Figure 1B . ST2 cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . As a control , cells were first solubilized and SF - IL - 17 was added only post lysis . Number of identified peptides ( unique + razor ) and iBAQ intensities for each protein in five independent experiments are shown . C . The stoichiometry of IL - 17RSC calculated as the ratio between iBAQ intensities of individual IL - 17RSC components to iBAQ intensity of IL - 17RC . The recruitment of kinases TBK1 and IKKε as compared to related kinases IKKα and IKKβ is significantly enhanced . Mean from five performed MS experiments is shown , the statistical significance was determined using unpaired two ‐ tailed Student ' s t ‐ test . D . ST2 cells were stimulated with IL - 17 ( 500 ng / ml ) , TNF ( 50 ng / ml ) , or IL - 1α ( 50 ng / ml ) for indicated time points and activation of signaling pathways was analyzed by immunoblotting . A representative of two independent experiments is shown .

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

Figure 1B. ST2 cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. As a control, cells were first solubilized and SF-IL-17 was added only post lysis. Number of identified peptides (unique + razor) and iBAQ intensities for each protein in five independent experiments are shown.C. The stoichiometry of IL-17RSC calculated as the ratio between iBAQ intensities of individual IL-17RSC components to iBAQ intensity of IL-17RC. The recruitment of kinases TBK1 and IKKε as compared to related kinases IKKα and IKKβ is significantly enhanced. Mean from five performed MS experiments is shown, the statistical significance was determined using unpaired two‐tailed Student's t‐test.D. ST2 cells were stimulated with IL-17 (500 ng/ml), TNF (50 ng/ml), or IL-1α (50 ng/ml) for indicated time points and activation of signaling pathways was analyzed by immunoblotting. A representative of two independent experiments is shown.


Figure 2ST2 cells were either left untreated ( A ) followed by stimulation with IL - 17 ( 500 ng / ml ) for 2 hours . Transcription response induced by IL - 17 stimulation as compared to unstimulated cells was analyzed by RNA sequencing . In red are transcripts considered to be significantly changed ( log2 fold change > 1 or < - 1 , - log10 Benjamini - Hochberg adjusted p - value > 2 , based on the analysis of three independent experiments ; tringle is used for transcripts with - log10 adjusted p - value > 100 ) . Names of several significantly upregulated transcripts are indicated . ST2 cells were pretreated with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) for 30 minutes ( B ) , followed by stimulation with IL - 17 ( 500 ng / ml ) for 2 hours . Transcription response induced by IL - 17 stimulation as compared to unstimulated cells was analyzed by RNA sequencing . In red are transcripts considered to be significantly changed ( log2 fold change > 1 or < - 1 , - log10 Benjamini - Hochberg adjusted p - value > 2 , based on the analysis of three independent experiments ; tringle is used for transcripts with - log10 adjusted p - value > 100 ) . Names of several significantly upregulated transcripts are indicated . D - E . Comparison of IL - 17 - induced transcriptional response in presence or absence of TBK1 / IKKε inhibitor . ( D ) Transcripts that are significantly changed in both conditions . ( E ) Transcripts that pass significance threshold for induction only in presence of TBK1 / IKKε inhibitor . Dashed lines indicate significantly upregulated transcripts ( log2 fold change > 1 ) ; red lines separates transcripts that are more upregulated upon IL - 17 stimulation in the presence of TBK1 / IKKε inhibitor as compared to IL - 17 alone . F . ST2 cells pretreated or not with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) were left unstimulated or stimulated with IL - 17 ( 500 ng / ml ) for 2 hours and induction of mRNA for selected genes was analyzed by real time PCR . Mean + SEM from five independent experiments is shown , statistical significance was determined using unpaired two ‐ tailed Student ' s t ‐ test . G . ST2 cells were pretreated or not with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) , stimulated with IL17 ( 500 ng / ml ) for indicated time points and analyzed by immunoblotting . H . ST2 wild type , TBK1 KO , IKKε KO , or cells lacking both kinases ( DKO ) were stimulated with IL - 17 ( 500 ng / ml ) as indicated and analyzed by immunoblotting . I . TBK1 / IKKε DKO cells reconstituted with either wild type or kinase dead mutant ( K38A ) version of both kinases were stimulated with IL - 17 ( 500 ng / ml ) as indicated and analyzed by immunoblotting .

