Figure 2B . Ptgfr mRNA levels in retinal microvessels of CKO - T and control mice ( n = 6 ) . INL , inner nuclear layer ; ONL , outer nuclear layer ; RGC , retinal ganglion cells . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test ( B , Data are represented as mean ± SEM . C . Representative images of OIR retinas from CKO - T and control mice on postnatal day 17 . The green color shows the isolectin B4 - stained vessels . The second - row panels are the enlarged images of white boxes from the first - row panels . The third - row images show neovascular tufts ( NV , white ) and the fourth - row images show the vaso - obliteration ( VO , white ) area . D . Quantitation of oxygen - induced retinal neovascularization in CKO - T and control mice ( n = 12 ) . E . Quantitation of retinal vaso - obliteration in CKO - T and control mice ( n = 12 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test D , E ) . Scale bars : 500 μm ( unmagnified image ) , 150 μm ( magnified image ) . Data are represented as mean ± SEM .

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

Figure 2B. Ptgfr mRNA levels in retinal microvessels of CKO-T and control mice (n=6). INL, inner nuclear layer; ONL, outer nuclear layer; RGC, retinal ganglion cells. Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test (B, Data are represented as mean ± SEM.C. Representative images of OIR retinas from CKO-T and control mice on postnatal day 17. The green color shows the isolectin B4-stained vessels. The second-row panels are the enlarged images of white boxes from the first-row panels. The third-row images show neovascular tufts (NV, white) and the fourth-row images show the vaso-obliteration (VO, white) area. D. Quantitation of oxygen-induced retinal neovascularization in CKO-T and control mice (n=12). E. Quantitation of retinal vaso-obliteration in CKO-T and control mice (n=12). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test D, E). Scale bars: 500 μm (unmagnified image), 150 μm (magnified image). Data are represented as mean ± SEM.


Figure 3A . Representative images of scratch - induced migration of PGF2α - treated HRMECs . Yellow solid lines represent scratch locations , dashed lines represent post - migration locations , and arrows indicate migration direction . B . Quantitation of the cell migration distance in A ( n = 4 ) . C . Effect of PGF2α treatment on HRMEC proliferation in a dose - dependent manner . ( n = 6 ) . Data information : One data point represented the mean value of three technical replicates from one independent biological replicate . Data were analyzed by Mann - Whitney test ( B , Kruskal - Wallis test with Dunn ' s multiple comparisons test ( C ) . Scale bars : 100 μm ( A ) Data are represented as mean ± SEM . D . Representative images of staining for BrdU incorporation in PGF2α - treated HRMECs . E . Quantification of BrdU + cells in D ( n = 4 ) . Data information : One data point represented the mean value of three technical replicates from one independent biological replicate . Data were analyzed by Mann - Whitney test Data are represented as mean ± SEM . F . Representative tube formation images of PGF2α - or vehicle ( DMSO ) - treated HRMECs . G - J . Quantitation of total tubule length , number of junctions , number of meshes , and percentage of mesh area ( n = 4 ) . Data information : One data point represented the mean value of three technical replicates from one independent biological replicate . Data were analyzed by Mann - Whitney test G , H , I , J , Scale bars : 200 μm ( F , Data are represented as mean ± SEM . K . Representative images of endothelial cell sprouting of PGF2α - treated mouse aortic rings . Yellow lines indicate the EC sprouting area . L . Quantification of the EC sprouting area ( n = 6 ) . Data information : One data point represented the mean value of three technical replicates from one independent biological replicate . Data were analyzed by Mann - Whitney test L ) Scale bars : 200 μm K ) . Data are represented as mean ± SEM .

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 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 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 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

Figure 3A. Representative images of scratch-induced migration of PGF2α-treated HRMECs. Yellow solid lines represent scratch locations, dashed lines represent post-migration locations, and arrows indicate migration direction. B. Quantitation of the cell migration distance in A (n=4). C. Effect of PGF2α treatment on HRMEC proliferation in a dose-dependent manner. (n=6). Data information: One data point represented the mean value of three technical replicates from one independent biological replicate. Data were analyzed by Mann-Whitney test (B, Kruskal-Wallis test with Dunn's multiple comparisons test (C). Scale bars: 100 μm (A) Data are represented as mean ± SEM.D. Representative images of staining for BrdU incorporation in PGF2α-treated HRMECs. E. Quantification of BrdU+ cells in D (n=4). Data information: One data point represented the mean value of three technical replicates from one independent biological replicate. Data were analyzed by Mann-Whitney test Data are represented as mean ± SEM.F. Representative tube formation images of PGF2α-or vehicle (DMSO)-treated HRMECs. G-J. Quantitation of total tubule length, number of junctions, number of meshes, and percentage of mesh area (n=4). Data information: One data point represented the mean value of three technical replicates from one independent biological replicate. Data were analyzed by Mann-Whitney test G, H, I, J, Scale bars: 200 μm (F, Data are represented as mean ± SEM.K. Representative images of endothelial cell sprouting of PGF2α-treated mouse aortic rings. Yellow lines indicate the EC sprouting area. L. Quantification of the EC sprouting area (n=6). Data information: One data point represented the mean value of three technical replicates from one independent biological replicate. Data were analyzed by Mann-Whitney test L) Scale bars: 200 μm K). Data are represented as mean ± SEM.


Figure 4A . Heat map of upregulated genes in PGF2α - treated HRMECs ( n = 3 samples ) . C . Effect of PGF2α treatment on ELR + CXC chemokine expression in HRMECs ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by unpaired Student ' s t - test ( C ) Data in C . D - G . Relative expression levels of enriched ELR + CXC chemokines in retinal microvascular ECs from patients with PDR ( n = 4 ) compared with those from normal subjects ( n = 7 ) ( Gene Expression Omnibus Database dataset GSE94019 ) . TPM stands for " Transcripts per kilobase million . " Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test ( D , E , F , G ) , Data are presented as the mean + SD . H . Effect of CXCR2 inhibitor SB265610 ( 1 μM ) on PGF2α - stimulated HRMEC migration . The yellow solid line indicates the original position of the scratch , the dashed line indicates the position after cell migration , and the arrows indicate the migratory direction . I . Quantitation of scratch - induced HRMEC migration in H ( n = 4 ) . J . Effect of SB265610 ( 1 μM ) on PGF2α - stimulated HRMEC proliferation ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test ( I , J , Data in I . J . are represented as mean ± SEM . Scale bar : 100 μm ( H ) K . Effect of CXCR2 inhibitor SB265610 ( 1μM ) on tube formation in PGF2α - treated HRMECs . L . Statistical graph of tube formation experiments in K , comparing the total tubule length , number of junctions , number of meshes , and percentage of mesh area ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test L ) . Data in L are represented as mean ± SEM . Scale bar : 200 μm ( K ) .

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

Figure 4A. Heat map of upregulated genes in PGF2α-treated HRMECs (n=3 samples).C. Effect of PGF2α treatment on ELR+ CXC chemokine expression in HRMECs (n=3). Data information: n.s. stands for "not significant." Data were analyzed by unpaired Student's t-test (C) Data in C.D-G. Relative expression levels of enriched ELR+ CXC chemokines in retinal microvascular ECs from patients with PDR (n=4) compared with those from normal subjects (n=7) (Gene Expression Omnibus Database dataset GSE94019). TPM stands for "Transcripts per kilobase million." Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test (D, E, F, G), Data are presented as the mean + SD.H. Effect of CXCR2 inhibitor SB265610 (1 μM) on PGF2α-stimulated HRMEC migration. The yellow solid line indicates the original position of the scratch, the dashed line indicates the position after cell migration, and the arrows indicate the migratory direction. I. Quantitation of scratch-induced HRMEC migration in H (n=4). J. Effect of SB265610 (1 μM) on PGF2α-stimulated HRMEC proliferation (n=4). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test (I, J, Data in I. J. are represented as mean ± SEM. Scale bar: 100 μm (H)K. Effect of CXCR2 inhibitor SB265610 (1μM) on tube formation in PGF2α-treated HRMECs. L. Statistical graph of tube formation experiments in K, comparing the total tubule length, number of junctions, number of meshes, and percentage of mesh area (n=4). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test L). Data in L are represented as mean ± SEM. Scale bar: 200 μm (K).


