was co - overexpressed with 2 μg of EED , 2 μg of SUZ12 , 2 μg of SUMO4 , 500 ng of UBC9 and 2 μg of CBX4 . Forty - eight hours post transfection , EZH2 and EZH2mut were IP with anti - EZH2 , followed by the incubation with 2 μg of mononucleosomes ( H3 . 1 ) and 20 μM of the cofactor S - adenosyl - L - methionine ( SAM ) to measure the methyltransferase activity of EZH2 . The IP reaction in the sixth group was incubated with 2 μg of mononucleosomes ( H3 . 1 K27M ) and 20 μM of SAM . Both total and IP samples were IB with anti - EZH2 , GAPDH , H3K27me3 and H3 . 1 antibodies . The purities of recombinant mononucleosomes which included H3 . 1 , H2B , H2A and H4 were verified by Coomassie blue staining . The expression ratios of SUMO4 - EZH2 and H3K27me3 were marked below the panel .

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E. In the first group, HEK293T cells within 6 cm dishes were transfected with empty vectors. In the second group, 3 μg of EZH2 was overexpressed. In the third group, 3 μg of EZH2 was co-overexpressed with 2 μg of EED and 2 μg of SUZ12. In the fourth and sixth group, 3 μg of EZH2 was co-overexpressed with 2 μg of EED, 2 μg of SUZ12, 2 μg of SUMO4, 500 ng of UBC9 and 2 μg of CBX4. In the fifth group, 3 μg of EZH2mut (Y731D) was co-overexpressed with 2 μg of EED, 2 μg of SUZ12, 2 μg of SUMO4, 500 ng of UBC9 and 2 μg of CBX4. Forty-eight hours post transfection, EZH2 and EZH2mut were IP with anti-EZH2, followed by the incubation with 2 μg of mononucleosomes (H3.1) and 20 μM of the cofactor S-adenosyl-L-methionine (SAM) to measure the methyltransferase activity of EZH2. The IP reaction in the sixth group was incubated with 2 μg of mononucleosomes (H3.1 K27M) and 20 μM of SAM. Both total and IP samples were IB with anti-EZH2, GAPDH, H3K27me3 and H3.1 antibodies. The purities of recombinant mononucleosomes which included H3.1, H2B, H2A and H4 were verified by Coomassie blue staining. The expression ratios of SUMO4-EZH2 and H3K27me3 were marked below the panel.


A . PHA - activated primary CD4 + T cells were infected with wildtype HIV - 1 viruses which harbored a GFP ORF after Nef gene , followed by infecting with shluc and shCBX4 lentiviruses respectively . The percentages of GFP - positive cells , which were HIV - 1 - expressing cells , were monitored every three days . Data information : Data represented mean ± SEM in biological triplicate . p - Values were calculated by two - way ANOVA with Sidak ' s multiple comparisons test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. PHA-activated primary CD4+ T cells were infected with wildtype HIV-1 viruses which harbored a GFP ORF after Nef gene, followed by infecting with shluc and shCBX4 lentiviruses respectively. The percentages of GFP-positive cells, which were HIV-1-expressing cells, were monitored every three days. Data information: Data represented mean ± SEM in biological triplicate. p-Values were calculated by two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.


B , C . Primary CD4 + T cells were treated as in ( A ) . On Day 6 post infection with HIV - 1 viruses and shRNA lentiviruses , ChIP assays with antibodies against H3K27me3 and H2AK119Ub were conducted in both shluc and shCBX4 groups . Data information : Data represented mean ± SEM in biological triplicate . p - Values were calculated by two - way ANOVA with Sidak ' s multiple comparisons test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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B, C. Primary CD4+ T cells were treated as in (A). On Day 6 post infection with HIV-1 viruses and shRNA lentiviruses, ChIP assays with antibodies against H3K27me3 and H2AK119Ub were conducted in both shluc and shCBX4 groups. Data information: Data represented mean ± SEM in biological triplicate. p-Values were calculated by two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.


D . PHA - activated CD4 + T cells were infected with wildtype HIV - 1 viruses as in ( A ) . One group of HIV - 1 - infected CD4 + T cells were left untreated . One group of HIV - 1 - infected cells were infected with shluc . Another group of HIV - 1 - infected cells were infected with shCBX4 . Two weeks later , one part of HIV - 1 - infected CD4 + T cells were reactivated by αCD3 / αCD28 / IL - 2 . One part of shluc - and shCBX4 - infected cells were co - stimulated with LRA JQ - 1 . GFP - positive cells in each group were measured by flow cytometry to indicate reactivated HIV - 1 - infected cells . Data information : Data represented mean ± SEM in biological triplicate . p - Values were calculated by one - way ANOVA with Tukey ' s multiple comparisons test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. PHA-activated CD4+ T cells were infected with wildtype HIV-1 viruses as in (A). One group of HIV-1-infected CD4+ T cells were left untreated. One group of HIV-1-infected cells were infected with shluc. Another group of HIV-1-infected cells were infected with shCBX4. Two weeks later, one part of HIV-1-infected CD4+ T cells were reactivated by αCD3/αCD28/IL-2. One part of shluc- and shCBX4-infected cells were co-stimulated with LRA JQ-1. GFP-positive cells in each group were measured by flow cytometry to indicate reactivated HIV-1-infected cells. Data information: Data represented mean ± SEM in biological triplicate. p-Values were calculated by one-way ANOVA with Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.


E - G . Primary CD4 + T cells , which contained latent HIV - 1 - infected cells , were isolated from three HIV - 1 - infected individuals . One group of cells were untreated . One group of cells were activated with αCD3 / αCD28 / IL - 2 . Another two groups of cells were transfected with siNC and siCBX4 utilizing 4D - Nucleofector System respectively . Three days post transfection , the amounts of intracellular HIV - 1 RNAs within each group were quantitated by RT - qPCR and represented as 103 copies per million CD4 + T cells . Data information : Data represented mean ± SEM in biological triplicate . p - Values were calculated by one - way ANOVA with Tukey ' s multiple comparisons test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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E-G. Primary CD4+ T cells, which contained latent HIV-1-infected cells, were isolated from three HIV-1-infected individuals. One group of cells were untreated. One group of cells were activated with αCD3/αCD28/IL-2. Another two groups of cells were transfected with siNC and siCBX4 utilizing 4D-Nucleofector System respectively. Three days post transfection, the amounts of intracellular HIV-1 RNAs within each group were quantitated by RT-qPCR and represented as 103 copies per million CD4+ T cells. Data information: Data represented mean ± SEM in biological triplicate. p-Values were calculated by one-way ANOVA with Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.


Fig . 1 . A co - culture assay based on the split TEV technique for monitoring NRG1 - ERBB4 signaling activity . ( B ) Dose - response assay using increasing numbers of Nrg1 type I β1a - expressing PC12 cells . For each 96 - well , 40 , 000 split TEV assay cells were co - plated with increasing numbers of Nrg1 - expressing cells , and incubated for 24h . Half - maximal activation is reached at 5 , 000 Nrg1 - expressing cells . The Z ' factor is 0 . 5 indicating a large separation band for this assay .

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Fig. 1. A co-culture assay based on the split TEV technique for monitoring NRG1-ERBB4 signaling activity.(B) Dose-response assay using increasing numbers of Nrg1 type I β1a-expressing PC12 cells. For each 96-well, 40,000 split TEV assay cells were co-plated with increasing numbers of Nrg1-expressing cells, and incubated for 24h. Half-maximal activation is reached at 5,000 Nrg1-expressing cells. The Z' factor is 0.5 indicating a large separation band for this assay.


Fig . 1 . A co - culture assay based on the split TEV technique for monitoring NRG1 - ERBB4 signaling activity . ( C ) Adding 10ng / ml EGFld resulted in a two - fold activation . Per 96 - well , 40 , 000 split TEV assay cells were co - plated with empty PC12 cells ( no Nrg1 expression ) , 10 , 000 Nrg1 - expressing cells , and 10 , 000 Nrg1 - expressing cells plus 10ng / ml EGFld .

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Fig. 1. A co-culture assay based on the split TEV technique for monitoring NRG1-ERBB4 signaling activity.(C) Adding 10ng/ml EGFld resulted in a two-fold activation. Per 96-well, 40,000 split TEV assay cells were co-plated with empty PC12 cells (no Nrg1 expression), 10,000 Nrg1-expressing cells, and 10,000 Nrg1-expressing cells plus 10ng/ml EGFld.