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 O O O O O O O O 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 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 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

Figure 2ST2 cells were either left untreated (A) followed by stimulation with IL-17 (500 ng/ml) for 2 hours. Transcription response induced by IL-17 stimulation as compared to unstimulated cells was analyzed by RNA sequencing. In red are transcripts considered to be significantly changed (log2 fold change > 1 or < -1, - log10 Benjamini-Hochberg adjusted p-value > 2, based on the analysis of three independent experiments; tringle is used for transcripts with - log10 adjusted p-value > 100). Names of several significantly upregulated transcripts are indicated.ST2 cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM) for 30 minutes (B), followed by stimulation with IL-17 (500 ng/ml) for 2 hours. Transcription response induced by IL-17 stimulation as compared to unstimulated cells was analyzed by RNA sequencing. In red are transcripts considered to be significantly changed (log2 fold change > 1 or < -1, - log10 Benjamini-Hochberg adjusted p-value > 2, based on the analysis of three independent experiments; tringle is used for transcripts with - log10 adjusted p-value > 100). Names of several significantly upregulated transcripts are indicated.D-E. Comparison of IL-17-induced transcriptional response in presence or absence of TBK1/IKKε inhibitor. (D) Transcripts that are significantly changed in both conditions. (E) Transcripts that pass significance threshold for induction only in presence of TBK1/IKKε inhibitor. Dashed lines indicate significantly upregulated transcripts (log2 fold change > 1); red lines separates transcripts that are more upregulated upon IL-17 stimulation in the presence of TBK1/IKKε inhibitor as compared to IL-17 alone.F. ST2 cells pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) were left unstimulated or stimulated with IL-17 (500 ng/ml) for 2 hours and induction of mRNA for selected genes was analyzed by real time PCR. Mean + SEM from five independent experiments is shown, statistical significance was determined using unpaired two‐tailed Student's t‐test.G. ST2 cells were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL17 (500 ng/ml) for indicated time points and analyzed by immunoblotting.H. ST2 wild type, TBK1 KO, IKKε KO, or cells lacking both kinases (DKO) were stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting.I. TBK1/IKKε DKO cells reconstituted with either wild type or kinase dead mutant (K38A) version of both kinases were stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting.


Figure 3ST2 wild type or TBK1 / IKKε DKO cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . ( A ) The heatmap shows the row - normalized Z - Score calculated from log2 transformed iBAQ intensities from five independent experiments . ST2 wild type or TBK1 / IKKε DKO cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . ( B ) The ratio between iBAQ intensities of selected IL - 17RSC components to iBAQ intensity of IL - 17RC . Mean + SEM from five independent experiments is shown , statistical significance was determined by two ‐ tailed Mann - Whitney test . C . ST2 wild type or TBK1 / IKKε DKO cells were stimulated with SF - IL - 17 ( 500 ng / ml ) for 15 minutes or were left unstimulated and IL - 17 was added post lysis . Lysates were subjected to anti - Flag immunoprecipitation to isolate IL - 17RSC and samples were analyzed by immunoblotting . ACT1 phosphorylation detected as upper band in WT cells is absent in TBK1 / IKKε DKO cells . D . Cells were pretreated with TAK1 inhibitor 7 - oxozeanol ( 2 µM ) and / or TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) , stimulated with IL - 17 ( 500 ng / ml ) for indicated time points and lysates were analyzed by immunoblotting . E . ST2 wild type or HOIP - deficient cells were pretreated with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) , stimulated with IL - 17 ( 500 ng / ml ) as indicated , solubilized , and analyzed by immunoblotting . * indicates unspecific band . F . ST2 cells pretreated or not with TBK1 / IKKε inhibitor MRT67307 were stimulated with SF - IL - 17 for 15 minutes or were left unstimulated and IL - 17 was added post lysis . IL - 17RSC was isolated and analyzed via immunoblotting .