Figure 5A . Effect of CXCR2 inhibitor SB265610 ( 1 μM ) on PGF2α - promoted sprouting of aortic rings . Yellow lines show the sprouting area . B . Quantitation of the sprouting areas in A ( n = 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test ( B , Scale bar : 200 μm ( A ) , image ) . Data are represented as mean ± SEM . C . mRNA expression of ELR + CXC chemokines in retinas from OIR mice during postnatal days 12 - 17 ( n = 4 - 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test image ) . Data are represented as mean ± SEM . D . Effect of PTGFR deficiency in ECs on ELR + CXC chemokine expression in retinas from OIR mice on postnatal day 16 ( n = 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test image ) . Data are represented as mean ± SEM . F . Retinal CXCL1 protein - expression levels in Cxcl1 - expressing lentivirus - injected CKO mice ( n = 3 ) . G . Quantitation of retinal CXCL1 protein in F ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test G , image ) . Data are represented as mean ± SEM . H . Representative images of oxygen - induced retinal angiogenesis in Cxcl1 - expressing lentivirus - injected CKO mice on postnatal day 17 . Green represents the isolectin B4 - stained vessels , the second - row panels display the enlarged images of white boxes in the first - row panels , the third - row images show neovascular tufts ( NV , white ) , and the fourth - row images show the vaso - obliteration ( VO , white ) area . I . Quantitation of oxygen - induced retinal neovascularization in H ( n = 8 ) . J . Quantitation of retinal vaso - obliteration in H ( n = 8 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test , I , J ) . Scale bar : 500 μm ( H , unmagnified image ) , 150 μm ( H , magnified image ) . Data are represented as mean ± SEM .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Effect of CXCR2 inhibitor SB265610 (1 μM) on PGF2α-promoted sprouting of aortic rings. Yellow lines show the sprouting area. B. Quantitation of the sprouting areas in A (n=6). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test (B, Scale bar: 200 μm (A), image).Data are represented as mean ± SEM.C. mRNA expression of ELR+ CXC chemokines in retinas from OIR mice during postnatal days 12-17 (n=4-6). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test image).Data are represented as mean ± SEM.D. Effect of PTGFR deficiency in ECs on ELR+ CXC chemokine expression in retinas from OIR mice on postnatal day 16 (n=6). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test image).Data are represented as mean ± SEM.F. Retinal CXCL1 protein-expression levels in Cxcl1-expressing lentivirus-injected CKO mice (n=3). G. Quantitation of retinal CXCL1 protein in F (n=3). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test G, image).Data are represented as mean ± SEM.H. Representative images of oxygen-induced retinal angiogenesis in Cxcl1-expressing lentivirus-injected CKO mice on postnatal day 17. Green represents the isolectin B4-stained vessels, the second-row panels display the enlarged images of white boxes in the first-row panels, the third-row images show neovascular tufts (NV, white), and the fourth-row images show the vaso-obliteration (VO, white) area. I. Quantitation of oxygen-induced retinal neovascularization in H (n=8). J. Quantitation of retinal vaso-obliteration in H (n=8). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test , I, J). Scale bar: 500 μm (H, unmagnified image), 150 μm (H, magnified image).Data are represented as mean ± SEM.


Figure 6A . Heat map of differentially expressed transcription factors in HRMECs in response to 500 nM PGF2α ( n = 3 ) . B . Relative expression levels of FOS in retinal microvascular ECs from patients with PDR ( n = 4 ) compared with the corresponding expression levels from normal subjects ( n = 7 ) ( Gene Expression Omnibus Database dataset GSE94019 ) . TPM stands for " Transcripts per kilobase million . " Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test ( B , Data in B are expressed as the mean + SD . C . Effect of FOS knockdown on 500 nM PGF2α - induced CXCL8 expression in HRMECs ( n = 4 ) . D . Effect of FOS knockdown on 500 nM PGF2α - induced HRMEC CXCL8 secretion in the culture medium ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test C , D , Data in C , D , are represented as mean ± SEM . E . and F . Effect of RA ( 1 μM , AP - 1 dimer inhibitor ) , SN - 50 ( 100 μg / mL , NFκB inhibitor ) , and Inca - 6 ( 2 . 5 μM , NFAT inhibitor ) on 500 nM PGF2α - induced CXCL8 expression in HRMECs and HRMEC CXCL8 secretion in the culture medium ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test E , F , Data in E , F , are represented as mean ± SEM . G . Representative images of scratch - induced migration of 500 nM PGF2α - treated HRMECs with or without FOS knockdown . The yellow solid line indicates the original position of the scratch , the dashed line indicates the position of the cells after migration , and the arrows represent the migratory direction . H . Quantitation of the cell - migration distance in H ( n = 4 ) . I . Effect of FOS knockdown on 500 nM PGF2α - induced HRMEC proliferation ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test ( H , I Scale bar : 100 μm ( G ) Data in H , I , are represented as mean ± SEM . J . Representative images of tube formation by 500 nM PGF2α - treated HRMECs with or without FOS knockdown . K . Quantitation of total tube length , number of junctions , number of meshes , and percentage of mesh area in J ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test K , Scale bar : 200 μm ( J , Data in K , are represented as mean ± SEM . L . Fos mRNA levels in retinas from OIR mice during postnatal days 12 - 17 ( n = 3 - 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test L , Data in , L , are represented as mean ± SEM . M . Effect of PTGFR deficiency in ECs on Fos expression in the retina from OIR mice on postnatal day 16 ( n = 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test M , Data in M , are represented as mean ± SEM . N . Effect of FOS inhibitor SR11302 ( 1 μM ) on 500 nM PGF2α - promoted sprouting of mouse aortic rings . Data information : Scale bar : 200 μm N ) . O . Quantitation of sprouting areas in N ( n = 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test O ) . Data in O , are represented as mean ± SEM . P . Effect of FOS inhibitor SR11302 ( 1 μM ) on 500 nM PGF2α - promoted CXCL1 secretion in mouse aortic ring culture medium ( n = 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test P ) Data in P are represented as mean ± SEM .

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

Figure 6A. Heat map of differentially expressed transcription factors in HRMECs in response to 500 nM PGF2α (n=3).B. Relative expression levels of FOS in retinal microvascular ECs from patients with PDR (n=4) compared with the corresponding expression levels from normal subjects (n=7) (Gene Expression Omnibus Database dataset GSE94019). TPM stands for "Transcripts per kilobase million." Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test (B, Data in B are expressed as the mean + SD.C. Effect of FOS knockdown on 500 nM PGF2α-induced CXCL8 expression in HRMECs (n=4). D. Effect of FOS knockdown on 500 nM PGF2α-induced HRMEC CXCL8 secretion in the culture medium (n=4). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test C, D, Data in C, D, are represented as mean ± SEM. E. and F. Effect of RA (1 μM, AP-1 dimer inhibitor), SN-50 (100 μg/mL, NFκB inhibitor), and Inca-6 (2.5 μM, NFAT inhibitor) on 500 nM PGF2α-induced CXCL8 expression in HRMECs and HRMEC CXCL8 secretion in the culture medium (n=4). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test E, F, Data in E, F, are represented as mean ± SEM. G. Representative images of scratch-induced migration of 500 nM PGF2α-treated HRMECs with or without FOS knockdown. The yellow solid line indicates the original position of the scratch, the dashed line indicates the position of the cells after migration, and the arrows represent the migratory direction. H. Quantitation of the cell-migration distance in H (n=4). I. Effect of FOS knockdown on 500 nM PGF2α-induced HRMEC proliferation (n=4). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test (H, I Scale bar: 100 μm (G) Data in H, I, are represented as mean ± SEM. J. Representative images of tube formation by 500 nM PGF2α-treated HRMECs with or without FOS knockdown. K. Quantitation of total tube length, number of junctions, number of meshes, and percentage of mesh area in J (n=4). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test K, Scale bar: 200 μm (J, Data in K, are represented as mean ± SEM. L. Fos mRNA levels in retinas from OIR mice during postnatal days 12-17 (n=3-4). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test L, Data in , L, are represented as mean ± SEM. M. Effect of PTGFR deficiency in ECs on Fos expression in the retina from OIR mice on postnatal day 16 (n=6). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test M, Data in M, are represented as mean ± SEM. N. Effect of FOS inhibitor SR11302 (1 μM) on 500 nM PGF2α-promoted sprouting of mouse aortic rings. Data information: Scale bar: 200 μm N).O. Quantitation of sprouting areas in N (n=6). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test O). Data in O, are represented as mean ± SEM. P. Effect of FOS inhibitor SR11302 (1 μM) on 500 nM PGF2α-promoted CXCL1 secretion in mouse aortic ring culture medium (n=6). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test P) Data in P are represented as mean ± SEM.


Figure 7B . Effect of U73122 ( 10 μM ) , BAPTA ( 50 μM ) , Y27632 ( 5 μM ) , and Ly294002 ( 15 μM ) treatment on 500 nM PGF2α - induced FOS gene expression in HRMECs ( n = 4 ) . C . Effect of KN93 ( 10 μM ) and RO - 318220 ( 250 nM ) treatment on 500 nM PGF2α - induced FOS expression in HRMECs ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test ( B , C Data are represented as mean ± SEM . D . Effect of SB203580 ( 2 μM ) , PD98059 ( 20 μM ) , and SP600125 ( 60 nM ) treatment on 500 nM PGF2α - induced FOS gene expression in HRMECs ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test Data are represented as mean ± SEM . E . Effect of U73122 ( 10 μM ) , BAPTA ( 50 μM ) , and KN93 ( 10 μM ) treatment on 500 nM PGF2α - induced p38 phosphorylation in HRMECs . F . Quantitation of the ratio of p38 phosphorylation to total p38 protein in E ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by unpaired student ' s t - test ( F , Data are represented as mean ± SEM . G . Effect of U73122 ( 10 μM ) , BAPTA ( 50 μM ) , and SB203580 ( 2 μM ) on the phosphorylation of CAMK2 isoforms in 500 nM PGF2α - treated HRMECs . H . Quantitation of the ratio of CAMK2G phosphorylation to total CAMK2G in G ( n = 3 ) . I . Quantitation of the ratio of phosphorylated CAMK2D to total CAMK2D in G ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by unpaired student ' s t - test H , I , Data are represented as mean ± SEM . J . Effect of CAMK2G or CAMK2D knockdown on 500 nM PGF2α - triggered p38 phosphorylation in HRMECs . K . Quantitation of the ratio of p38 phosphorylation to total p38 protein in J ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by unpaired student ' s t - test Data are represented as mean ± SEM . L . Effect of CAMK2G or CAMK2D knockdown on 500 nM PGF2α - induced FOS expression in HRMECs ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test L ) Data are represented as mean ± SEM . M . Effect of U73122 ( 10 μM ) , BAPTA ( 50 μM ) , KN93 ( 10 μM ) , and SB203580 ( 2 μM ) on ELK - 1 phosphorylation in 500 nM PGF2α - treated HRMECs . N . Quantitation of the ratio of phosphorylated ELK - 1 to total ELK - 1 in M ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by unpaired student ' s t - test N ) . Data are represented as mean ± SEM .