Fig . 1 . A co - culture assay based on the split TEV technique for monitoring NRG1 - ERBB4 signaling activity . ( D ) Lapatinib antagonizes ERBB4 - PIK3R1 signaling in a dose - dependent manner . Per 96 - well , 40 , 000 split TEV assay cells were incubated with increasing amounts of lapatinib , followed by co - plating 10 , 000 Nrg1 type I β1a expressing cells . The inset depicts the IC 50 value in µM . After compound / stimulus addition , each assay was incubated for 24h . RLU , relative luciferase units ; Fluc , firefly luciferase activity ( black line ) ; Rluc , Renilla luciferase activity ( grey line ) ; n = 6 ; data are shown as mean , error bars represent SEM .

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Fig. 1. A co-culture assay based on the split TEV technique for monitoring NRG1-ERBB4 signaling activity.(D) Lapatinib antagonizes ERBB4-PIK3R1 signaling in a dose-dependent manner. Per 96-well, 40,000 split TEV assay cells were incubated with increasing amounts of lapatinib, followed by co-plating 10,000 Nrg1 type I β1a expressing cells. The inset depicts the IC 50 value in µM. After compound/stimulus addition, each assay was incubated for 24h. RLU, relative luciferase units; Fluc, firefly luciferase activity (black line); Rluc, Renilla luciferase activity (grey line); n=6; data are shown as mean, error bars represent SEM.


Fig . 2 . Spironolactone is the primary candidate recovered from the co - culture screen . ( B ) Graphic visualisation of the primary screen data of the NIH - NCC library set 1 . All counts ( 320 compounds and 64 controls ) from the Nrg1 - ERBB4 - PIK3R1 split TEV compound screen were plotted against the Z - score using the Mondrian programme , with pathway activators displaying high values and inhibitors low values . For the secondary analysis , we selected all candidates that were at least three standard deviations away from the mean . EGFld positive and Lapatinib / CI - 1033 negative controls are shown in red .

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Fig. 2. Spironolactone is the primary candidate recovered from the co-culture screen.(B) Graphic visualisation of the primary screen data of the NIH-NCC library set 1. All counts (320 compounds and 64 controls) from the Nrg1-ERBB4-PIK3R1 split TEV compound screen were plotted against the Z-score using the Mondrian programme, with pathway activators displaying high values and inhibitors low values. For the secondary analysis, we selected all candidates that were at least three standard deviations away from the mean. EGFld positive and Lapatinib/CI-1033 negative controls are shown in red.


Fig . 2 . Spironolactone is the primary candidate recovered from the co - culture screen . ( C and D ) Spironolactone antagonizes Nrg1 - ERBB4 - PIK3R1 signaling . In dose - response assays using ERBB4 - NTEV - tevS - GV and PIK3R1 - CTEV plasmids transfected into PC12 cells , spironolactone was administered at increasing concentrations before seeding Nrg1 type I ( C ) expressing PC12 cells .

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Fig. 2. Spironolactone is the primary candidate recovered from the co-culture screen.(C and D) Spironolactone antagonizes Nrg1-ERBB4-PIK3R1 signaling. In dose-response assays using ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids transfected into PC12 cells, spironolactone was administered at increasing concentrations before seeding Nrg1 type I (C) expressing PC12 cells.


Fig . 2 . Spironolactone is the primary candidate recovered from the co - culture screen . ( C and D ) Spironolactone antagonizes Nrg1 - ERBB4 - PIK3R1 signaling . In dose - response assays using ERBB4 - NTEV - tevS - GV and PIK3R1 - CTEV plasmids transfected into PC12 cells , spironolactone was administered at increasing concentrations before seeding Nrg1 type III ( D ) expressing PC12 cells .

Fig. 2. Spironolactone is the primary candidate recovered from the co-culture screen. (C and D) Spironolactone antagonizes Nrg1-ERBB4-PIK3R1 signaling. In dose-response assays using ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids transfected into PC12 cells, spironolactone was administered at increasing concentrations before seeding Nrg1 type III (D) expressing PC12 cells.


Fig . 2 . Spironolactone is the primary candidate recovered from the co - culture screen . ( E ) Spironolactone inhibits ERBB4 receptor dimerization . Dose - dependent dimerization of the ERBB4 receptor was analyzed using a split TEV assay encompassing ERBB4 - NTEV - tevS - GV and ERBB4 - CTEV plasmids transfected into PC12 cells . 10 ng / ml EGFld was applied as Nrg1 stimulus . Fluc , firefly luciferase activity ( black lines ) ; Rluc , Renilla luciferase activity ( grey lines , indicating toxicity levels ) ; n = 6 ; data are shown as mean , error bars represent SEM . The insets depict IC 50 values in µM .

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Fig. 2. Spironolactone is the primary candidate recovered from the co-culture screen.(E) Spironolactone inhibits ERBB4 receptor dimerization. Dose-dependent dimerization of the ERBB4 receptor was analyzed using a split TEV assay encompassing ERBB4-NTEV-tevS-GV and ERBB4-CTEV plasmids transfected into PC12 cells. 10 ng/ml EGFld was applied as Nrg1 stimulus. Fluc, firefly luciferase activity (black lines); Rluc, Renilla luciferase activity (grey lines, indicating toxicity levels); n=6; data are shown as mean, error bars represent SEM. The insets depict IC 50 values in µM.


Fig . 3 . Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment . ( A ) Spironolactone , molecular structure shown left , inhibits ERBB4 / PIK3R1 split TEV assay activity ( black line ) , with an IC 50 value of 2 . 76 µM .

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Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment.(A) Spironolactone, molecular structure shown left, inhibits ERBB4/PIK3R1 split TEV assay activity (black line), with an IC 50 value of 2.76 µM.


Fig . 3 . Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment . ( B - D ) The spironolactone metabolites ( B ) canrenone . Note that only spironolactone bears a thio - ketone group attached to the sterol core structure . All ERBB4 / PIK3R1 assays were run in a single - culture assay mode using 10ng / ml EGFld as functional Nrg1 stimulus , and ERBB4 - NTEV - tevS - GV and PIK3R1 - CTEV plasmids were transfected into PC12 cells ( indicated by icon ) . Fluc , firefly luciferase activity reporting ERBB4 - PIK3R1 assay activity ( black lines ) ; Rluc , Renilla luciferase activity ( grey lines ) assessing viability ; data are shown as mean , error bars represent SEM ; n = 6 .

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Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment.(B-D) The spironolactone metabolites (B) canrenone. Note that only spironolactone bears a thio-ketone group attached to the sterol core structure. All ERBB4/PIK3R1 assays were run in a single-culture assay mode using 10ng/ml EGFld as functional Nrg1 stimulus, and ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids were transfected into PC12 cells (indicated by icon). Fluc, firefly luciferase activity reporting ERBB4-PIK3R1 assay activity (black lines); Rluc, Renilla luciferase activity (grey lines) assessing viability; data are shown as mean, error bars represent SEM; n=6.


Fig . 3 . Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment . ( B - D ) The spironolactone metabolites ( C ) 7α - thiomethyl - spironolactone . Note that only spironolactone bears a thio - ketone group attached to the sterol core structure . All ERBB4 / PIK3R1 assays were run in a single - culture assay mode using 10ng / ml EGFld as functional Nrg1 stimulus , and ERBB4 - NTEV - tevS - GV and PIK3R1 - CTEV plasmids were transfected into PC12 cells ( indicated by icon ) . Fluc , firefly luciferase activity reporting ERBB4 - PIK3R1 assay activity ( black lines ) ; Rluc , Renilla luciferase activity ( grey lines ) assessing viability ; data are shown as mean , error bars represent SEM ; n = 6 .

O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment. (B-D) The spironolactone metabolites(C) 7α-thiomethyl-spironolactone. Note that only spironolactone bears a thio-ketone group attached to the sterol core structure. All ERBB4/PIK3R1 assays were run in a single-culture assay mode using 10ng/ml EGFld as functional Nrg1 stimulus, and ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids were transfected into PC12 cells (indicated by icon). Fluc, firefly luciferase activity reporting ERBB4-PIK3R1 assay activity (black lines); Rluc, Renilla luciferase activity (grey lines) assessing viability; data are shown as mean, error bars represent SEM; n=6.


Fig . 3 . Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment . ( B - D ) The spironolactone metabolites ( D ) eplerenone do not attenuate the ERBB4 / PIK3R1 assay activity ( black lines ) . Note that only spironolactone bears a thio - ketone group attached to the sterol core structure . All ERBB4 / PIK3R1 assays were run in a single - culture assay mode using 10ng / ml EGFld as functional Nrg1 stimulus , and ERBB4 - NTEV - tevS - GV and PIK3R1 - CTEV plasmids were transfected into PC12 cells ( indicated by icon ) . Fluc , firefly luciferase activity reporting ERBB4 - PIK3R1 assay activity ( black lines ) ; Rluc , Renilla luciferase activity ( grey lines ) assessing viability ; data are shown as mean , error bars represent SEM ; n = 6 .