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Figure 3ST2 wild type or TBK1/IKKε DKO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (A) The heatmap shows the row-normalized Z-Score calculated from log2 transformed iBAQ intensities from five independent experiments.ST2 wild type or TBK1/IKKε DKO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (B) The ratio between iBAQ intensities of selected IL-17RSC components to iBAQ intensity of IL-17RC. Mean + SEM from five independent experiments is shown, statistical significance was determined by two‐tailed Mann-Whitney test.C. ST2 wild type or TBK1/IKKε DKO cells were stimulated with SF-IL-17 (500 ng/ml) for 15 minutes or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC and samples were analyzed by immunoblotting. ACT1 phosphorylation detected as upper band in WT cells is absent in TBK1/IKKε DKO cells.D. Cells were pretreated with TAK1 inhibitor 7-oxozeanol (2 µM) and/or TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL-17 (500 ng/ml) for indicated time points and lysates were analyzed by immunoblotting.E. ST2 wild type or HOIP-deficient cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL-17 (500 ng/ml) as indicated, solubilized, and analyzed by immunoblotting. * indicates unspecific band.F. ST2 cells pretreated or not with TBK1/IKKε inhibitor MRT67307 were stimulated with SF-IL-17 for 15 minutes or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated and analyzed via immunoblotting.


Figure 4A . ST2 wild type and ACT1 - deficient cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . As a control , cells were first solubilized and SF - IL - 17 was added only post lysis . Number of identified peptides ( unique + razor ) and iBAQ intensities for each protein in two independent experiments are shown . B . ST2 wild type , ACT1 KO or TRAF6 KO cells were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . Lysates were subjected to anti - Flag immunoprecipitation to isolate IL - 17RSC and samples were analyzed by immunoblotting . C . ST2 cells deficient in TRAF6 reconstituted with TRAF6 ( WT ) or enzymatically inactive TRAF6 ( C70A ) were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . Number of identified peptides ( unique + razor ) and iBAQ intensities for each protein in two independent experiments are shown . D . TRAF6 deficient cells reconstituted with TRAF6 ( WT ) , TRAF6 ( C70A ) or empty vector were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . IL - 17RSC was isolated from lysates by anti - Flag immunoprecipitation and analyzed by immunoblotting . Short and long exposure of the same membrane stained for Act1 are shown for lysate and IP samples , respectively . TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) and stimulated with IL - 17 ( 500 ng / ml ) for indicated time . ( E ) The activation of signaling pathways analyzed by immunoblotting . TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) and stimulated with IL - 17 ( 500 ng / ml ) for indicated time . The induction of mRNA for selected genes analyzed by real time PCR . Mean + SEM from four independent experiments is shown , statistical significance was determined using unpaired two ‐ tailed Student ' s t ‐ test . G . ST2 cells wild type or NEMO KO were stimulated with IL - 17 ( 500 ng / ml ) for indicated time points , solubilized and analyzed by immunoblotting . ST2 wild type or NEMO KO cells were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . Lysates were subjected to anti - Flag immunoprecipitation to isolate IL - 17RSC . Samples were analyzed by immunoblotting ( H ) . ST2 wild type or NEMO KO cells were stimulated with SF - IL - 17 or were left unstimulated and IL - 17 was added post lysis . results from four independent experiments were quantified by densitometry and mean + SEM from four independent experiments is shown ( I ) .

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Figure 4A. ST2 wild type and ACT1-deficient cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. As a control, cells were first solubilized and SF-IL-17 was added only post lysis. Number of identified peptides (unique + razor) and iBAQ intensities for each protein in two independent experiments are shown.B. ST2 wild type, ACT1 KO or TRAF6 KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC and samples were analyzed by immunoblotting.C. ST2 cells deficient in TRAF6 reconstituted with TRAF6(WT) or enzymatically inactive TRAF6(C70A) were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. Number of identified peptides (unique + razor) and iBAQ intensities for each protein in two independent experiments are shown.D. TRAF6 deficient cells reconstituted with TRAF6(WT), TRAF6(C70A) or empty vector were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated from lysates by anti-Flag immunoprecipitation and analyzed by immunoblotting. Short and long exposure of the same membrane stained for Act1 are shown for lysate and IP samples, respectively.TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) and stimulated with IL-17 (500 ng/ml) for indicated time. (E) The activation of signaling pathways analyzed by immunoblotting.TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) and stimulated with IL-17 (500 ng/ml) for indicated time. The induction of mRNA for selected genes analyzed by real time PCR. Mean + SEM from four independent experiments is shown, statistical significance was determined using unpaired two‐tailed Student's t‐test.G. ST2 cells wild type or NEMO KO were stimulated with IL-17 (500 ng/ml) for indicated time points, solubilized and analyzed by immunoblotting.ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC. Samples were analyzed by immunoblotting (H).ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 or were left unstimulated and IL-17 was added post lysis. results from four independent experiments were quantified by densitometry and mean + SEM from four independent experiments is shown (I).