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 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 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Effect of U73122 (10 μM), BAPTA (50 μM), Y27632 (5 μM), and Ly294002 (15 μM) treatment on 500 nM PGF2α-induced FOS gene expression in HRMECs (n=4). C. Effect of KN93 (10 μM) and RO-318220 (250 nM) treatment on 500 nM PGF2α-induced FOS expression in HRMECs (n=4). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test (B, C Data are represented as mean ± SEM.D. Effect of SB203580 (2 μM), PD98059 (20 μM), and SP600125 (60 nM) treatment on 500 nM PGF2α-induced FOS gene expression in HRMECs (n=4). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test Data are represented as mean ± SEM.E. Effect of U73122 (10 μM), BAPTA (50 μM), and KN93 (10 μM) treatment on 500 nM PGF2α-induced p38 phosphorylation in HRMECs. F. Quantitation of the ratio of p38 phosphorylation to total p38 protein in E (n=3). Data information: n.s. stands for "not significant." Data were analyzed by unpaired student's t-test (F, Data are represented as mean ± SEM.G. Effect of U73122 (10 μM), BAPTA (50 μM), and SB203580 (2 μM) on the phosphorylation of CAMK2 isoforms in 500 nM PGF2α-treated HRMECs. H. Quantitation of the ratio of CAMK2G phosphorylation to total CAMK2G in G (n=3). I. Quantitation of the ratio of phosphorylated CAMK2D to total CAMK2D in G (n=3). Data information: n.s. stands for "not significant." Data were analyzed by unpaired student's t-test H, I, Data are represented as mean ± SEM.J. Effect of CAMK2G or CAMK2D knockdown on 500 nM PGF2α-triggered p38 phosphorylation in HRMECs. K. Quantitation of the ratio of p38 phosphorylation to total p38 protein in J (n=3). Data information: n.s. stands for "not significant." Data were analyzed by unpaired student's t-test Data are represented as mean ± SEM.L. Effect of CAMK2G or CAMK2D knockdown on 500 nM PGF2α-induced FOS expression in HRMECs (n=4). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test L) Data are represented as mean ± SEM.M. Effect of U73122 (10 μM), BAPTA (50 μM), KN93 (10 μM), and SB203580 (2 μM) on ELK-1 phosphorylation in 500 nM PGF2α-treated HRMECs. N. Quantitation of the ratio of phosphorylated ELK-1 to total ELK-1 in M (n=3). Data information: n.s. stands for "not significant." Data were analyzed by unpaired student's t-test N). Data are represented as mean ± SEM.


Figure 8B . Representative images of OIR retinas in WT and Cxcr2 - / - mice with or without AL8810 treatment . The green color shows the isolectin B4 - stained vessels , the second - row panels display the enlarged images of white boxes in the first - row panels , the third - row images show neovascular tufts ( NV , white ) , and the fourth - row images show the vaso - obliteration ( VO , white ) area . C . Quantitation of oxygen - induced retinal neovascularization in WT and Cxcr2 - / - mice with or without AL8810 treatment ( n = 12 ) . D . Quantitation of retinal vaso - obliteration in WT and Cxcr2 - / - mice with or without AL8810 treatment ( n = 12 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test ( C , D ) Scale bar : 500 μm ( unmagnified image ) , 150 μm ( magnified image ) . Data are represented as mean ± SEM . E . Effect of AL8810 on retinal Fos expression in WT and Cxcr2 - / - OIR mice on postnatal day 16 ( n = 6 ) . F . Effect of AL8810 on retinal Cxcl1 expression in WT and Cxcr2 - / - OIR mice on postnatal day 16 ( n = 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Mann - Whitney test ( E , F ) . Data are represented as mean ± SEM .

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 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

Figure 8B. Representative images of OIR retinas in WT and Cxcr2-/- mice with or without AL8810 treatment. The green color shows the isolectin B4-stained vessels, the second-row panels display the enlarged images of white boxes in the first-row panels, the third-row images show neovascular tufts (NV, white), and the fourth-row images show the vaso-obliteration (VO, white) area. C. Quantitation of oxygen-induced retinal neovascularization in WT and Cxcr2-/- mice with or without AL8810 treatment (n=12). D. Quantitation of retinal vaso-obliteration in WT and Cxcr2-/- mice with or without AL8810 treatment (n=12). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test (C, D) Scale bar: 500 μm (unmagnified image), 150 μm (magnified image). Data are represented as mean ± SEM.E. Effect of AL8810 on retinal Fos expression in WT and Cxcr2-/- OIR mice on postnatal day 16 (n=6). F. Effect of AL8810 on retinal Cxcl1 expression in WT and Cxcr2-/- OIR mice on postnatal day 16 (n=6). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Mann-Whitney test (E, F). Data are represented as mean ± SEM.


Figure 1b , Representative confocal images of NeuN ( green ) and Npas4 ( red ) immunostaining performed 1h after the last injection of cocaine or saline ( scale bar : 50 µm ) . c , Quantifications of Npas4 - positive cells ( fold change normalized to the 30min saline control group ) in the NAc and dStr . Mean + SEM ; n = 4 mice per group . 10 images ( 5 per hemisphere ) per mouse for the NAc and 10 images ( 5 per hemisphere ) for the dStr . p * * < 0 . 01 , unpaired Student ' s t - test .

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

Figure 1b, Representative confocal images of NeuN (green) and Npas4 (red) immunostaining performed 1h after the last injection of cocaine or saline (scale bar: 50 µm).c, Quantifications of Npas4-positive cells (fold change normalized to the 30min saline control group) in the NAc and dStr. Mean + SEM; n = 4 mice per group. 10 images (5 per hemisphere) per mouse for the NAc and 10 images (5 per hemisphere) for the dStr. p** < 0.01, unpaired Student's t-test.


Figure 2c , MSN cultures at DIV11 were treated for 2h with either 10 µM of the D1 - agonist SKF - 38393 ( SKF ) alone or with SKF and 10 µM of the selective D1 - antagonist SCH - 23390 ( SCH ) . qPCR was performed to determine the relative mRNA levels of the immediate early genes cFos and Arc . Mean + SEM ; one - way ANOVA with Holm - Sidak post - hoc correction ; p * * < 0 . 01 , p * * * * < 0 . 0001 ; n = 3 independent cultures as biological replicates . d , MSNs were depolarized with potassium chloride ( HiK , 55 mM ) or treated with the D1 - agonist SKF - 38393 ( 10 µM ) for 1h , 2h or 4h . Subsequently , qPCR was performed to assess immediate early gene mRNA levels . Mean + SEM ; two - way ANOVA with Bonferroni post - hoc test ; indicated p - values for induction over control for each treatment ; p * < 0 . 05 , p * * < 0 . 01 , p * * * < 0 . 001 ; n = 4 independent cultures as biological replicates . g , Differential induction of Npas4 through HiK and not forskolin in hiPSC - derived forebrain organoids . Organoids were treated with HiK ( 55 mM ) or the PKA activator forskolin plus TTX ( Fsk , 10 µM ; TTX 1 µM ) for 1h before qPCR analysis was performed to assess mRNA levels of Npas4 and Arc . Mean + SEM ; n = 7 organoids in control condition , n = 5 organoids for HiK , n = 5 organoids for Fsk / TTX . One - way ANOVA with Tukey ' s post - hoc test , ns = not significant , p * < 0 . 05 , p * * * < 0 . 001 .

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 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

Figure 2c, MSN cultures at DIV11 were treated for 2h with either 10 µM of the D1-agonist SKF-38393 (SKF) alone or with SKF and 10 µM of the selective D1-antagonist SCH-23390 (SCH). qPCR was performed to determine the relative mRNA levels of the immediate early genes cFos and Arc. Mean + SEM; one-way ANOVA with Holm-Sidak post-hoc correction; p** < 0.01, p**** < 0.0001; n = 3 independent cultures as biological replicates.d, MSNs were depolarized with potassium chloride (HiK, 55 mM) or treated with the D1-agonist SKF-38393 (10 µM) for 1h, 2h or 4h. Subsequently, qPCR was performed to assess immediate early gene mRNA levels. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test; indicated p-values for induction over control for each treatment; p* < 0.05, p** < 0.01, p***<0.001; n = 4 independent cultures as biological replicates.g, Differential induction of Npas4 through HiK and not forskolin in hiPSC-derived forebrain organoids. Organoids were treated with HiK (55 mM) or the PKA activator forskolin plus TTX (Fsk, 10 µM; TTX 1 µM) for 1h before qPCR analysis was performed to assess mRNA levels of Npas4 and Arc. Mean + SEM; n = 7 organoids in control condition, n = 5 organoids for HiK, n = 5 organoids for Fsk/TTX. One-way ANOVA with Tukey's post-hoc test, ns = not significant, p* < 0.05, p***<0.001.