O O O O 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-CONTROLLED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O 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-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment.(B-D) The spironolactone metabolites (D) eplerenone do not attenuate the ERBB4/PIK3R1 assay activity (black lines). Note that only spironolactone bears a thio-ketone group attached to the sterol core structure. All ERBB4/PIK3R1 assays were run in a single-culture assay mode using 10ng/ml EGFld as functional Nrg1 stimulus, and ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids were transfected into PC12 cells (indicated by icon). Fluc, firefly luciferase activity reporting ERBB4-PIK3R1 assay activity (black lines); Rluc, Renilla luciferase activity (grey lines) assessing viability; data are shown as mean, error bars represent SEM; n=6.


Fig . 3 . Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment . ( F ) Heat map showing the IC 50 values obtained from individual ERBB dimerization split TEV assays . All single dose - response assays can be found in Fig EV4 ; the combination ERBB4 - ERBB4 is shown in Fig 2E . Fluc , firefly luciferase activity ( black lines ) ; Rluc , Renilla luciferase activity ( grey lines , indicating toxicity levels ) ; n = 6 ; data are shown as mean , error bars represent SEM . The insets depict IC 50 values in µM .

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Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment.(F) Heat map showing the IC 50 values obtained from individual ERBB dimerization split TEV assays. All single dose-response assays can be found in Fig EV4; the combination ERBB4-ERBB4 is shown in Fig 2E. Fluc, firefly luciferase activity (black lines); Rluc, Renilla luciferase activity (grey lines, indicating toxicity levels); n=6; data are shown as mean, error bars represent SEM. The insets depict IC 50 values in µM.


Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( A ) Spironolactone reduces ERBB4 levels . T - 47D cells were stimulated with 10 ng / ml EGFld , 10 µM lapatinib and 10 µM spironolactone for 5 min as indicated . Cell lysates were probed for ERBB4 phosphorylation levels at Tyr1056 and Tyr1284 .

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Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(A) Spironolactone reduces ERBB4 levels. T-47D cells were stimulated with 10 ng/ml EGFld, 10 µM lapatinib and 10 µM spironolactone for 5 min as indicated. Cell lysates were probed for ERBB4 phosphorylation levels at Tyr1056 and Tyr1284.


Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( B ) Quantification of band intensities for phospho - ERBB4 levels ( n = 4 per condition ) shown in ( A ) using ImageJ . Phosphorylation levels are normalized to protein levels of ERBB4 . Data shown as mean , error bars represent SD ; t - test , with * , p = 0 . 0356 for p - ERBB4 ( Y1056 ) , and * * , p = 0 . 0079 for p - ERBB4 ( Y1284 ) .

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Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(B) Quantification of band intensities for phospho-ERBB4 levels (n=4 per condition) shown in (A) using ImageJ. Phosphorylation levels are normalized to protein levels of ERBB4. Data shown as mean, error bars represent SD; t-test, with *, p=0.0356 for p-ERBB4 (Y1056), and **, p=0.0079 for p-ERBB4 (Y1284).


Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( D ) Spironolactone reduces phospho - Erbb4 levels in Nrg1 - tg mice . Mice were treated with spironolactone for 21 days and sacrificed for western blot analysis . Lysates were probed with indicated antibodies .

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Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(D) Spironolactone reduces phospho-Erbb4 levels in Nrg1-tg mice. Mice were treated with spironolactone for 21 days and sacrificed for western blot analysis. Lysates were probed with indicated antibodies.


Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( E ) Quantification of band intensities for phospho - Erbb4 and phospho - Limk1 levels shown in ( D ) using ImageJ . Phosphorylation levels are normalized to protein levels of Erbb4 and Limk1 . Data shown as mean , error bars represent SD ; t - test , with * , p = 0 . 0330 for p - Erbb4 , and p = 0 . 0201 for p - Limk1 .

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Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo. (E) Quantification of band intensities for phospho-Erbb4 and phospho-Limk1 levels shown in (D) using ImageJ. Phosphorylation levels are normalized to protein levels of Erbb4 and Limk1. Data shown as mean, error bars represent SD; t-test, with *, p=0.0330 for p-Erbb4, and p=0.0201 for p-Limk1.


Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( F ) Canrenone ( applied as 10 µM ) showed no effects on frequencies and amplitudes of sIPSCs in pyramidal neurons of the prefrontal cortex ( PFC ) . Representative traces of sIPSCs ( upper ) and a histogram of mean sIPSC ( lower ) are shown for both before and after addition of canrenone .

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Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo. (F) Canrenone (applied as 10 µM) showed no effects on frequencies and amplitudes of sIPSCs in pyramidal neurons of the prefrontal cortex (PFC). Representative traces of sIPSCs (upper) and a histogram of mean sIPSC (lower) are shown for both before and after addition of canrenone.


Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( G ) Spironolactone ( applied as 10 µM ) significantly increases frequencies ( n = 12 ; p = 0 . 0454 ) and amplitudes ( n = 12 , p = 0 . 0478 ) of sIPSCs in pyramidal neurons of PFC . Representative traces of sIPSCs ( upper ) and a histogram of mean sIPSC ( lower ) are shown for both before and after addition of spironolactone .

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Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(G) Spironolactone (applied as 10 µM) significantly increases frequencies (n=12; p=0.0454) and amplitudes (n=12, p=0.0478) of sIPSCs in pyramidal neurons of PFC. Representative traces of sIPSCs (upper) and a histogram of mean sIPSC (lower) are shown for both before and after addition of spironolactone.


Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( H ) Spironolactone increases amplitudes of evoked IPSCs in pyramidal neurons of PFC ( n = 15 , p = 0 . 0286 ) . Spironolactone ( 5 µM ) was applied in the presence of canrenone ( 10 µM ) . For ( H , I ) , sample recording ( upper ) and a histogram of averaged eIPSC ( lower ) is shown for both before and after drug application . For ( F - I ) , the numbers displayed inside the histogram bars indicate the number of recorded slices / number of animals . Data are shown as mean , error bars represent SEM ( n = 4 ) ; paired t - test , with * , p ≤ 0 . 05 ; Spiro , spironolactone ; Canre , canrenone ; Lap , lapatinib .

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Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(H) Spironolactone increases amplitudes of evoked IPSCs in pyramidal neurons of PFC (n=15, p=0.0286). Spironolactone (5 µM) was applied in the presence of canrenone (10 µM).For (H, I), sample recording (upper) and a histogram of averaged eIPSC (lower) is shown for both before and after drug application. For (F-I), the numbers displayed inside the histogram bars indicate the number of recorded slices / number of animals. Data are shown as mean, error bars represent SEM (n=4); paired t-test, with *, p≤0.05; Spiro, spironolactone; Canre, canrenone; Lap, lapatinib.


Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( I ) Lapatinib increases amplitudes of evoked IPSCs in pyramidal neurons of PFC ( n = 15 , p = 0 . 0124 ) . Lapatinib was applied in 5 µM . For ( H , I ) , sample recording ( upper ) and a histogram of averaged eIPSC ( lower ) is shown for both before and after drug application . For ( F - I ) , the numbers displayed inside the histogram bars indicate the number of recorded slices / number of animals . Data are shown as mean , error bars represent SEM ( n = 4 ) ; paired t - test , with * , p ≤ 0 . 05 ; Spiro , spironolactone ; Canre , canrenone ; Lap , lapatinib .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O

Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(I) Lapatinib increases amplitudes of evoked IPSCs in pyramidal neurons of PFC (n=15, p=0.0124). Lapatinib was applied in 5 µM. For (H, I), sample recording (upper) and a histogram of averaged eIPSC (lower) is shown for both before and after drug application. For (F-I), the numbers displayed inside the histogram bars indicate the number of recorded slices / number of animals. Data are shown as mean, error bars represent SEM (n=4); paired t-test, with *, p≤0.05; Spiro, spironolactone; Canre, canrenone; Lap, lapatinib.


Fig . 5 . Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1 - tg mice . ( B ) Nrg1 - tg mice travelled longer distances in the open field arena ( effect of genotype F ( 1 , 44 ) = 10 . 53 ; p = 0 . 0022 ; 2 - way ANOVA ) . Bonferroni post - hoc analysis revealed a significant genotype - dependent difference between vehicle - treated groups ( p = 0 . 0044 ) but not in spironolactone - treated groups ( p = 0 . 3783 ) . Genotype differences were abolished upon spironolactone treatment .

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Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.(B) Nrg1-tg mice travelled longer distances in the open field arena (effect of genotype F (1,44) =10.53; p=0.0022; 2-way ANOVA). Bonferroni post-hoc analysis revealed a significant genotype-dependent difference between vehicle-treated groups (p=0.0044) but not in spironolactone-treated groups (p=0.3783). Genotype differences were abolished upon spironolactone treatment.