Figure 5ST2 wild type or NEMO KO cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . ( A ) The heatmap shows the row - normalized Z - Score calculated from log2 transformed iBAQ values from two independent experiments . ST2 wild type or NEMO KO cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . ( B ) The ratio between iBAQ intensities of selected IL - 17RSC components to iBAQ intensity of IL - 17RC . Mean + SEM from two independent experiments is shown . ST2 wild type or NEMO KO cells were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . Lysates were subjected to anti - Flag immunoprecipitation to isolate IL - 17RSC ( C ) and analyzed by immunoblotting . ST2 wild type or NEMO KO cells were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . Lysates were tested for activation of signaling pathways ( D ) and analyzed by immunoblotting . E . ST2 cells were pretreated with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) or IKKα / IKKβ inhibitor TPCA1 ( 10 µM ) , stimulated with IL - 17 ( 500 ng / ml ) as indicated and analyzed by immunoblotting . F . ST2 wild type or NEMO KO cells were stimulated with TNF ( 50 ng / ml ) for indicated time points and lysates were analyzed by immunoblotting .

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Figure 5ST2 wild type or NEMO KO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (A) The heatmap shows the row-normalized Z-Score calculated from log2 transformed iBAQ values from two independent experiments.ST2 wild type or NEMO KO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (B) The ratio between iBAQ intensities of selected IL-17RSC components to iBAQ intensity of IL-17RC. Mean + SEM from two independent experiments is shown.ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC (C) and analyzed by immunoblotting.ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were tested for activation of signaling pathways (D) and analyzed by immunoblotting.E. ST2 cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM) or IKKα/IKKβ inhibitor TPCA1 (10 µM), stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting.F. ST2 wild type or NEMO KO cells were stimulated with TNF (50 ng/ml) for indicated time points and lysates were analyzed by immunoblotting.


Figure 6B . Analysis of ACT1 structure using three different algorithms predicting disordered regions . Detected phosphorylation sites are all in the disordered region of the protein . ACT1 KO cells were reconstituted with either wild type ACT1 or indicated mutants or ACT1 with all nine identified phospho - sites mutated to alanines ( 9ST mut ) . Cells were stimulated with IL - 17 ( 500 ng / ml ) as indicated and lysates were analyzed by immunoblotting . * indicates nonspecific band . ACT1 KO cells were reconstituted with either wild type ACT1 or indicated mutants or ACT1 with all nine identified phospho - sites mutated to alanines ( 9ST mut ) . Cells were stimulated with IL - 17 ( 500 ng / ml ) as indicated and lysates were analyzed by immunoblotting . * indicates nonspecific band . E . ACT1 KO cells expressing either ACT1 ( WT ) or ACT1 ( 9ST mut ) were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . IL - 17RSC was isolated via anti - Flag immunoprecipitation and analyzed by immunoblotting . F . ACT1 KO cells were reconstituted with either ACT1 ( WT ) or ACT1 ( Δ20 - 380 ) . Cells were stimulated with IL - 17 ( 500 ng / ml ) as indicated and lysates were analyzed by immunoblotting . * indicates nonspecific band . G . Analysis of iBAQ intensity of ACT1 normalized to iBAQ intensity of IL - 17RC . Data are composite of all 14 MS analyses of IL - 17RSC from wild type ST2 cells performed in this study . Mean + SEM is shown .

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Figure 6B. Analysis of ACT1 structure using three different algorithms predicting disordered regions. Detected phosphorylation sites are all in the disordered region of the protein.ACT1 KO cells were reconstituted with either wild type ACT1 or indicated mutants or ACT1 with all nine identified phospho-sites mutated to alanines (9ST mut). Cells were stimulated with IL-17 (500 ng/ml) as indicated and lysates were analyzed by immunoblotting. * indicates nonspecific band.ACT1 KO cells were reconstituted with either wild type ACT1 or indicated mutants or ACT1 with all nine identified phospho-sites mutated to alanines (9ST mut). Cells were stimulated with IL-17 (500 ng/ml) as indicated and lysates were analyzed by immunoblotting. * indicates nonspecific band.E. ACT1 KO cells expressing either ACT1(WT) or ACT1(9ST mut) were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated via anti-Flag immunoprecipitation and analyzed by immunoblotting.F. ACT1 KO cells were reconstituted with either ACT1(WT) or ACT1(Δ20-380). Cells were stimulated with IL-17 (500 ng/ml) as indicated and lysates were analyzed by immunoblotting. * indicates nonspecific band.G. Analysis of iBAQ intensity of ACT1 normalized to iBAQ intensity of IL-17RC. Data are composite of all 14 MS analyses of IL-17RSC from wild type ST2 cells performed in this study. Mean + SEM is shown.