Figure 3MSN cultures were stimulated with HiK ( 55 mM ) for 1h in the presence of various pharmacologicals to dissect the synapse - to - nucleus cascades mediating transcription of Npas4 , Fosb and Arc . Values were normalized to the induced condition to compare for differences in induction between treatments . Mean + SEM and one - way ANOVA with Bonferroni post - hoc correction were used in all experiments . p * < 0 . 05 , p * * < 0 . 01 , p * * * < 0 . 001 , p * * * * < 0 . 0001 . n - number indicated in each panel . a , co - incubation with the NMDA receptor blocker APV ( 100 mM , applied 5min before ) reveals that all IEG inductions through HiK in MSNs depend on NDMA receptor activity . Mean + SEM ; n = 4 independent cultures as biological replicates . b , blockade of voltage - dependent calcium channels through verapamil ( 30 µM , 5min before ) and TTX ( 1 µM , 5min , to prevent verapamil - induced bursting ) leads to significant reduction in gene - inductions for Npas4 and Fosb . Mean + SEM ; n = 4 independent cultures as biological replicates . c , inhibition of ERK1 / 2 through UO126 ( 10 µM , 20min before ) leads to significant blockade of mRNA induction for Arc and Fosb , but not Npas4 . Mean + SEM ; n = 3 independent cultures as biological replicates . d , inhibition of p38alpha and p38beta through SB203580 or SB202190 respectively ( both 10 µM and 20min before ) leads to significant reductions in mRNA induction for Arc and Fosb , but not Npas4 . Mean + SEM ; n = 3 independent cultures as biological replicates . e , blockade of PKA activity through H - 89 ( 10 µM , 20min before ) leads to reduced induction of Arc , unaffected induction of Fosb and a superinduction of Npas4 . Mean + SEM ; n = 3 independent cultures as biological replicates . f , inhibition of calcineurin through co - application of Cyclosporin and FK506 ( 1 µM each , 20min before ) leads to unaffected induction of mRNAs for Arc and Fosb , but significantly reduced levels of Npas4 . Mean + SEM ; n = 3 independent cultures as biological replicates . MSN cultures were stimulated with HiK ( 55 mM ) for 1h in the presence of various pharmacologicals to dissect the synapse - to - nucleus cascades mediating transcription of Npas4 , Fosb and Arc . Values were normalized to the induced condition to compare for differences in induction between treatments . Mean + SEM and one - way ANOVA with Bonferroni post - hoc correction were used in all experiments . p * < 0 . 05 , p * * < 0 . 01 , p * * * < 0 . 001 , p * * * * < 0 . 0001 . n - number indicated in each panel . g , blockade of CaMKII / IV through application of KN - 93 ( 1 µM , 20min before ) leads to significantly reduced mRNA inductions of Arc , Fosb and Npas4 . Mean + SEM ; n = 4 independent cultures as biological replicates . h , co - application of HiK and SKF reveals synergistic effects on mRNA induction of Fosb but not Arc or Npas4 , Mean + SEM ; n = 6 independent cultures as biological replicates . investigating the role of nuclear calcium signaling in mRNA induction of Arc , Fosb and Npas4 . k , immunohistochemical analysis of MSNs infected with mCherry - NLS as control or the competitive nuclear Ca2 + / CaM antagonist CaMBP4 - mCherry . Infection on DIV4 , fixation on DIV11 . D32 = anti - Darpp - 32 . scale bar = 10 µm . l , MSNs expressing rAAV - mCherry - control or CaMBP4 ( BP4 ) were treated with 55mM HiK for 1h and qPCR analysis was performed for the indicated IEGs . Mean + SEM ; two - way ANOVA with Bonferroni post - hoc test , ns = not significant , p * * * * < 0 . 0001 . n = 5 independent cultures as biological replicates . m , immunohistochemical analysis of MSNs infected with mCherry - NLS as control or the nuclear Ca2 + - buffer Parvalbumin - NLS - mCherry . Infection on DIV4 , fixation on DIV11 . D32 = anti - Darpp - 32 . n , MSNs expressing rAAV - mCherry - control or PV - NLS were treated with 55mM HiK for 1h and qPCR analysis was performed for the indicated IEGs . Mean + SEM ; two - way ANOVA with Bonferroni post - hoc test , p * < 0 . 05 , p * * < 0 . 01 , p * * * * < 0 . 0001 . n = 3 independent cultures as biological replicates .

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

Figure 3MSN cultures were stimulated with HiK (55 mM) for 1h in the presence of various pharmacologicals to dissect the synapse-to-nucleus cascades mediating transcription of Npas4, Fosb and Arc. Values were normalized to the induced condition to compare for differences in induction between treatments. Mean + SEM and one-way ANOVA with Bonferroni post-hoc correction were used in all experiments. p* < 0.05, p** < 0.01, p***<0.001, p****<0.0001. n-number indicated in each panel. a, co-incubation with the NMDA receptor blocker APV (100 mM, applied 5min before) reveals that all IEG inductions through HiK in MSNs depend on NDMA receptor activity. Mean + SEM; n = 4 independent cultures as biological replicates. b, blockade of voltage-dependent calcium channels through verapamil (30 µM, 5min before) and TTX (1 µM, 5min, to prevent verapamil-induced bursting) leads to significant reduction in gene-inductions for Npas4 and Fosb. Mean + SEM; n =4 independent cultures as biological replicates. c, inhibition of ERK1/2 through UO126 (10 µM, 20min before) leads to significant blockade of mRNA induction for Arc and Fosb, but not Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. d, inhibition of p38alpha and p38beta through SB203580 or SB202190 respectively (both 10 µM and 20min before) leads to significant reductions in mRNA induction for Arc and Fosb, but not Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. e, blockade of PKA activity through H-89 (10 µM, 20min before) leads to reduced induction of Arc, unaffected induction of Fosb and a superinduction of Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. f, inhibition of calcineurin through co-application of Cyclosporin and FK506 (1 µM each, 20min before) leads to unaffected induction of mRNAs for Arc and Fosb, but significantly reduced levels of Npas4. Mean + SEM; n = 3 independent cultures as biological replicates.MSN cultures were stimulated with HiK (55 mM) for 1h in the presence of various pharmacologicals to dissect the synapse-to-nucleus cascades mediating transcription of Npas4, Fosb and Arc. Values were normalized to the induced condition to compare for differences in induction between treatments. Mean + SEM and one-way ANOVA with Bonferroni post-hoc correction were used in all experiments. p* < 0.05, p** < 0.01, p***<0.001, p****<0.0001. n-number indicated in each panel. g, blockade of CaMKII/IV through application of KN-93 (1 µM, 20min before) leads to significantly reduced mRNA inductions of Arc, Fosb and Npas4. Mean + SEM; n = 4 independent cultures as biological replicates. h, co-application of HiK and SKF reveals synergistic effects on mRNA induction of Fosb but not Arc or Npas4, Mean + SEM; n = 6 independent cultures as biological replicates. investigating the role of nuclear calcium signaling in mRNA induction of Arc, Fosb and Npas4. k, immunohistochemical analysis of MSNs infected with mCherry-NLS as control or the competitive nuclear Ca2+/CaM antagonist CaMBP4-mCherry. Infection on DIV4, fixation on DIV11. D32 = anti-Darpp-32. scale bar = 10 µm. l, MSNs expressing rAAV-mCherry-control or CaMBP4 (BP4) were treated with 55mM HiK for 1h and qPCR analysis was performed for the indicated IEGs. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test, ns = not significant, p****<0.0001. n = 5 independent cultures as biological replicates. m, immunohistochemical analysis of MSNs infected with mCherry-NLS as control or the nuclear Ca2+-buffer Parvalbumin-NLS-mCherry. Infection on DIV4, fixation on DIV11. D32 = anti-Darpp-32. n, MSNs expressing rAAV-mCherry-control or PV-NLS were treated with 55mM HiK for 1h and qPCR analysis was performed for the indicated IEGs. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test, p* < 0.05, p** < 0.01, p****<0.0001. n = 3 independent cultures as biological replicates.


Figure 4e , Npas4 controls activity - dependent upregulation of genes for glutamatergic synapse formation and Ca2 + channels . MSNs were infected with rAAVs expressing shRNA - scrambled ( control ) or shNpas4 ( Npas4 knockdown ) on DIV5 . On DIV 12 , cultures were stimulated with HiK ( 55 mM ) for 1h or 6h and mRNA levels were analysed by qPCR . mRNA level = fold change in mRNA compared to shscrambled unstimulated control . Mean + SEM ; two - way ANOVA with Bonferroni post - hoc test , p * < 0 . 05 , p * * * < 0 . 001 . n = 5 independent cultures as biological replicates .

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 4e, Npas4 controls activity-dependent upregulation of genes for glutamatergic synapse formation and Ca2+ channels. MSNs were infected with rAAVs expressing shRNA-scrambled (control) or shNpas4 (Npas4 knockdown) on DIV5. On DIV 12, cultures were stimulated with HiK (55 mM) for 1h or 6h and mRNA levels were analysed by qPCR. mRNA level = fold change in mRNA compared to shscrambled unstimulated control. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test, p* < 0.05, p***<0.001. n = 5 independent cultures as biological replicates.


Figure 5e , Confocal images of tdTomato - labelled D1 - MSNs expressing shscr - GFP or shNpas4 - GFP ( left panels ; scale bar : 20 µm ) . 3D volume - renderings of MSN dendrites from the NAc shell ( right panels ; scale bar : 2 µm ) . g , Confocal images of tdTomato - labelled D2 - MSNs expressing shscr - GFP or shNpas4 - GFP ( left panels ; scale bar : 20 µm ) . 3D volume - renderings of MSN dendrites from the NAc shell ( right panels ; scale bar : 2 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 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

Figure 5e, Confocal images of tdTomato-labelled D1-MSNs expressing shscr-GFP or shNpas4-GFP (left panels; scale bar: 20 µm). 3D volume-renderings of MSN dendrites from the NAc shell (right panels; scale bar: 2 µm).g, Confocal images of tdTomato-labelled D2-MSNs expressing shscr-GFP or shNpas4-GFP (left panels; scale bar: 20 µm). 3D volume-renderings of MSN dendrites from the NAc shell (right panels; scale bar: 2 µm).