Fig . 5 . Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1 - tg mice . ( C ) When vehicle treated animal were analyzed in 1 min intervals , transgenic mice showed an increased activity throughout the entire test ( effect of genotype F ( 1 , 22 ) = 9 . 27 ; p = 0 . 0060 ; 2 - way ANOVA ) , most prominent in intervals 2 , 7 , and 8 ( p = 0 . 0427 , p = 0 . 0385 and p = 0 . 000036 , respectively , Bonferroni post - hoc test ) .

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Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.(C) When vehicle treated animal were analyzed in 1 min intervals, transgenic mice showed an increased activity throughout the entire test (effect of genotype F (1,22) =9.27; p=0.0060; 2-way ANOVA), most prominent in intervals 2, 7, and 8 (p=0.0427, p=0.0385 and p=0.000036, respectively, Bonferroni post-hoc test).


Fig . 5 . Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1 - tg mice . ( D ) There was no significant difference between the genotypes when treated with spironolactone ( effect of genotype F ( 1 , 22 ) = 2 . 19 ; p = 0 . 1535 ; 2 - way ANOVA ) . However , the interaction of genotype and treatment was significant ( F ( 9 , 198 ) = 1 . 94 ; p = 0 . 0481 ; 2 - way ANOVA ) .

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Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.(D) There was no significant difference between the genotypes when treated with spironolactone (effect of genotype F (1,22) =2.19; p=0.1535; 2-way ANOVA). However, the interaction of genotype and treatment was significant (F (9,198) =1.94; p=0.0481; 2-way ANOVA).


Fig . 5 . Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1 - tg mice . ( E ) Nrg1 - tg mice treated with spironolactone spent more time in the light compartment during the light - dark test ( interaction gene × treatment F ( 1 , 41 ) = 4 . 90 ; p = 0 . 0324 ; 2 - way ANOVA , and p = 0 . 0219 , Bonferroni post - test ) .

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Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.(E) Nrg1-tg mice treated with spironolactone spent more time in the light compartment during the light-dark test (interaction gene × treatment F (1,41) =4.90; p=0.0324; 2-way ANOVA, and p=0.0219, Bonferroni post-test).


Fig . 5 . Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1 - tg mice . ( F ) In the Y - maze test , transgenic mice performed less alterations ( effect of genotype F ( 1 , 44 ) = 11 . 50 ; p = 0 . 0015 ; 2 - way ANOVA ) . The Bonferroni test confirmed this phenotype in vehicle - treated groups ( p = 0 . 0011 ) , but not in spironolactone - treated animals ( p = 0 . 5950 ) . Spironolactone treatment had a significant effect on the number of alterations ( F ( 1 , 44 ) = 4 . 12 ; p = 0 . 0484 ; 2 - way ANOVA ) .

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Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice. (F) In the Y-maze test, transgenic mice performed less alterations (effect of genotype F (1,44) =11.50; p=0.0015; 2-way ANOVA). The Bonferroni test confirmed this phenotype in vehicle-treated groups (p=0.0011), but not in spironolactone-treated animals (p=0.5950). Spironolactone treatment had a significant effect on the number of alterations (F (1,44) =4.12; p=0.0484; 2-way ANOVA).


Fig . 5 . Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1 - tg mice . ( G ) Spironolactone treatment significantly enhanced PPI in Nrg1 - tg mice ( effect of treatment F ( 1 , 21 ) = 5 . 07 ; p = 0 . 0325 ; 2 - way repeated measures ANOVA ) . Data are shown as mean , error bars represent SEM . * , p < 0 . 05 , * * , p < 0 . 01 and * * * , p < 0 . 001 , Bonferroni test following 2 - way ANOVA ; Spiro , spironolactone ; Veh , vehicle . n = 12 per genotype and treatment with an exception of ( E ) ( Nrg1 - tg vehicle , n = 11 ; Nrg1 - tg Spiro , n = 10 ; wt vehicle , n = 12 ; wt Spiro , n = 12 ) and ( G ) ( Nrg1 - tg vehicle , n = 11 ; Nrg1 - tg Spiro , n = 12 ; wt vehicle , n = 12 ; wt Spiro , n = 12 ) .

O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.(G) Spironolactone treatment significantly enhanced PPI in Nrg1-tg mice (effect of treatment F (1,21) =5.07; p=0.0325; 2-way repeated measures ANOVA). Data are shown as mean, error bars represent SEM. *, p<0.05, **, p<0.01 and ***, p<0.001, Bonferroni test following 2-way ANOVA; Spiro, spironolactone; Veh, vehicle. n=12 per genotype and treatment with an exception of (E) (Nrg1-tg vehicle, n=11; Nrg1-tg Spiro, n=10; wt vehicle, n=12; wt Spiro, n=12) and (G) (Nrg1-tg vehicle, n=11; Nrg1-tg Spiro, n=12; wt vehicle, n=12; wt Spiro, n=12).


Autophagy is activated in dying nurse cells during late oogenesis in D . melanogaster . ( A - C ) Confocal micrographs of egg chambers of flies expressing the UASp - GFP - mCherry - DrAtg8a transgene exclusively in the germline ( genotype : UASp - GFP - mCherry - DrAtg8a / + ; nanos - VP - 16 Gal4 / + ) . Expression of UASp - GFP - mCherry - DrAtg8a exhibits punctate staining pattern ( arrows ) . Note that during developmental stages early 12 ( A ) and late 12 ( B ) , the punctate dots are yellow ( merge ) . In late stage 13 ( C ) , large red dots are evident , indicating the presence of acidic compartments ( autolysosomes ; arrows ) . The arrowheads in A point to the physical connection between the oocyte and the nurse cells during early stage 12 ( not observed during late stage 12 ) .

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Autophagy is activated in dying nurse cells during late oogenesis in D. melanogaster. (A-C) Confocal micrographs of egg chambers of flies expressing the UASp-GFP-mCherry-DrAtg8a transgene exclusively in the germline (genotype: UASp-GFP-mCherry-DrAtg8a/+; nanos-VP-16 Gal4/+). Expression of UASp-GFP-mCherry-DrAtg8a exhibits punctate staining pattern (arrows). Note that during developmental stages early 12 (A) and late 12 (B), the punctate dots are yellow (merge). In late stage 13 (C), large red dots are evident, indicating the presence of acidic compartments (autolysosomes; arrows). The arrowheads in A point to the physical connection between the oocyte and the nurse cells during early stage 12 (not observed during late stage 12).


( D and E ) Transmission electron micrographs of nurse cells in stage 12 ( D ) and in stage 13 ( E ) egg chambers . Autophagosomes ( inset in D ) and autolysosomes ( arrow in E ) are evident in the remaining nurse cell cytoplasm ( arrow in D indicates actin bundles ) . Note that the autolysosome shown in E contains condensed material resembling the material of the condensed and fragmented nurse cell nucleus ( NCN ) , providing evidence for nuclear

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D and E) Transmission electron micrographs of nurse cells in stage 12 (D) and in stage 13 (E) egg chambers. Autophagosomes (inset in D) and autolysosomes (arrow in E) are evident in the remaining nurse cell cytoplasm (arrow in D indicates actin bundles). Note that the autolysosome shown in E contains condensed material resembling the material of the condensed and fragmented nurse cell nucleus (NCN), providing evidence for nuclear autophagy


( F ) Nurse cell of a late stage 13 egg chamber expressing UASp - mCherry - DrAtg8a exclusively in the germline ( genotype : UASp - mCherry - DrAtg8a / + ; nanos - VP - 16 Gal4 / + ) . mCherry - DrAtg8a puncta are attached to or located adjacent to the fragmented nucleus ( arrows and insets ) . Hoechst staining ( blue ) was performed to visualize the nuclei . Nurse cells are outlined with a white line . FC , follicle cell ; FCN , follicle cell nucleus ; OC , oocyte . Bars : ( A - C ) 10 µm ; ( D - F ) 1 µm .

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(F) Nurse cell of a late stage 13 egg chamber expressing UASp-mCherry-DrAtg8a exclusively in the germline (genotype: UASp-mCherry-DrAtg8a/+; nanos-VP-16 Gal4/+). mCherry-DrAtg8a puncta are attached to or located adjacent to the fragmented nucleus (arrows and insets). Hoechst staining (blue) was performed to visualize the nuclei. Nurse cells are outlined with a white line. FC, follicle cell; FCN, follicle cell nucleus; OC, oocyte. Bars: (A-C) 10 µm; (D-F) 1 µm.