Figure 2B , single optical plane in the HPuHG of wildtype mice and Alk1EndoKO mice showing dramatic vascular defect in Alk1EndoKO mice . Severely tangled and tortuous vasculatures were outlined in white dotted rectangles . C , 8 μm skin section of Cre - control ( CT ) and Alk1EndoKO mice stained for Cdh5 ( red ) , Ki67 ( green ) , Erg ( white ) , and Dapi ( blue ) . Examples of Cdh5 / Ki67 double positive cells were enlarged in the white dotted inset for visualization purposes . D - G , Error bars represent standard deviation . Each bar represents one individual mouse , and each dot represents measurement from one image . N = 3 mice per group , and n = 9 - 16 images per mouse . Restricted maximum likelihood ( REML ) analysis with unbounded variance components was used as statistical test and individual mouse was treated as random effect to take into the account of mouse - to - mouse variability . D , Quantification of number of ECs in HPuHG area from C . E , Percentage of proliferative ECs ( percentage of Ki67 + Cdh5 + cells in total Cdh5 + cells ) in HPuHG from C . F , Percentage of proliferative ECs ( percentage of Ki67 + / Cdh5 + cells in total skin Cdh5 + cells ) from C G , Percentage of proliferative ECs ( percentage of BrdU + Cdh5 + cells in total Cdh5 + cells ) in total skin from PD17 - 24 BrdU - pulsed CT and Alk1EndoKO

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 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, single optical plane in the HPuHG of wildtype mice and Alk1EndoKO mice showing dramatic vascular defect in Alk1EndoKO mice. Severely tangled and tortuous vasculatures were outlined in white dotted rectangles.C, 8 μm skin section of Cre- control (CT) and Alk1EndoKO mice stained for Cdh5 (red), Ki67 (green), Erg (white), and Dapi (blue). Examples of Cdh5/Ki67 double positive cells were enlarged in the white dotted inset for visualization purposes.D-G, Error bars represent standard deviation. Each bar represents one individual mouse, and each dot represents measurement from one image. N = 3 mice per group, and n = 9-16 images per mouse. Restricted maximum likelihood (REML) analysis with unbounded variance components was used as statistical test and individual mouse was treated as random effect to take into the account of mouse-to-mouse variability. D, Quantification of number of ECs in HPuHG area from C. E, Percentage of proliferative ECs (percentage of Ki67+ Cdh5+ cells in total Cdh5+ cells) in HPuHG from C. F, Percentage of proliferative ECs (percentage of Ki67+/Cdh5+ cells in total skin Cdh5+ cells) from C G, Percentage of proliferative ECs (percentage of BrdU+ Cdh5+ cells in total Cdh5+ cells) in total skin from PD17-24 BrdU-pulsed CT and Alk1EndoKO mice


Figure 3A - C , Immunofluorescence staining images of 12 μm skin section from PD17 induced PD20 telogen CT , PD25 early anagen CT , and PD25 telogen Alk1EndoKO mice stained for Endomucin ( EMCN , red ) , CD31 ( green ) , and Dapi ( blue ) . HPuHG region was outlined in yellow dotted lines . D - F , Error bars represent standard deviation . Each bar represents one individual mouse , and each dot represents measurement from one image . N = 3 mice per group , and n = 8 - 9 images per mouse . Restricted maximum likelihood ( REML ) analysis with unbounded variance components was used to calculate p - values and individual mouse was treated as random effect to take into the account of mouse - to - mouse variability . D , Quantification of EMCN + / CD31 + area from images like those in A - C , shown as percentage of BV in the HPuHG . E , Quantification of absolute EMCN + area ( e . g . BV area ) in HPuHG in images like those in A - C . F , Quantification of EMCN - / CD31 + area from images like those in A - C as LV area in the HPuHG . G - J , Maximum projection through optical Z - stacks of 60 μm skin sections from mice TM - induced at PD17 and sacrificed at PD20 telogen CT , PD25 early anagen CT , PD32 Anagen CT , and PD25 telogen Alk1EndoKO mice stained for VEGFR3 ( red ) , LYVE1 ( green ) , and Dapi ( blue ) . Lymphatic capillaries were outlined in white dotted lines . K , Quantification of lymphatic capillary caliber from images like those in G - J in PD20 telogen CT , PD25 early anagen CT , and PD25 telogen Alk1EndoKO mice Error bars represent standard deviation . Each bar represents one individual mouse , and each dot represents measurement from one lymphatic capillary . N = 3 mice per group , and n = 9 - 25 measurements per mouse . Restricted maximum likelihood ( REML ) analysis with unbounded variance components was used to calculate p - values and individual mouse was treated as random effect to take into the account of mouse - to - mouse variability .