Figure 6a - d , Scatter plot of passive and active membrane properties of MSNs in ex vivo slice preparations from shRNA - AAV - infected mice , plotted for MSNs as D1 - positive ( D1 + ) , D1 - negative ( D1 - ) and those two populations pooled ( all ) . a , rheobase ; mean ± SEM . b , firing rate estimated at twice the value of the rheobase ; mean ± SEM . c , input / output gain values recorded in shscr - or shNpas4 - injected mice , mean ± SEM . The increased rheobase and lower input - output gain indicate a lower excitability for MSNs in the Npas4 - knockdown group . d , facilitation at corticostriatal synapses examined in MSNs with paired - pulse stimulation at 20 and 4 Hz . Representative current traces and scatter plots of paired - pulse experiments illustrate a potent facilitation in MSNs in mice injected with scr - shRNA and a significant decrease of the facilitation in mice injected with shNpas4 , with a stronger effect in D1 - negative MSNs . Mean ± SEM . Data information : For n - numbers , please see Table EV1 . Mann - Whitney - Test , p * < 0 . 05 , p * * < 0 . 01 .

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

Figure 6a-d, Scatter plot of passive and active membrane properties of MSNs in ex vivo slice preparations from shRNA-AAV-infected mice, plotted for MSNs as D1-positive (D1+), D1-negative (D1-) and those two populations pooled (all). a, rheobase; mean ± SEM. b, firing rate estimated at twice the value of the rheobase; mean ± SEM. c, input/output gain values recorded in shscr- or shNpas4-injected mice, mean ± SEM. The increased rheobase and lower input-output gain indicate a lower excitability for MSNs in the Npas4-knockdown group. d, facilitation at corticostriatal synapses examined in MSNs with paired-pulse stimulation at 20 and 4 Hz. Representative current traces and scatter plots of paired-pulse experiments illustrate a potent facilitation in MSNs in mice injected with scr-shRNA and a significant decrease of the facilitation in mice injected with shNpas4, with a stronger effect in D1-negative MSNs. Mean ± SEM. Data information: For n-numbers, please see Table EV1. Mann-Whitney-Test, p*<0.05, p**<0.01.


Figure 7b , cocaine - induced hyperlocomotion is attenuated by Npas4 - knockdown while locomotor behaviour after saline injection is unaffected on all days . Mean + SEM ; two - way ANOVA followed by Bonferroni post hoc test . p * < 0 . 05 , p * * < 0 . 01 , ns = not significant . n = 12 - 16 animals per condition .

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

Figure 7b, cocaine-induced hyperlocomotion is attenuated by Npas4-knockdown while locomotor behaviour after saline injection is unaffected on all days. Mean + SEM; two-way ANOVA followed by Bonferroni post hoc test. p* < 0.05, p** < 0.01, ns = not significant. n = 12 - 16 animals per condition.


Figure 1 ( B - D ) Quantification of normalized bouton number ( ( B ) , total number of boutons / muscle surface area ( μm2 × 1 , 000 ) ) , normalized axon length ( C ) and normalized branching ( D ) of NMJ 6 / 7 in A2 - A3 of the indicated genotypes . Bars show mean ± s . e . m . Multiple comparisons were performed using one - way ANOVA with a post - hoc Sidak - Bonferroni correction . ( n . s . = not significant ; p < 0 . 05 = * ; p < 0 . 001 = * * * ; p < 0 . 0001 = * * * * Data information : In ( B - D bars are labelled with the number of replicates .

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 1(B-D) Quantification of normalized bouton number ((B), total number of boutons/muscle surface area (μm2 × 1,000)), normalized axon length (C) and normalized branching (D) of NMJ 6/7 in A2-A3 of the indicated genotypes. Bars show mean ± s.e.m. Multiple comparisons were performed using one-way ANOVA with a post-hoc Sidak-Bonferroni correction. (n.s. = not significant; p<0.05 = *; p<0.001= ***; p<0.0001=**** Data information: In (B-D bars are labelled with the number of replicates.


Figure 2 ( B , C ) Western Blot showing the protein enrichments upon pulldown of the same probes as in ( A ) incubated with increasing concentrations of purified recombinant GST - Ythdf ( B ) and His - NT - Fmr1 proteins ( C ) . Quantification of the m6A / A signal intensity is shown below the blot as median + / - SEM of the triplicates . Both proteins bind more efficiently upon methylation . ( D ) Results of fluorescence polarization ( FP ) assay using Cy5 labelled RNA probes containing four AAACU m6A consensus sites , with or without the methylation , incubated with GST - Ythdf and His - NT - Fmr1 . The relative FP values from three independent experiments including standard deviation were plotted using GraphPad Prism 8 . Binding constants ( kd values ) were determined by fitting a Michaelis - Menten non - linear regression onto the relative FP values in GraphPad Prism 8 , if applicable . The respective binding constants are given in the table next to the graphs .

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

Figure 2(B, C) Western Blot showing the protein enrichments upon pulldown of the same probes as in (A) incubated with increasing concentrations of purified recombinant GST-Ythdf (B) and His-NT-Fmr1 proteins (C). Quantification of the m6A/A signal intensity is shown below the blot as median +/- SEM of the triplicates. Both proteins bind more efficiently upon methylation.(D) Results of fluorescence polarization (FP) assay using Cy5 labelled RNA probes containing four AAACU m6A consensus sites, with or without the methylation, incubated with GST-Ythdf and His-NT-Fmr1. The relative FP values from three independent experiments including standard deviation were plotted using GraphPad Prism 8. Binding constants (kd values) were determined by fitting a Michaelis-Menten non-linear regression onto the relative FP values in GraphPad Prism 8, if applicable. The respective binding constants are given in the table next to the graphs.


Figure 3 ( B - D ) Quantification of normalized bouton number ( ( B ) , total number of boutons / muscle surface area ( μm2 × 1 , 000 ) ) , normalized axon length ( C ) and normalized branching ( D ) of NMJ 6 / 7 in A2 - A3 of the indicated genotypes . Bars show mean ± s . e . m . Multiple comparisons were performed using one - way ANOVA with a post - hoc Sidak - Bonferroni correction ( n . s . = not significant ; p < 0 . 05 = * ; p < 0 . 01 = * * ; p < 0 . 001 = * * * ; p < 0 . 0001 = * * * * ) . Data information : In ( B - D , , bars are labelled with the number of replicates .

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

Figure 3(B-D) Quantification of normalized bouton number ((B), total number of boutons/muscle surface area (μm2 × 1,000)), normalized axon length (C) and normalized branching (D) of NMJ 6/7 in A2-A3 of the indicated genotypes. Bars show mean ± s.e.m. Multiple comparisons were performed using one-way ANOVA with a post-hoc Sidak-Bonferroni correction (n.s. = not significant; p<0.05 = *; p<0.01 = **; p<0.001= ***; p<0.0001=****). Data information: In (B-D, , bars are labelled with the number of replicates.


Figure 4 ( B ) Co - immunoprecipitation experiment in S2R + cells co - expressing FlagMyc - tagged Ythdf and Myc - tagged Fmr1 . Ythdf was used as a bait via its Flag tag . The lysate was treated with Benzonase as indicated to remove interactions enabled by RNA .

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

Figure 4(B) Co-immunoprecipitation experiment in S2R+ cells co-expressing FlagMyc-tagged Ythdf and Myc-tagged Fmr1. Ythdf was used as a bait via its Flag tag. The lysate was treated with Benzonase as indicated to remove interactions enabled by RNA.


Figure 5Pulldown of biotinylated RNA probes of ( A ) repetitive GGACU sequences incubated with 4 pmol His - NT - Fmr1 and increasing concentrations of GST - Ythdf The indicated P - values were determined by unpaired , two - tailed Student ' s t - test .

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

Figure 5Pulldown of biotinylated RNA probes of (A) repetitive GGACU sequences incubated with 4 pmol His-NT-Fmr1 and increasing concentrations of GST-Ythdf The indicated P-values were determined by unpaired, two-tailed Student's t-test.


Figure 6 ( A ) Representative Western blot analysis of protein extracts of late pupae heads ( 85 - 95h ) from control , Ythdf # 0 / Ythdf # 0 and Fmr1 ∆ 50 / Fmr1 ∆ 50 flies . The membranes were probed with anti - Profilin or anti - Actin antibodies . ( B ) Quantification of Profilin protein levels ( mean ± SD ) obtained from three independent protein - extraction and Western blot analysis as in ( A ) . Quantification was performed using Fiji . p < 0 . 005 = * * ; p < 0 . 05 = * , measured with unpaired 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

Figure 6(A) Representative Western blot analysis of protein extracts of late pupae heads (85-95h) from control, Ythdf#0/ Ythdf#0 and Fmr1∆50/Fmr1∆50 flies. The membranes were probed with anti-Profilin or anti-Actin antibodies. (B) Quantification of Profilin protein levels (mean ± SD) obtained from three independent protein-extraction and Western blot analysis as in (A). Quantification was performed using Fiji. p<0.005 = **; p<0.05 = *, measured with unpaired t-test.