Genetic inhibition of autophagy in the germline prevents DNA fragmentation . Confocal micrographs of stage 13 / 14 egg chambers after TUNEL staining . ( A ) Wild - type ( wt ) stage 13 egg chamber contains nurse cell nuclei exhibiting fragmented DNA ( positive TUNEL staining ; arrows ) . ( B ) Wild - type stage 14 egg chambers do not contain nurse cells . Arrows point to the respiratory appendages ( RA ) . ( C - E ) Stage 14 atg1 − / − ( C ) , atg13 − / − ( D ) , and vps34 − / − ( E ) germline mutant chambers have persisting nurse cell nuclei that do not contain fragmented DNA ( negative TUNEL staining ; arrows ) . Nurse cells are outlined with a white line . Draq5 / Hoechst staining ( blue ) was performed to visualize the nuclei . PhC , phase contrast . Bars , 50 µm .

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Genetic inhibition of autophagy in the germline prevents DNA fragmentation. Confocal micrographs of stage 13/14 egg chambers after TUNEL staining. (A) Wild-type (wt) stage 13 egg chamber contains nurse cell nuclei exhibiting fragmented DNA (positive TUNEL staining; arrows). (B) Wild-type stage 14 egg chambers do not contain nurse cells. Arrows point to the respiratory appendages (RA). (C-E) Stage 14 atg1−/− (C), atg13−/− (D), and vps34−/− (E) germline mutant chambers have persisting nurse cell nuclei that do not contain fragmented DNA (negative TUNEL staining; arrows). Nurse cells are outlined with a white line. Draq5/Hoechst staining (blue) was performed to visualize the nuclei. PhC, phase contrast. Bars, 50 µm.


Germline autophagy mutant egg chambers exhibit reduced expression of cleaved caspase - 3 . Confocal micrographs of stage 13 / 14 egg chambers stained for cleaved caspase - 3 ( casp - 3 ) . ( A ) Wild - type ( wt ) late stage 13 egg chamber showing cleaved caspase - 3 staining in the degenerating nurse cell cluster . ( B ) In wild - type stage 14 egg chamber , nurse cells are completely degraded ( arrows ) . ( C - E ) atg1 − / − ( C ) , atg13 − / − ( D ) , and vps34 − / − ( E ) germline mutant stage 14 egg chambers exhibit significantly reduced staining for cleaved caspase - 3 and contain persisting nurse cell nuclei . Nurse cells are outlined with a white line . Draq5 / Hoechst staining ( blue ) was performed to visualize the nuclei . PhC , phase contrast . ( F ) Quantification of mean intensity of cleaved caspase - 3 staining observed in the nurse cells of stage 12 - 14 egg chambers in wild - type and germline autophagy mutant egg chambers . Wild type ( WT ) : three independent experiments , n = 30 egg chambers ; atg1 − / − GLCs : four independent experiments , n = 30 egg chambers ; atg13 − / − GLCs : four independent experiments , n = 30 egg chambers ; vps34 − / − GLCs : three independent experiments , n = 30 egg chambers . Data are presented as mean ± SD . Difference was significant with P < 0 . 001 for all values versus wild type . Bars , 20 µm .

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Germline autophagy mutant egg chambers exhibit reduced expression of cleaved caspase-3. Confocal micrographs of stage 13/14 egg chambers stained for cleaved caspase-3(casp-3). (A) Wild-type (wt) late stage 13 egg chamber showing cleaved caspase-3 staining in the degenerating nurse cell cluster. (B) In wild-type stage 14 egg chamber, nurse cells are completely degraded (arrows). (C-E) atg1−/− (C), atg13−/− (D), and vps34−/− (E) germline mutant stage 14 egg chambers exhibit significantly reduced staining for cleaved caspase-3 and contain persisting nurse cell nuclei. Nurse cells are outlined with a white line. Draq5/Hoechst staining (blue) was performed to visualize the nuclei. PhC, phase contrast. (F) Quantification of mean intensity of cleaved caspase-3 staining observed in the nurse cells of stage 12-14 egg chambers in wild-type and germline autophagy mutant egg chambers. Wild type (WT): three independent experiments, n = 30 egg chambers; atg1−/− GLCs: four independent experiments, n = 30 egg chambers; atg13−/− GLCs: four independent experiments, n = 30 egg chambers; vps34−/− GLCs: three independent experiments, n = 30 egg chambers. Data are presented as mean ± SD. Difference was significant with P < 0.001 for all values versus wild type. Bars, 20 µm.


dBruce colocalizes with the autophagic marker Atg8a - GFP in the nurse cells during late oogenesis and is accumulated in autophagy germline mutants . Confocal micrographs of late stage egg chambers expressing Atg8a - GFP and stained for dBruce . ( A - C ) Stage 10 ( A ) , early stage 12 ( B ) , and late stage 12 ( C ) . dBruce colocalizes with Atg8a - GFP in punctuate structures during stages 12 and 13 ( insets in B and insets and arrows in C ) and not during stage 10 ( insets in A ) .

B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O 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-MEASURED_VAR O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

dBruce colocalizes with the autophagic marker Atg8a-GFP in the nurse cells during late oogenesis and is accumulated in autophagy germline mutants. Confocal micrographs of late stage egg chambers expressing Atg8a-GFP and stained for dBruce. (A-C) Stage 10 (A), early stage 12 (B), and late stage 12 (C). dBruce colocalizes with Atg8a-GFP in punctuate structures during stages 12 and 13 (insets in B and insets and arrows in C) and not during stage 10 (insets in A).


( D - G ) Confocal micrographs of stage 14 wild - type ( D ) and autophagy mutant egg chambers ( E - G ) stained for dBruce . dBruce is accumulated in atg1 , atg13 , and vps34 mutant nurse cells ( arrows ) . Draq5 / Hoechst staining ( blue ) was performed to visualize the nuclei . Nurse cells are outlined with a white line . OC , oocyte ; PhC , phase contrast . Bars , 10 µm .

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(D-G) Confocal micrographs of stage 14 wild-type (D) and autophagy mutant egg chambers (E-G) stained for dBruce. dBruce is accumulated in atg1, atg13, and vps34 mutant nurse cells (arrows). Draq5/Hoechst staining (blue) was performed to visualize the nuclei. Nurse cells are outlined with a white line. OC, oocyte; PhC, phase contrast. Bars, 10 µm.


dBruce is required for nurse cell survival by controlling DNA fragmentation during oogenesis . ( A ) Confocal micrographs of dBrucee00984 mutant ovarioles stained for TUNEL and DNA . Arrows point to degenerating stage 8 / 9 egg chambers that are TUNEL positive .

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dBruce is required for nurse cell survival by controlling DNA fragmentation during oogenesis. (A) Confocal micrographs of dBrucee00984 mutant ovarioles stained for TUNEL and DNA. Arrows point to degenerating stage 8/9 egg chambers that are TUNEL positive.


( B and C ) Confocal micrographs of vps34 / dBruceE81 and atg , dBruceE81 double mutant egg chambers stained for TUNEL ( green and red , respectively ) and DNA . ( B ) vps34 − / − GLCs ; dBruceE81 / dBruceE81 stage 14 mutant egg chamber contain persisting TUNEL - positive nurse cell nuclei ( outlined ; arrow ) . ( C ) atg1 − / − GLCs , dBruceE81 / dBruceE81 stage 14 mutant egg chamber contain persisting TUNEL - positive nurse cell nuclei . atg1 mutant nurse cells are identified by the lack of GFP staining ( outlined ; arrow ) . Draq5 / Hoechst staining ( blue ) was performed to visualize the nuclei . PhC , phase contrast . Bars , 50 µm .

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(B and C) Confocal micrographs of vps34/dBruceE81 and atg, dBruceE81 double mutant egg chambers stained for TUNEL (green and red, respectively) and DNA. (B) vps34−/−GLCs; dBruceE81/dBruceE81 stage 14 mutant egg chamber contain persisting TUNEL-positive nurse cell nuclei (outlined; arrow). (C) atg1−/−GLCs, dBruceE81/dBruceE81 stage 14 mutant egg chamber contain persisting TUNEL-positive nurse cell nuclei. atg1 mutant nurse cells are identified by the lack of GFP staining (outlined; arrow). Draq5/Hoechst staining (blue) was performed to visualize the nuclei. PhC, phase contrast. Bars, 50 µm.


Labelling scheme and representative DNA fibers from U2OS cells transfected with siLuc or siChk1 .

Labelling scheme and representative DNA fibers from U2OS cells transfected with siLuc or siChk1.


Western Blot of Chk1 in U2OS cells , 48 h after transfection with siRNA . Actin was used as loading control .

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Western Blot of Chk1 in U2OS cells, 48 h after transfection with siRNA. Actin was used as loading control.