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 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

Figure 3A-C, Immunofluorescence staining images of 12 μm skin section from PD17 induced PD20 telogen CT, PD25 early anagen CT, and PD25 telogen Alk1EndoKO mice stained for Endomucin (EMCN, red), CD31 (green), and Dapi (blue). HPuHG region was outlined in yellow dotted lines. D-F, Error bars represent standard deviation. Each bar represents one individual mouse, and each dot represents measurement from one image. N = 3 mice per group, and n = 8-9 images per mouse. Restricted maximum likelihood (REML) analysis with unbounded variance components was used to calculate p-values and individual mouse was treated as random effect to take into the account of mouse-to-mouse variability. D, Quantification of EMCN+/CD31+ area from images like those in A-C, shown as percentage of BV in the HPuHG. E, Quantification of absolute EMCN+ area (e.g. BV area) in HPuHG in images like those in A-C. F, Quantification of EMCN-/CD31+ area from images like those in A-C as LV area in the HPuHG.G-J, Maximum projection through optical Z-stacks of 60 μm skin sections from mice TM-induced at PD17 and sacrificed at PD20 telogen CT, PD25 early anagen CT, PD32 Anagen CT, and PD25 telogen Alk1EndoKO mice stained for VEGFR3 (red), LYVE1 (green), and Dapi (blue). Lymphatic capillaries were outlined in white dotted lines. K, Quantification of lymphatic capillary caliber from images like those in G-J in PD20 telogen CT, PD25 early anagen CT, and PD25 telogen Alk1EndoKO mice Error bars represent standard deviation. Each bar represents one individual mouse, and each dot represents measurement from one lymphatic capillary. N = 3 mice per group, and n = 9-25 measurements per mouse. Restricted maximum likelihood (REML) analysis with unbounded variance components was used to calculate p-values and individual mouse was treated as random effect to take into the account of mouse-to-mouse variability.


Figure 4B , Immunofluorescence image of 12 μm skin section from mice treated as shown in A , stained for VEGFR3 ( green ) and Dapi ( blue ) . C , Quantification of tdTomato + / VEGFR3 + area from images like those in B , shows high induction efficiency . Error bars represent standard deviation . Each bar represents one individual mouse , and each dot represents measurement from one image . N = 3 mice , and n = 7 - 8 images per mouse . F , Quantification of lymphatic capillary caliber from images like those in D in CT and Alk1LymphKO mice Error bars represent standard deviation . Each bar represents one individual mouse , and each dot represents measurement of one lymphatic capillary . N = 3 mice per group , and n = 13 - 16 measurements per mouse . Restricted maximum likelihood ( REML ) analysis with unbounded variance components was used as statistical test and individual mouse was treated as random effect to take into the account of mouse - to - mouse variability . G , Immunofluorescence images of 12 μm skin section of PD17 induced PD25 CT and Alk1LymphKO mice stained for Ki67 ( red ) , Itga6 ( green ) and Dapi ( blue ) showing representative hair follicle . Hair follicles were outlined with yellow dotted lines . Note Ki67 + cells in the hair germ ( HG ) area indicative of HFSC activation . Telogen hair follicle is defined as no Ki67 in the hair germ . Anagen I is defined as ≤ 7 Ki67 + cells in the unenlarged hair germ . Anagen II is defined as > 7 Ki67 + cells in the hair germ or enlarged hair germ with changed shape that starts to enclose the DP . Experiments were repeated at least 2 times with at least 3 skin sections / each mouse analyzed with N = 6 / 4 mice / group . See Table 1 for quantification detail .