Figure 7 ( D ) Quantification of futsch mRNA levels obtained from isolated 3rd instar larvae brains of the indicated genotypes via real - time PCR analysis . Bars show average ± SD of biological triplicates . Bars are labelled with the number of replicates . ( F ) Quantification of normalized bouton number ( total number of boutons / muscle surface area ( μm2 × 1 , 000 ) ) of NMJ 6 / 7 in hemisegment A3 of the indicated genotypes . Bars show mean ± s . e . m . Multiple comparisons were performed using one - way ANOVA with a post - hoc Sidak - Bonferroni correction ( n . s . = not significant ; p < 0 . 01 = * * ; p < 0 . 0001 = * * * * ) . Bars are labelled with the number of replicates .

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

Figure 7(D) Quantification of futsch mRNA levels obtained from isolated 3rd instar larvae brains of the indicated genotypes via real-time PCR analysis. Bars show average ± SD of biological triplicates. Bars are labelled with the number of replicates.(F) Quantification of normalized bouton number (total number of boutons/muscle surface area (μm2 × 1,000)) of NMJ 6/7 in hemisegment A3 of the indicated genotypes. Bars show mean ± s.e.m. Multiple comparisons were performed using one-way ANOVA with a post-hoc Sidak-Bonferroni correction (n.s. = not significant; p<0.01 = **; p<0.0001=****). Bars are labelled with the number of replicates.


Figure 8E - G ) Representative Western blot analysis of the protein level ( E ) and quantification of the translation efficiency of S2R + cells transfected with the wildtype or mutated futsch reporter constructs upon control or Mettl3 KD ( F ) or Ythdf KD ( G ) . LacZ dsRNA was used for the control KD . The translation efficiency was calculated as the ratio of the relative GFP protein level and GFP mRNA level . mCherry served as a normalization control for both the protein and mRNA level . Bars show mean ± s . e . m of biological triplicates . P values were determined with a Student ' s t - test . ( n . s . = not significant ; p < 0 . 05 = * ; p < 0 . 01 = * * ) .

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

Figure 8E-G) Representative Western blot analysis of the protein level (E) and quantification of the translation efficiency of S2R+ cells transfected with the wildtype or mutated futsch reporter constructs upon control or Mettl3 KD (F) or Ythdf KD (G). LacZ dsRNA was used for the control KD. The translation efficiency was calculated as the ratio of the relative GFP protein level and GFP mRNA level. mCherry served as a normalization control for both the protein and mRNA level. Bars show mean ± s.e.m of biological triplicates. P values were determined with a Student's t-test. (n.s. = not significant; p<0.05 = *; p<0.01 = **).


Figure 2 ( B ) Pol II ChIP - seq was performed in six different tissues shown in the left panel ( scale bar - 100 µm ) . The middle and the right panels show the read count normalized Pol II ChIP - seq tracks ( RPM ) from the six tissues at individual genes . For each gene , grey and red tracks indicate non - expressing tissues and expressing tissue , respectively . The middle panel shows the Pol II profile at two non - paused genes , which have Pol II only in the expressing tissues . The expression is limited to specific tissues as shown in the in situ images from BDGP . The right panel shows the Pol II profile at a paused gene , which has Pol II in all observed tissues , although the expression is limited to specific tissues as shown in the in situ images from BDGP . Paused Pol II is generally highest in the tissue with the highest expression . We also found systematic differences between samples , thus some tissues have generally higher enrichments than others , presumably because they are easier to crosslink .

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

Figure 2(B) Pol II ChIP-seq was performed in six different tissues shown in the left panel (scale bar - 100 µm). The middle and the right panels show the read count normalized Pol II ChIP-seq tracks (RPM) from the six tissues at individual genes. For each gene, grey and red tracks indicate non-expressing tissues and expressing tissue, respectively. The middle panel shows the Pol II profile at two non-paused genes, which have Pol II only in the expressing tissues. The expression is limited to specific tissues as shown in the in situ images from BDGP. The right panel shows the Pol II profile at a paused gene, which has Pol II in all observed tissues, although the expression is limited to specific tissues as shown in the in situ images from BDGP. Paused Pol II is generally highest in the tissue with the highest expression. We also found systematic differences between samples, thus some tissues have generally higher enrichments than others, presumably because they are easier to crosslink.


Figure 3 ( C , D ) Normalized gene expression levels ( read count normalized for each cell , log2 ) in different tissues , from the scRNA - seq experiment , for a TATA group gene , and a highly paused group gene , are shown . ( C ) The TATA gene , Ccp84Aa , shows noisy expression in the epidermis , without detectable background expression in non - expressing tissues . ( D ) The highly paused gene , Gip , shows very robust expression in crystal cells , but has high background expression in the non - expressing tissues .

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

Figure 3(C,D) Normalized gene expression levels (read count normalized for each cell, log2) in different tissues, from the scRNA-seq experiment, for a TATA group gene, and a highly paused group gene, are shown. (C) The TATA gene, Ccp84Aa, shows noisy expression in the epidermis, without detectable background expression in non-expressing tissues. (D) The highly paused gene, Gip, shows very robust expression in crystal cells, but has high background expression in the non-expressing tissues.


Figure 4C , D ) ATAC - seq chromatin accessibility was measured in different tissues isolated from 14 - 17 h embryos using INTACT . ( C ) Read - count normalized ATAC ( RPM ) signal at individual genes from the TATA group ( Ccp84Aa ) and highly paused gene group ( ect ) are shown . ( D ) Read - count normalized ATAC signals ( RPM ) from different tissues were calculated for each gene from 150 bp upstream of the TSS to the TSS . Paused genes show higher average accessibility across all tissues compared to the TATA genes ( Wilcoxon two - sided test , * P < 2 . 2 * 10 - 16 ) .

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

Figure 4C, D) ATAC-seq chromatin accessibility was measured in different tissues isolated from 14-17 h embryos using INTACT. (C) Read-count normalized ATAC (RPM) signal at individual genes from the TATA group (Ccp84Aa) and highly paused gene group (ect) are shown. (D) Read-count normalized ATAC signals (RPM) from different tissues were calculated for each gene from 150 bp upstream of the TSS to the TSS. Paused genes show higher average accessibility across all tissues compared to the TATA genes (Wilcoxon two-sided test, *P <2.2*10-16).


Figure 1 ( B ) Scatterplots represent SILAC / SUMO - IP quantification after EGF treatment from three biological replicates . Each dot represents a protein that is either present in the same ratio between untreated and EGF treated cells ( dark blue dots ) or is significantly more present in one of the samples ( red and yellow dots ) . ( C ) Left panel : Bar graph depicting mass spectrometry results of 11 proteins with altered SUMOylation ( significant hits with P < 0 . 0001 ) , as well as the three non - changing proteins SUMO2 , RanGAP1 and TRIM28 . Right panel : Proteins highlighted in bold in the left panel were validated by SUMO - IP / immunoblotting from serum starved HeLa cells without or with 10 min EGF stimulation . ( D ) Time course experiment : serum starved HeLa cells were treated with 100 nM EGF , samples were harvested at indicated times and subjected to SUMO2 IP followed by immunoblotting with the indicated antibodies . IRF2BP1 , IRF2BP2 and TRIM24 are rapidly but transiently deSUMOylated upon EGF treatment .

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

Figure 1(B) Scatterplots represent SILAC/SUMO-IP quantification after EGF treatment from three biological replicates. Each dot represents a protein that is either present in the same ratio between untreated and EGF treated cells (dark blue dots) or is significantly more present in one of the samples (red and yellow dots).(C) Left panel: Bar graph depicting mass spectrometry results of 11 proteins with altered SUMOylation (significant hits with P<0.0001), as well as the three non-changing proteins SUMO2, RanGAP1 and TRIM28. Right panel: Proteins highlighted in bold in the left panel were validated by SUMO-IP/immunoblotting from serum starved HeLa cells without or with 10 min EGF stimulation.(D) Time course experiment: serum starved HeLa cells were treated with 100 nM EGF, samples were harvested at indicated times and subjected to SUMO2 IP followed by immunoblotting with the indicated antibodies. IRF2BP1, IRF2BP2 and TRIM24 are rapidly but transiently deSUMOylated upon EGF treatment.


Figure 2 ( B ) Identification of the endogenous SUMO - sites in IRF2BP1 ( left panel ) and IRF2BP2 ( right panel ) . HeLa cells were transfected with HA - tagged wildtype ( WT ) or mutant ( KR ) proteins , endogenous SUMO - IPs were performed and the HA - signal was analyzed by immunoblotting . Mutation of the C - terminal SUMO - site abolished SUMOylation of IRF2BP1 and IRF2BP2 in HeLa cells . ( E ) Stable HeLa cells expressing pIRES - empty vector , IRF2BP1 WT or IRF2BP1 K579R were treated with siRNA against endogenous IRF2BP1 or non - targeting ( nt ) siRNA . Exogenous siRNA - resistant IRF2BP1 was expressed at low levels similar to endogenous IRF2BP1 . * refers to an unspecific band . ( F ) Wt and mutant IRF2BP1 localizes in the nucleus . After knockdown of endogenous IRF2BP1 , stable IRF2BP1 ( WT or K579R ) cell lines were immunostained for IRF2BP1 . Exogenous IRF2BP1 variants show a similar nuclear localization . Scale bar = 10 µm . ( G ) IRF2BP1 wt and mutant associate with chromatin to a similar extent . HeLa cells were lysed in 0 . 075 % NP40 ( Input ) . After centrifugation , the nuclei were incubated and vortexed with a nuclear extract ( NE ) buffer containing 170 mM NaCl . The eluates were collected and the procedure was repeated using a NE buffer with higher salt concentrations , first 290 mM , then 420 mM . Wildtype IRF2BP1 , the SUMO - deficient K579R mutant and the SUMOylated wildtype form ( * ) all behave similarly .