Control ( siLuc ) and Chk1 - depleted ( siChk1 ) U20S cells were labeled with CldU and IdU for 10 and 30 min , respectively . Mean ( + SEM ) track lengths ( top ) and CldU / IdU ratios ( bottom ) are shown . Data come from 6 independent experiments and a total of 579 ( siLuc ) and 590 ( siChk1 ) fibers were scored .

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Control (siLuc) and Chk1-depleted (siChk1) U20S cells were labeled with CldU and IdU for 10 and 30 min, respectively. Mean (+SEM) track lengths (top) and CldU/IdU ratios (bottom) are shown. Data come from 6 independent experiments and a total of 579 (siLuc) and 590 (siChk1) fibers were scored.


Western Blot of phospho - serine - 296 - Chk1 ( p - Chk1 ) in U2OS cells . Cells were treated 1 . 5 h with the Chk1 inhibitor Gö6976 ( Chk1i ) or DMSO , irradiated with 100J / m2 UV to induce high levels of p - Chk1 , and collected 1 . 5 h afterwards . Actin was used as a loading control .

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Western Blot of phospho-serine-296-Chk1 (p-Chk1) in U2OS cells. Cells were treated 1.5 h with the Chk1 inhibitor Gö6976 (Chk1i) or DMSO, irradiated with 100J/m2 UV to induce high levels of p-Chk1, and collected 1.5 h afterwards. Actin was used as a loading control.


U2OS cells treated with DMSO ( control ) or Chk1i were labeled with CldU and IdU for 10 and 30 min , respectively . Data come from 3 independent experiments and a total of 304 ( DMSO ) and 312 ( Chk1i ) fibers were scored .

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U2OS cells treated with DMSO (control) or Chk1i were labeled with CldU and IdU for 10 and 30 min, respectively. Data come from 3 independent experiments and a total of 304 (DMSO) and 312 (Chk1i) fibers were scored.


U2OS cells were labeled with CldU for 10 min , followed by increasing IdU labelling times and subjected to DNA stretching . ( A ) Labelling scheme and representative DNA fibers . Scale bars : 5 μm .

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U2OS cells were labeled with CldU for 10 min, followed by increasing IdU labelling times and subjected to DNA stretching. (A) Labelling scheme and representative DNA fibers. Scale bars: 5 μm.


U2OS cells were labeled with CldU for 10 min , followed by increasing IdU labelling times and subjected to DNA stretching . IdU track length after 10 - 80 min . Theoretical IdU track lengths are shown in red ; the theoretical 10 - min points were considered equal to the experimental values obtained for siLuc or siChk1 , and the following theoretical points were calculated proportionally . Data were calculated as the mean ( + SD ) of mean IdU track lengths ( B ) n = 4 for IdU times 20 - 60 min or n = 3 for IdU times 10 and 80 min ; for each experiment and condition 60 - 100 fibers were scored .

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U2OS cells were labeled with CldU for 10 min, followed by increasing IdU labelling times and subjected to DNA stretching. IdU track length after 10-80 min. Theoretical IdU track lengths are shown in red; the theoretical 10-min points were considered equal to the experimental values obtained for siLuc or siChk1, and the following theoretical points were calculated proportionally. Data were calculated as the mean (+SD) of mean IdU track lengths (B) n=4 for IdU times 20-60 min or n=3 for IdU times 10 and 80 min; for each experiment and condition 60-100 fibers were scored.


U2OS cells were labeled with CldU for 10 min , followed by increasing IdU labelling times and subjected to DNA stretching . Fork speed for each IdU labelling time . Data were calculated as the mean ( + SD ) of mean fork speeds ( C ) . n = 4 for IdU times 20 - 60 min or n = 3 for IdU times 10 and 80 min ; for each experiment and condition 60 - 100 fibers were scored .

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U2OS cells were labeled with CldU for 10 min, followed by increasing IdU labelling times and subjected to DNA stretching. Fork speed for each IdU labelling time. Data were calculated as the mean (+SD) of mean fork speeds (C). n=4 for IdU times 20-60 min or n=3 for IdU times 10 and 80 min; for each experiment and condition 60-100 fibers were scored.


DNA combing from HCT116 cells labeled with CldU and IdU for 20 min . The upper left panel shows the labelling scheme . The upper right panel shows representative sister replication forks that emanate from the same origin ( ORI = origin of replication ) ; a pair of sister forks is considered asymmetric if their lengths ( CldU + IdU ) differ in more than 33 % . The percentage of asymmetric forks ( lower left panel ; a Western Blot of Chk1 and Ku80 , loading control , performed in parallel to the combing experiments , is shown ) and the lengths of each pair of forks ( lower right panel ; points outside of the dotted lines correspond to asymmetric forks ) are shown . Data ( siLuc , n = 131 ; siChk1 , n = 117 ) come from 2 independent experiments ; the numbers indicate the mean ( + SD ) percentage of asymmetric forks .

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DNA combing from HCT116 cells labeled with CldU and IdU for 20 min. The upper left panel shows the labelling scheme. The upper right panel shows representative sister replication forks that emanate from the same origin (ORI=origin of replication); a pair of sister forks is considered asymmetric if their lengths (CldU+IdU) differ in more than 33%. The percentage of asymmetric forks (lower left panel; a Western Blot of Chk1 and Ku80, loading control, performed in parallel to the combing experiments, is shown) and the lengths of each pair of forks (lower right panel; points outside of the dotted lines correspond to asymmetric forks) are shown. Data (siLuc, n=131; siChk1, n=117) come from 2 independent experiments; the numbers indicate the mean (+SD) percentage of asymmetric forks.


Labelling protocol used here and in all figures that follow , except otherwise indicated . The lower panels show representative images and schemes of an ongoing fork and of origins that fired during the CldU ( 1st ) or IdU ( 2nd ) pulses . Gray dotted lines represent unlabeled DNA . Percentage of origins was calculated as : ( 1st + 2nd pulse origins ) / total number of fibers .

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Labelling protocol used here and in all figures that follow, except otherwise indicated. The lower panels show representative images and schemes of an ongoing fork and of origins that fired during the CldU (1st) or IdU (2nd) pulses. Gray dotted lines represent unlabeled DNA. Percentage of origins was calculated as: (1st+2nd pulse origins)/total number of fibers.


IdU track lengths from U2OS cells ( Rosc , roscovitine ) . 180 - 200 DNA fibers obtained from 2 independent experiments were measured for each condition . The upper panel shows a Western Blot of Polη , 48 h after transfection with siRNA . Actin was used as a loading control .

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IdU track lengths from U2OS cells (Rosc, roscovitine). 180-200 DNA fibers obtained from 2 independent experiments were measured for each condition. The upper panel shows a Western Blot of Polη, 48 h after transfection with siRNA. Actin was used as a loading control.


Percentage of origin firing ( mean + SD ) from DNA fibers in ( A ) ( n = 2 ) . The dotted line represents the mean percentage of origin firing in control samples .

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Percentage of origin firing (mean+SD) from DNA fibers in (A) (n=2). The dotted line represents the mean percentage of origin firing in control samples.


Representative images of siChk1 - depleted U2OS cells showing nuclear localization of GFP - Polη - WT / S687A / S687D . Numbers indicate percentage of cells showing nuclear GFP - Polη ( mean + SD , n = 2 ) . Scale bar : 100 μm .

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Representative images of siChk1-depleted U2OS cells showing nuclear localization of GFP-Polη-WT/S687A/S687D. Numbers indicate percentage of cells showing nuclear GFP-Polη (mean+SD, n=2). Scale bar: 100 μm.


Western Blot of GFP - Polη in U2OS cells , 48 / 24 h after transfection with siRNA / GFP ( - Polη ) . Actin was used as loading control .

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Western Blot of GFP-Polη in U2OS cells, 48/24 h after transfection with siRNA/GFP(-Polη). Actin was used as loading control.


IdU track lengths from U2OS cells transfected with GFP ( - ) or GFP - Polη - WT / S687A / S687D . > 180 fibers obtained from 2 independent experiments were measured for each condition .

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IdU track lengths from U2OS cells transfected with GFP (-) or GFP-Polη-WT/S687A/S687D. >180 fibers obtained from 2 independent experiments were measured for each condition.


Number of foci per cell after CSK extraction in siLuc and siChk1 - depleted U2OS cells transfected with GFP - Polη - WT / S687A / S687D . > 150 cells / sample were analyzed in 4 independent experiments . p < 0 . 05 was considered significant .

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Number of foci per cell after CSK extraction in siLuc and siChk1-depleted U2OS cells transfected with GFP-Polη-WT/S687A/S687D. >150 cells/sample were analyzed in 4 independent experiments. p<0.05 was considered significant.


Representative images of cells with GFP - Polη foci used for quantification in ( E ) . The number of foci per cell for each representative nucleus is indicated . Scale bar : 10 μm .