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 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 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

Figure 4B, Immunofluorescence image of 12 μm skin section from mice treated as shown in A, stained for VEGFR3 (green) and Dapi (blue). C, Quantification of tdTomato+/VEGFR3+ area from images like those in B, shows high induction efficiency. Error bars represent standard deviation. Each bar represents one individual mouse, and each dot represents measurement from one image. N = 3 mice, and n = 7-8 images per mouse.F, Quantification of lymphatic capillary caliber from images like those in D in CT and Alk1LymphKO mice Error bars represent standard deviation. Each bar represents one individual mouse, and each dot represents measurement of one lymphatic capillary. N = 3 mice per group, and n = 13-16 measurements per mouse. Restricted maximum likelihood (REML) analysis with unbounded variance components was used as statistical test and individual mouse was treated as random effect to take into the account of mouse-to-mouse variability.G, Immunofluorescence images of 12 μm skin section of PD17 induced PD25 CT and Alk1LymphKO mice stained for Ki67 (red), Itga6 (green) and Dapi (blue) showing representative hair follicle. Hair follicles were outlined with yellow dotted lines. Note Ki67+ cells in the hair germ (HG) area indicative of HFSC activation. Telogen hair follicle is defined as no Ki67 in the hair germ. Anagen I is defined as ≤7 Ki67+ cells in the unenlarged hair germ. Anagen II is defined as >7 Ki67+ cells in the hair germ or enlarged hair germ with changed shape that starts to enclose the DP. Experiments were repeated at least 2 times with at least 3 skin sections/each mouse analyzed with N = 6/4 mice/group. See Table 1 for quantification detail.


Figure 5B , Immunofluorescence image of 12 μm skin section from CT and Alk1EndoKO mice used for RNA - seq stained for Ki67 ( red ) and Dapi ( blue ) show that HFSCs are in quiescence .

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Figure 5B, Immunofluorescence image of 12 μm skin section from CT and Alk1EndoKO mice used for RNA-seq stained for Ki67 (red) and Dapi (blue) show that HFSCs are in quiescence.


Figure 6C , Images of 20 μm skin section of PD20 and PD25 BMP4 - LacZ ; Alk1f / f ; Cdh5 - CreERT2 negative , injected with tamoxifen TM at PD17 ( labelled as CT ) , and PD20 / PD25 Cdh5CreERT2 ; Alk1f / f ; BMP4 - LacZ mice ( labelled as Alk1EndoKO ) TM injected at PD17 , all stained for CD31 ( IHC method , brown ) and X - gal ( blue ) . Hair follicles ( HF ) were outlined with black dotted lines . Arrows point to X - Gal + vascular structure in HPuHG area . G - J , Immunofluorescence images of 12 μm skin section of PD17 induced stained for ( Top panel ) Ki67 ( red ) , Itga6 ( green ) , and Dapi ( blue ) and ( Bottom panel ) CD31 ( red ) and Dapi ( blue ) . HPuHG region was outlined with white dotted lines . K , Quantification of hair follicle percentage of PD17 - induced PD25 CT , Alk1EndoKO , Alk1 BMP4dKO , and BMP4EndoKO at telogen , anagen I , and anagen II stages . Telogen hair follicle is defined as no Ki67 in the hair germ . Anagen I is defined as ≤ 7 Ki67 + cells in the unenlarged hair germ . Anagen II is defined as > 7 Ki67 + cells in the hair germ or enlarged hair germ with changed shape that starts to enclose the DP . P - value was generated from Chi - square test . See Table 1 for quantification detail .

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

Figure 6C, Images of 20 μm skin section of PD20 and PD25 BMP4-LacZ; Alk1f/f; Cdh5-CreERT2 negative, injected with tamoxifen TM at PD17 (labelled as CT), and PD20/PD25 Cdh5CreERT2; Alk1f/f; BMP4-LacZ mice (labelled as Alk1EndoKO) TM injected at PD17, all stained for CD31 (IHC method, brown) and X-gal (blue). Hair follicles (HF) were outlined with black dotted lines. Arrows point to X-Gal+ vascular structure in HPuHG area.G-J, Immunofluorescence images of 12 μm skin section of PD17 induced stained for (Top panel) Ki67 (red), Itga6 (green), and Dapi (blue) and (Bottom panel) CD31 (red) and Dapi (blue). HPuHG region was outlined with white dotted lines.K, Quantification of hair follicle percentage of PD17-induced PD25 CT, Alk1EndoKO, Alk1 BMP4dKO, and BMP4EndoKO at telogen, anagen I, and anagen II stages. Telogen hair follicle is defined as no Ki67 in the hair germ. Anagen I is defined as ≤7 Ki67+ cells in the unenlarged hair germ. Anagen II is defined as >7 Ki67+ cells in the hair germ or enlarged hair germ with changed shape that starts to enclose the DP. P-value was generated from Chi-square test. See Table 1 for quantification detail.