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

Figure 2(B) Identification of the endogenous SUMO-sites in IRF2BP1 (left panel) and IRF2BP2 (right panel). HeLa cells were transfected with HA-tagged wildtype (WT) or mutant (KR) proteins, endogenous SUMO-IPs were performed and the HA-signal was analyzed by immunoblotting. Mutation of the C-terminal SUMO-site abolished SUMOylation of IRF2BP1 and IRF2BP2 in HeLa cells.(E) Stable HeLa cells expressing pIRES-empty vector, IRF2BP1 WT or IRF2BP1 K579R were treated with siRNA against endogenous IRF2BP1 or non-targeting (nt) siRNA. Exogenous siRNA-resistant IRF2BP1 was expressed at low levels similar to endogenous IRF2BP1. * refers to an unspecific band.(F) Wt and mutant IRF2BP1 localizes in the nucleus. After knockdown of endogenous IRF2BP1, stable IRF2BP1 (WT or K579R) cell lines were immunostained for IRF2BP1. Exogenous IRF2BP1 variants show a similar nuclear localization. Scale bar = 10 µm.(G) IRF2BP1 wt and mutant associate with chromatin to a similar extent. HeLa cells were lysed in 0.075 % NP40 (Input). After centrifugation, the nuclei were incubated and vortexed with a nuclear extract (NE) buffer containing 170 mM NaCl. The eluates were collected and the procedure was repeated using a NE buffer with higher salt concentrations, first 290 mM, then 420 mM. Wildtype IRF2BP1, the SUMO-deficient K579R mutant and the SUMOylated wildtype form (*) all behave similarly.


Figure 3 ( A ) Stable IRF2BP1 cell lines ( knocked - down for endogenous IRF2BP1 ) were used to perform a microarray experiment under serum starvation and upon treatment with EGF for 1 hour . A subset of 38 EGF - dependent genes are differentially regulated in IRF2BP1 wildtype cells compared to IRF2BP1 K579R cell lines ( at least 1 . 5 - fold ) . Among them are DUSP1 ( arrow ) , ATF3 , Fos and Egr2 . Each microarray was performed in triplicates , the bars show log2 values of the fold changes for wt cells ( black bars ) and for KR cells ( grey bars ) . ( B ) Chromatin IP reveals association of human IRF2BP1 with the proximal DUSP1 promoter in HeLa cells . Gene architecture of human DUSP1 . The primers at - 243 / - 67 ( " - 67 " ) , - 473 / - 224 ( " - 224 " ) and - 1170 / - 961 ( " - 916 " ) relative to the TSS were used for ChIP experiments . IRF2BP1 and IRF2BP2 bind to the promoter region of DUSP1 between nucleotides - 243 and - 67 . ( C ) ChIP / qPCR experiments reveal preferential IRF2BP1 binding to the promoter region of DUSP1 between - 243 and - 67 . Data show mean ( bar ) and individual data points from 2 biological replicates . ( D ) IRF2BP1 wildtype and the K579R mutant both bind the DUSP1 promoter in the absence or presence of EGF . Stable cell lines expressing IRF2BP1 wildtype or K579R were knocked - down for endogenous IRF2BP1 , followed by IRF2BP1 ChIP and DUSP1 qPCR of its promoter region - 243 and - 67 . Data show means + / - SEM from 3 biological replicates . ( E ) Q - PCR data after EGF treatment in IR2BP1 wildtype and K579R cell lines after knockdown of the endogenous IRF2BP1 . Data show means + / - SEM from 3 biological replicates . ( F ) IRF2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1 were analysed for DUSP1 transcription after EGF treatment by q - PCR ( left panel ) , data show means + / − SEM from 5 biological replicates ( left panel ) , and IRF2BP1 protein levels . As expected , IRF2BP1 V578A is not SUMOylated ( right panel ) . * refers to an unspecific band . ( G ) Q - PCR data for immediate early genes after EGF treatment in the IR2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1 . Data show means + / − SEM from 4 biological replicates .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3(A) Stable IRF2BP1 cell lines (knocked-down for endogenous IRF2BP1) were used to perform a microarray experiment under serum starvation and upon treatment with EGF for 1 hour. A subset of 38 EGF-dependent genes are differentially regulated in IRF2BP1 wildtype cells compared to IRF2BP1 K579R cell lines (at least 1.5-fold). Among them are DUSP1 (arrow), ATF3, Fos and Egr2. Each microarray was performed in triplicates, the bars show log2 values of the fold changes for wt cells (black bars) and for KR cells (grey bars).(B) Chromatin IP reveals association of human IRF2BP1 with the proximal DUSP1 promoter in HeLa cells. Gene architecture of human DUSP1. The primers at -243/-67 ("-67"), -473/-224 ("-224") and -1170/-961 ("-916") relative to the TSS were used for ChIP experiments. IRF2BP1 and IRF2BP2 bind to the promoter region of DUSP1 between nucleotides -243 and -67.(C) ChIP/qPCR experiments reveal preferential IRF2BP1 binding to the promoter region of DUSP1 between -243 and -67. Data show mean (bar) and individual data points from 2 biological replicates.(D) IRF2BP1 wildtype and the K579R mutant both bind the DUSP1 promoter in the absence or presence of EGF. Stable cell lines expressing IRF2BP1 wildtype or K579R were knocked-down for endogenous IRF2BP1, followed by IRF2BP1 ChIP and DUSP1 qPCR of its promoter region -243 and -67. Data show means +/- SEM from 3 biological replicates.(E) Q-PCR data after EGF treatment in IR2BP1 wildtype and K579R cell lines after knockdown of the endogenous IRF2BP1. Data show means +/- SEM from 3 biological replicates.(F) IRF2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1 were analysed for DUSP1 transcription after EGF treatment by q-PCR (left panel), data show means +/− SEM from 5 biological replicates (left panel), and IRF2BP1 protein levels. As expected, IRF2BP1 V578A is not SUMOylated (right panel). * refers to an unspecific band.(G) Q-PCR data for immediate early genes after EGF treatment in the IR2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1. Data show means +/− SEM from 4 biological replicates.


Figure 4 ( A ) Immunoblotting of HeLa lysates at indicated times after EGF treatment reveals enhanced DUSP1 and ATF3 expression upon knockdown of IRF2BP1 . Uba2 and beta - actin serve as independent loading controls . ( C ) Wound healing assay : HeLa cells were incubated with nt or IRF2BP1 siRNA for 2 days , grown to 90 % confluency and analysed for wound closure with or without addition of 100 ng / ml EGF . Data show means of the relative wound density + / - SEM from 4 biological replicates . ( D ) Proliferation assay : HeLa cells upon knockdown of IRF2BP1 and IRF2BP1 were analysed for cell density over 6 days in growth medium . One representative biological experiment is shown with means + / - SEM from 5 technical replicates . ( E ) IRF2BP1 , IRF2BP2 , IRF2BP1 + IRF2BP2 were knocked - down in HeLa cells for 72 hours and gene expression data were recorded by microarray analysis ; non - targeting siRNAs were used as a control . IRF2BP1 and IRF2BP2 have distinct and overlapping functions . Experiments were done in triplicates . ( G ) Validation of two candidate genes : IRF2BP1 and IRF2BP2 were knocked down for 72 hours in HeLa cells , and cell lysates were analyzed by immunoblotting with the indicated antibodies . ( H ) FACS - based analysis of EGFR surface expression : IRF2BP1 and IRF2BP2 were knocked down for 48 hours in HeLa cells , cells were serum starved for 16 hours , stained with fluorescent anti - EGFR antibodies and analyzed by flow cytometry . Shown are means of 3 independent experiments + / - SEM .

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

Figure 4(A) Immunoblotting of HeLa lysates at indicated times after EGF treatment reveals enhanced DUSP1 and ATF3 expression upon knockdown of IRF2BP1. Uba2 and beta-actin serve as independent loading controls.(C) Wound healing assay: HeLa cells were incubated with nt or IRF2BP1 siRNA for 2 days, grown to 90% confluency and analysed for wound closure with or without addition of 100 ng/ml EGF. Data show means of the relative wound density +/- SEM from 4 biological replicates.(D) Proliferation assay: HeLa cells upon knockdown of IRF2BP1 and IRF2BP1 were analysed for cell density over 6 days in growth medium. One representative biological experiment is shown with means +/-SEM from 5 technical replicates.(E) IRF2BP1, IRF2BP2, IRF2BP1+IRF2BP2 were knocked-down in HeLa cells for 72 hours and gene expression data were recorded by microarray analysis; non-targeting siRNAs were used as a control. IRF2BP1 and IRF2BP2 have distinct and overlapping functions. Experiments were done in triplicates.(G) Validation of two candidate genes: IRF2BP1 and IRF2BP2 were knocked down for 72 hours in HeLa cells, and cell lysates were analyzed by immunoblotting with the indicated antibodies.(H) FACS-based analysis of EGFR surface expression: IRF2BP1 and IRF2BP2 were knocked down for 48 hours in HeLa cells, cells were serum starved for 16 hours, stained with fluorescent anti-EGFR antibodies and analyzed by flow cytometry. Shown are means of 3 independent experiments +/-SEM.