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Representative images of cells with GFP-Polη foci used for quantification in (E). The number of foci per cell for each representative nucleus is indicated. Scale bar: 10 μm.


IdU track lengths and percentage of origin firing ( mean + SD ) from U2OS cells treated or not with a CDC7 inhibitor ( CDC7i ) . > 380 ( track length ) and > 600 ( origin frequency ) fibers obtained from 4 ( track length ) or 3 ( origin frequency ) independent experiments were measured for each condition .

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IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells treated or not with a CDC7 inhibitor (CDC7i). >380 (track length) and >600 (origin frequency) fibers obtained from 4 (track length) or 3 (origin frequency) independent experiments were measured for each condition.


IdU track lengths and percentage of origin firing ( mean + SD ) from U2OS cells transfected or not with siCDT1 . Track lengths from > 290 fibers obtained from 3 independent experiments were measured for each condition . To calculate origin frequency > 700 DNA fibers from 3 independent experiments were scored .

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IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells transfected or not with siCDT1. Track lengths from >290 fibers obtained from 3 independent experiments were measured for each condition. To calculate origin frequency >700 DNA fibers from 3 independent experiments were scored.


IdU track lengths and percentage of origin firing ( mean + SD ) from U2OS cells supplemented or not with nucleosides ( NS ) . > 195 ( track length ) and > 350 ( origin frequency ) fibers obtained from 2 independent experiments were measured for each condition .

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IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells supplemented or not with nucleosides (NS). >195 (track length) and >350 (origin frequency) fibers obtained from 2 independent experiments were measured for each condition.


IdU track lengths and percentage of origin firing ( mean + SD ) from U2OS cells transfected or not with GFP - Polη . > 180 ( track length ) and > 400 ( origin frequency ) DNA fibers obtained from 2 independent experiments were measured for each condition .

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IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells transfected or not with GFP-Polη. >180 (track length) and >400 (origin frequency) DNA fibers obtained from 2 independent experiments were measured for each condition.


Percentage of γH2AX - positive U2OS cells ( mean + SD ) after 5 h treatment with the Chk1 inhibitor Gö6976 ( Chk1i ) , in the presence of 5 μM NS , 1 , 25 μM CDC7i or 10 μM roscovitine . > 900 ( DMSO ) or > 2000 ( Chk1i ) cells / sample were analyzed in 2 independent experiments . Total γH2AX was used to perform the statistics . Representative images of data shown in ( B ) . Scale bar : 50 μm .

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Percentage of γH2AX-positive U2OS cells (mean+SD) after 5 h treatment with the Chk1 inhibitor Gö6976 (Chk1i), in the presence of 5 μM NS, 1,25 μM CDC7i or 10 μM roscovitine. >900 (DMSO) or >2000 (Chk1i) cells/sample were analyzed in 2 independent experiments. Total γH2AX was used to perform the statistics. Representative images of data shown in (B). Scale bar: 50 μm.


DSB accumulation measured by neutral comet assay after 8 h treatment with Chk1i , in the presence of 5 μM NS , siCDC7 or 10 μM roscovitine . 85 - 170 cells / sample were analyzed in 2 independent experiments . Representative images of data shown in ( D ) . Scale bar : 25 μm .

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DSB accumulation measured by neutral comet assay after 8 h treatment with Chk1i, in the presence of 5 μM NS, siCDC7 or 10 μM roscovitine. 85-170 cells/sample were analyzed in 2 independent experiments. Representative images of data shown in (D). Scale bar: 25 μm.


Percentage of parental ssDNA - positive U2OS cells ( mean + SD ) after 5 h treatment with Chk1i , in the presence of 5 μM NS , 1 , 25 μM CDC7i or 10 μM roscovitine . > 450 cells / sample were analyzed in 2 independent experiments . The induction of ssDNA on control samples was null . Representative images of data shown in ( F ) . Scale bar : 25 μm .

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Percentage of parental ssDNA-positive U2OS cells (mean+SD) after 5 h treatment with Chk1i, in the presence of 5 μM NS, 1,25 μM CDC7i or 10 μM roscovitine. >450 cells/sample were analyzed in 2 independent experiments. The induction of ssDNA on control samples was null. Representative images of data shown in (F). Scale bar: 25 μm.


Sensitivity of U2OS cells to Chk1 inhibition , roscovitine ( 5 μM ) and Polη depletion . Cell number was determined 4 days after a 24 h treatment ( 5 days in total ) with Chk1i ± Rosc . Data represent the mean ( + SD ) of 3 independent experiments .

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Sensitivity of U2OS cells to Chk1 inhibition, roscovitine (5 μM) and Polη depletion. Cell number was determined 4 days after a 24 h treatment (5 days in total) with Chk1i±Rosc. Data represent the mean (+SD) of 3 independent experiments.


Sensitivity of U2OS cells transduced with shRNA targeting Chk1 ( shChk1 ) or non - targeting shRNA ( shScr ) and Lenti - GFP - Polη or Lenti - GFP ( - ) . Cell number was determined 7 days after transduction ( 5 days after maximal downregulation ) . Data represent the mean ( + SD ) of 3 independent experiments .

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Sensitivity of U2OS cells transduced with shRNA targeting Chk1 (shChk1) or non-targeting shRNA (shScr) and Lenti-GFP-Polη or Lenti-GFP (-). Cell number was determined 7 days after transduction (5 days after maximal downregulation). Data represent the mean (+SD) of 3 independent experiments.


Western Blot of CDC45 and Chk1 in U2OS cells after an extraction with CSK buffer to separate the insoluble and soluble fractions . Roscovitine ( 25μM ) was added 2 h before harvesting . H2B was used as a loading control .

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Western Blot of CDC45 and Chk1 in U2OS cells after an extraction with CSK buffer to separate the insoluble and soluble fractions. Roscovitine (25μM) was added 2 h before harvesting. H2B was used as a loading control.


IdU track lengths from U2OS cells . 200 DNA fibers obtained from 2 independent experiments were measured for each condition .

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IdU track lengths from U2OS cells. 200 DNA fibers obtained from 2 independent experiments were measured for each condition.


IdU track lengths from U2OS cells . > 170 DNA fibers obtained from 2 independent experiments were measured for each condition . For comparison , the data showing that Polη is required for roscovitine - dependent rescue of fork elongation in Chk1 - depleted cells ( Fig 3B ) is shown on the right . The lower panel shows the corresponding Western Blot of Chk1 , Polη and CDC45 . Actin was used as a loading control .

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IdU track lengths from U2OS cells. >170 DNA fibers obtained from 2 independent experiments were measured for each condition. For comparison, the data showing that Polη is required for roscovitine-dependent rescue of fork elongation in Chk1-depleted cells (Fig 3B) is shown on the right. The lower panel shows the corresponding Western Blot of Chk1, Polη and CDC45. Actin was used as a loading control.


Percentage of origin firing ( mean + SD ) from DNA fibers in C ( n = 2 ) . > 490 fibers per condition were scored .

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Percentage of origin firing (mean+SD) from DNA fibers in C (n=2). >490 fibers per condition were scored.


Sensitivity of U2OS cells to Chk1 and CDC45 depletion . Cell number was determined 7 days after transfection ( 5 days after maximal downregulation ) . Data represent the mean ( + SD ) of 2 independent experiments . Representative image of data shown in ( E ) . Scale bar : 100 μm .

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Sensitivity of U2OS cells to Chk1 and CDC45 depletion. Cell number was determined 7 days after transfection (5 days after maximal downregulation). Data represent the mean (+SD) of 2 independent experiments. Representative image of data shown in (E). Scale bar: 100 μm.


Sensitivity of U2OS cells to Chk1 inhibition and roscovitine ( 5 μM ) . Cell number was determined 4 days after a 24 h treatment ( 5 days in total ) with Chk1i ± Rosc . Data represent the mean ( + SD ) of 2 independent experiments . Representative image of data shown in ( G ) . Scale bar : 100 μm .

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Sensitivity of U2OS cells to Chk1 inhibition and roscovitine (5 μM). Cell number was determined 4 days after a 24 h treatment (5 days in total) with Chk1i±Rosc. Data represent the mean (+SD) of 2 independent experiments. Representative image of data shown in (G). Scale bar: 100 μm.


Flow cytometry analysis of PD - L1 expression in ( A ) KPC and ( B ) Pan02 cells following treatment with gemcitabine and RT . Gemcitabine was diluted in DMSO ( vehicle ) and DMSO was used for the ' untreated ' control group ( mean ± SD , n = 3 , One - way ANOVA , Bonferroni test ) .