Figure 1 ( C ) All eight subunits of the CCT chaperonin complex were enriched in U2OS - A3A samples . Proteins detected by MS are displayed by fold enrichment ( log2 ) from U2OS - A3A cells compared to parental cells ( x - axis ) , and by protein abundance within the U2OS - A3A samples ( y - axis ) . Significantly enriched proteins ( p < 0 . 05 by one - tailed t - test , n = 3 ) are denoted in dark blue . Components of the CCT complex are highlighted and labeled .

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

Figure 1(C) All eight subunits of the CCT chaperonin complex were enriched in U2OS-A3A samples. Proteins detected by MS are displayed by fold enrichment (log2) from U2OS-A3A cells compared to parental cells (x-axis), and by protein abundance within the U2OS-A3A samples (y-axis). Significantly enriched proteins (p<0.05 by one-tailed t-test, n=3) are denoted in dark blue. Components of the CCT complex are highlighted and labeled.


Figure 2 ( A ) A3A co - precipitates with CCT complex subunits . U2OS and HepaRG cells with dox - inducible A3A - HA transgenes were treated with dox prior to immunoprecipitation of HA . HA immunoprecipitates were analyzed by immunoblot with antibodies for HA and subunits of the CCT complex . ( C ) A3A interacts with CCT in leukemia cells . HA immunoprecipitation in hematopoietic cell lines with inducible A3A - HA was analyzed by immunoblot . HA , CCT1 , and CCT5 antibodies were used to evaluate co - precipitation of CCT subunits with A3A .

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

Figure 2(A) A3A co-precipitates with CCT complex subunits. U2OS and HepaRG cells with dox-inducible A3A-HA transgenes were treated with dox prior to immunoprecipitation of HA. HA immunoprecipitates were analyzed by immunoblot with antibodies for HA and subunits of the CCT complex.(C) A3A interacts with CCT in leukemia cells. HA immunoprecipitation in hematopoietic cell lines with inducible A3A-HA was analyzed by immunoblot. HA, CCT1, and CCT5 antibodies were used to evaluate co-precipitation of CCT subunits with A3A.


Figure 3 ( A ) CCT co - precipitates with A3A but not A3B or A3G . 293T cells were transfected with empty vector ( V ) , A3A - HA , A3B - HA , or A3G - HA . CCT IP was performed from transfected cells and evaluated by immunoblot analysis using antibodies for CCT5 and HA . ( B ) A3A , but not other APOBEC3 enzymes , interacts with CCT complex subunits . After transfection of 293T cells with HA - tagged APOBEC3 expression plasmids , IP was performed with anti - HA antibody and analyzed by immunoblot using antibodies to HA and CCT subunits . Arrows mark location of each APOBEC3 protein on HA immunoblot . ( F ) CCT co - precipitates with endogenous A3A . Immunoprecipitation of the CCT complex in BICR6 cells was analyzed by immunoblot using antibodies to endogenous A3A and CCT1 . Chemiluminescent exposure times described as short and long are indicated beside the blot . IgG antibody was used as a control throughout .

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

Figure 3(A) CCT co-precipitates with A3A but not A3B or A3G. 293T cells were transfected with empty vector (V), A3A-HA, A3B-HA, or A3G-HA. CCT IP was performed from transfected cells and evaluated by immunoblot analysis using antibodies for CCT5 and HA.(B) A3A, but not other APOBEC3 enzymes, interacts with CCT complex subunits. After transfection of 293T cells with HA-tagged APOBEC3 expression plasmids, IP was performed with anti-HA antibody and analyzed by immunoblot using antibodies to HA and CCT subunits. Arrows mark location of each APOBEC3 protein on HA immunoblot.(F) CCT co-precipitates with endogenous A3A. Immunoprecipitation of the CCT complex in BICR6 cells was analyzed by immunoblot using antibodies to endogenous A3A and CCT1. Chemiluminescent exposure times described as short and long are indicated beside the blot. IgG antibody was used as a control throughout.


Figure 4 ( A ) Knockdown of one CCT complex member results in depletion of other CCT subunits . Following siCCT4 or siCCT7 transfection , cell lysates were evaluated by immunoblot for expression of additional CCT subunits by endogenous antibodies for CCT1 , CCT4 , CCT5 , and CCT7 . GAPDH was used as a loading control . ( B ) Decreased cell viability upon CCT depletion and A3A expression . CCT4 ( left ) and CCT7 ( right ) subunits were depleted by siRNA transfection in U2OS cells with dox - inducible A3A transgenes . Immunoblot analysis of cell lysates showed decreased expression of targeted CCT subunit , but no alteration in A3A expression . Scr indicates non - targeted siRNA , used as control . Ku - 86 is a loading control . Following siRNA transfection to deplete CCT subunits , U2OS - A3A cells were treated with dox . Viability was determined by colorimetric change after addition of a water - soluble tetrazolium salt . Percent viability was normalized to untreated controls . Statistical analysis was performed using a paired two - tailed t - test , n = 3 biological replicates ; error bars , SEM . ( D ) Viability of cells with catalytically inactive A3A . U2OS - A3A - C106S cells were induced with dox to express the catalytically inactive A3A mutant ( C106S ) , depleted of CCT4 by siRNA , or combination . Cells were subsequently evaluated by live - dead staining assessed by FACS . Bar chart shows quantitation of FACS results averaged over three biological replicates . Statistical analysis was performed using a paired two - tailed t - test , n = 3 ; error bars , SEM . Immunoblot analysis of cell lysates showed decreased expression of CCT4 after siRNA targeting . Ku - 86 is a loading control . ( E ) Increased DNA damage signaling upon CCT depletion and A3A expression . Depletion of CCT7 in K562 - A3A cells was achieved by siRNA targeting . Expression of A3A was induced by dox treatment . Intracellular staining with anti - γH2AX antibody was analyzed by flow cytometry . Bar chart shows quantification of FACS results obtained over three biological replicates . Statistical analysis was performed using a two - tailed t - test , n = 3 ; error bars , SEM . Immunoblot analysis of cell lysates showed decreased expression of CCT7 after siRNA targeting and A3A expression upon dox treatment . Ku - 86 is a loading control . * * p - value < 0 . 01 , * * * p - value < 0 . 01 , * * * * p - value < 0 . 0001 , n . s . non - significant .

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

Figure 4(A) Knockdown of one CCT complex member results in depletion of other CCT subunits. Following siCCT4 or siCCT7 transfection, cell lysates were evaluated by immunoblot for expression of additional CCT subunits by endogenous antibodies for CCT1, CCT4, CCT5, and CCT7. GAPDH was used as a loading control.(B) Decreased cell viability upon CCT depletion and A3A expression. CCT4 (left) and CCT7 (right) subunits were depleted by siRNA transfection in U2OS cells with dox-inducible A3A transgenes. Immunoblot analysis of cell lysates showed decreased expression of targeted CCT subunit, but no alteration in A3A expression. Scr indicates non-targeted siRNA, used as control. Ku-86 is a loading control. Following siRNA transfection to deplete CCT subunits, U2OS-A3A cells were treated with dox. Viability was determined by colorimetric change after addition of a water-soluble tetrazolium salt. Percent viability was normalized to untreated controls. Statistical analysis was performed using a paired two-tailed t-test, n=3 biological replicates; error bars, SEM.(D) Viability of cells with catalytically inactive A3A. U2OS-A3A-C106S cells were induced with dox to express the catalytically inactive A3A mutant (C106S), depleted of CCT4 by siRNA, or combination. Cells were subsequently evaluated by live-dead staining assessed by FACS. Bar chart shows quantitation of FACS results averaged over three biological replicates. Statistical analysis was performed using a paired two-tailed t-test, n=3; error bars, SEM. Immunoblot analysis of cell lysates showed decreased expression of CCT4 after siRNA targeting. Ku-86 is a loading control.(E) Increased DNA damage signaling upon CCT depletion and A3A expression. Depletion of CCT7 in K562-A3A cells was achieved by siRNA targeting. Expression of A3A was induced by dox treatment. Intracellular staining with anti-γH2AX antibody was analyzed by flow cytometry. Bar chart shows quantification of FACS results obtained over three biological replicates. Statistical analysis was performed using a two-tailed t-test, n=3; error bars, SEM. Immunoblot analysis of cell lysates showed decreased expression of CCT7 after siRNA targeting and A3A expression upon dox treatment. Ku-86 is a loading control. **p-value <0.01, ***p-value <0.01, ****p-value <0.0001, n.s. non-significant.


Figure 5 ( C ) Quantification of APOBEC3 mutational signatures among three tumor types that have previously been associated with high APOBEC3 activity . Breast , bladder , and cervical cancer samples from the TCGA database were divided into CCT - mutated and non - mutated genomes , as above . The number of mutations contributing to SBS 2 and SBS 13 in each tumor type was quantified and displayed as an average . Comparison by Wilcoxon rank sum test yielded p - values denoted .

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

Figure 5(C) Quantification of APOBEC3 mutational signatures among three tumor types that have previously been associated with high APOBEC3 activity. Breast, bladder, and cervical cancer samples from the TCGA database were divided into CCT-mutated and non-mutated genomes, as above. The number of mutations contributing to SBS 2 and SBS 13 in each tumor type was quantified and displayed as an average. Comparison by Wilcoxon rank sum test yielded p-values denoted.