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Flow cytometry analysis of PD-L1 expression in (A) KPC and (B) Pan02 cells following treatment with gemcitabine and RT. Gemcitabine was diluted in DMSO (vehicle) and DMSO was used for the 'untreated' control group (mean ± SD, n=3, One-way ANOVA, Bonferroni test).


Western blot analysis of indicated proteins in both KPC ( C ) and Pan02 ( D ) cells following radiation and chemotherapy ± AG490 , a JAK / Stat kinase inhibitor . Actin represents loading control .

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Western blot analysis of indicated proteins in both KPC (C) and Pan02 (D) cells following radiation and chemotherapy ± AG490, a JAK/Stat kinase inhibitor. Actin represents loading control.


Flow cytometry analysis of PD - L1 after gemcitabine and RT ( as described above ) in KPC ( E ) and Pan02 ( F ) following Stat1 downregulation by siRNA ( mean ± SD , n = 3 , Student ' s t - test ) . MFI , mean fluorescence index ;

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Flow cytometry analysis of PD-L1 after gemcitabine and RT (as described above) in KPC (E) and Pan02 (F) following Stat1 downregulation by siRNA (mean ± SD, n=3, Student's t-test). MFI, mean fluorescence index;


( A ) Tumor growth delay was measured in the different groups , as indicated ( n = 6 mice per group ) . Anti - PD - L1 was given at days 0 3 , 6 and 9 ( black arrows ) . The average time ( days ) for tumors to reach a volume of 400 mm3 from day 0 is shown ( means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) . No weight loss was observed in the in vivo

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(A) Tumor growth delay was measured in the different groups, as indicated (n=6 mice per group). Anti-PD-L1 was given at days 0 3, 6 and 9 (black arrows). The average time (days) for tumors to reach a volume of 400 mm3 from day 0 is shown (means ± SD, n=1, One-way ANOVA, Bonferroni test). No weight loss was observed in the in vivo experiment


( B ) Quantitative data on the percentage of gated cells are shown ( n = 5 per group ; means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) .

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(B) Quantitative data on the percentage of gated cells are shown (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test).


( A ) Tumor growth delay was measured by treating tumor with RT ( 12 Gy or 5x3 Gy daily ; initiated at day 0 ) and anti - PD - L1 alone or combination of RT ( n = 6 mice per group ) . Anti - PD - L1 was given at days 0 , 3 , 6 and 9 ( black arrows ) . The radiosensitizing effect of anti - PD - L1 after 12 Gy and 5x3 Gy was assessed as shown by the capped lines ( means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) . No weight loss was observed in the in vivo experiment .

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(A) Tumor growth delay was measured by treating tumor with RT (12 Gy or 5x3 Gy daily; initiated at day 0) and anti-PD-L1 alone or combination of RT (n=6 mice per group). Anti-PD-L1 was given at days 0, 3, 6 and 9 (black arrows). The radiosensitizing effect of anti-PD-L1 after 12 Gy and 5x3 Gy was assessed as shown by the capped lines (means ± SD, n=1, One-way ANOVA, Bonferroni test). No weight loss was observed in the in vivo experiment.


( A ) KPC tumor growth delay after treatment with either 12 Gy ( day 0 ) , anti - PD - L1 ( days 0 , 3 , 6 and 9 ; black arrows ) , anti - CD8 alone ( days 0 3 , 6 and 9 ) or their combinations , as indicated .

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(A) KPC tumor growth delay after treatment with either 12 Gy (day 0), anti-PD-L1 (days 0, 3, 6 and 9; black arrows), anti-CD8 alone (days 0 3, 6 and 9) or their combinations, as indicated.


( B ) KPC tumor growth delay after treatment with either 5x3 Gy ( day 0 - 4 ) , anti - PD - L1 alone ( days 0 , 3 , 6 and 9 ; black arrows ) , anti - CD8 alone ( days 0 , 3 , 6 and 9 ) or their combinations , as indicated .

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(B) KPC tumor growth delay after treatment with either 5x3 Gy (day 0-4), anti-PD-L1 alone (days 0, 3, 6 and 9; black arrows), anti-CD8 alone (days 0, 3, 6 and 9) or their combinations, as indicated.


( C ) KPC tumor growth delay after either simultaneous ( day 0 , 3 , 6 and 9 ; black arrows - upper row ) or sequential ( day 6 , 9 , 13 and 16 ; black arrows - lower row ) addition of anti - PD - L1 to RT ( day 0 ) . The average time ( days ) for tumors to reach a volume of 400 mm3 from day 0 for Figure 4A - C is also shown ( means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) . No weight loss was observed in the in vivo experiments .

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(C) KPC tumor growth delay after either simultaneous (day 0, 3, 6 and 9; black arrows-upper row) or sequential (day 6, 9, 13 and 16; black arrows-lower row) addition of anti-PD-L1 to RT (day 0). The average time (days) for tumors to reach a volume of 400 mm3 from day 0 for Figure 4A-C is also shown (means ± SD, n=1, One-way ANOVA, Bonferroni test). No weight loss was observed in the in vivo experiments.


( A ) Mice were treated with either gemcitabine ( day 0 , 3 ) , anti - PD - L1 ( day 4 , 7 , 10 and 13 ; black arrows ) , 12 Gy ( day 4 ) alone or in combinations , as indicated ( n = 8 mice per group ) . The average time ( days ) for tumors to reach a volume of 400 mm3 from day 0 is shown ( means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) No weight loss was observed in the in vivo experiment .

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(A) Mice were treated with either gemcitabine (day 0, 3), anti-PD-L1 (day 4, 7, 10 and 13; black arrows), 12 Gy (day 4) alone or in combinations, as indicated (n=8 mice per group). The average time (days) for tumors to reach a volume of 400 mm3 from day 0 is shown (means ± SD, n=1, One-way ANOVA, Bonferroni test) No weight loss was observed in the in vivo experiment.


RT plus anti - PD - L1 ( B ) reverses T - cell exhaustion . Bar graphs show quantitation of T cell activation markers in tumor samples based on flow cytometry analysis , as indicated ( n = 5 per group ; means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) .

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RT plus anti-PD-L1 (B) reverses T-cell exhaustion . Bar graphs show quantitation of T cell activation markers in tumor samples based on flow cytometry analysis, as indicated (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test).


RT plus anti - PD - L1 ( C ) increases CD8 : Treg ratio . Bar graphs show ratio of CD8 : Treg ratio in tumor samples based on flow cytometry analysis , as indicated ( n = 5 per group ; means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) .

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RT plus anti-PD-L1 (C) increases CD8:Treg ratio. Bar graphs show ratio of CD8:Treg ratio in tumor samples based on flow cytometry analysis, as indicated (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test).


( A ) Representative images of livers from the different treatment groups , as indicated . The corresponding liver weights are shown below ( n = 6 mice per group ) .

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(A) Representative images of livers from the different treatment groups, as indicated. The corresponding liver weights are shown below (n=6 mice per group).


( B ) Flow cytometry analysis of CD45 + CD8 + , CD11b + Gr1 + and CD4 + CD25 + FOXP3 + cell populations in the livers of the mice from the different treatment groups , as indicated ( n = 5 per group ; ( means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) .

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(B) Flow cytometry analysis of CD45+CD8+, CD11b+Gr1+ and CD4+CD25+FOXP3+ cell populations in the livers of the mice from the different treatment groups, as indicated (n=5 per group; (means ± SD, n=1, One-way ANOVA, Bonferroni test).


( C ) Co - immunoprecipitation of Cdc4 using either unphosphorylated or Clb5 - Cdk1 - phosphorylated Far1 ( 85 - 150 ) as bait . The experiment was repeated twice , a representative example is shown .

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(C) Co-immunoprecipitation of Cdc4 using either unphosphorylated or Clb5-Cdk1-phosphorylated Far1(85-150) as bait. The experiment was repeated twice, a representative example is shown.


( D ) Time - lapse fluorescence images showing the expression and degradation of wild - type and S87A S91A mutant Far1 ( 1 - 150 ) - GFP and nuclear - cytoplasmic shuttling of Whi5 - mCherry . Nuclear export of Whi5 - mCherry takes place at 0 min ( Start point ) and was used to synchronize the cells for quantitative analysis .

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(D) Time-lapse fluorescence images showing the expression and degradation of wild-type and S87A S91A mutant Far1(1-150)-GFP and nuclear-cytoplasmic shuttling of Whi5-mCherry. Nuclear export of Whi5-mCherry takes place at 0 min (Start point) and was used to synchronize the cells for quantitative analysis.


( E , F ) Mean ± SEM fluorescence intensities of the indicated Far1 ( 1 - 150 ) - GFP mutants over the cell cycle .

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(E, F) Mean±SEM fluorescence intensities of the indicated Far1(1-150)-GFP mutants over the cell cycle.