Figure 1 ( A ) Laser injured DRG axons showing growth cone regeneration ( upper panels ) and regenerative failure ( lower panels ) . Arrows as indicated . ( B ) Upper graph shows percentage of regenerating axons of DIV 1 - 2 adult DRG neurons in the presence of p110 inhibitors , 2 h after injury . Lower graph shows time taken to regenerate a new growth cone . Inhibitors were added 1hr before axons were injured , and maintained throughout : LY294002 , 20 μM . A66 5 μM . XL - 147 5 μM . TGX221 500 nM . IC - 87114 10μM . Idelalisib 500 nM . Upper graph data are shown as the mean + / - SEM . P values indicate statistical significance analysed by Fishers exact ( upper graph ) or Kruskal - Wallis test ( lower graph ) . ( C ) Examples of static vs . growing uninjured axons treated with p110α or δ inhibitors as indicated . Both arrows in bottom right image indicate growing axons . ( D ) Percentage of uninjured , growing axons in the presence of p110 inhibitors . Inhibitors were added as for ( B ) . Lower graph shows length grown in 2 h . Data are shown as the mean + / - SEM . P values indicate significance measured by Fishers exact test ( upper graph ) or ANOVA with Tukey ' s post - hoc analysis ( lower graph ) . ( E ) Adult DRG neurons in microfluidic compartmental chambers . Cell bodies on the left side , axons extending through microchannels on the right . Lower panels are schematics . ( F ) Upper graph shows percentage of regenerating axons in microfluidic chambers . p110α or δ inhibitors were applied to either soma or axons . Lower graph shows time taken to regenerate a new growth cone . Inhibitors were added 1hr before axons were injured , and maintained throughout : A66 5 μM . Idelalisib 500 nM . Upper graph data are shown as the mean + / - SEM . P values indicate statistical significance as measured by Fishers exact test ( upper graph ) or by Kruskal - Wallis test ( lower graph ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 1(A) Laser injured DRG axons showing growth cone regeneration (upper panels) and regenerative failure (lower panels). Arrows as indicated.(B) Upper graph shows percentage of regenerating axons of DIV 1-2 adult DRG neurons in the presence of p110 inhibitors, 2 h after injury. Lower graph shows time taken to regenerate a new growth cone. Inhibitors were added 1hr before axons were injured, and maintained throughout: LY294002, 20 μM. A66 5 μM. XL-147 5 μM. TGX221 500 nM. IC-87114 10μM. Idelalisib 500 nM. Upper graph data are shown as the mean +/- SEM. P values indicate statistical significance analysed by Fishers exact (upper graph) or Kruskal-Wallis test (lower graph).(C) Examples of static vs. growing uninjured axons treated with p110α or δ inhibitors as indicated. Both arrows in bottom right image indicate growing axons.(D) Percentage of uninjured, growing axons in the presence of p110 inhibitors. Inhibitors were added as for (B). Lower graph shows length grown in 2 h. Data are shown as the mean +/- SEM. P values indicate significance measured by Fishers exact test (upper graph) or ANOVA with Tukey's post-hoc analysis (lower graph).(E) Adult DRG neurons in microfluidic compartmental chambers. Cell bodies on the left side, axons extending through microchannels on the right. Lower panels are schematics.(F) Upper graph shows percentage of regenerating axons in microfluidic chambers. p110α or δ inhibitors were applied to either soma or axons. Lower graph shows time taken to regenerate a new growth cone. Inhibitors were added 1hr before axons were injured, and maintained throughout: A66 5 μM. Idelalisib 500 nM. Upper graph data are shown as the mean +/- SEM. P values indicate statistical significance as measured by Fishers exact test (upper graph) or by Kruskal-Wallis test (lower graph).


Figure 2 ( A ) DIV 3 cortical neurons immunolabelled for PIP3 . Spectrum heatmap shows fluorescence intensity . ( B ) DIV 8 cortical neuron transfected with GFP and immunolabelled for PIP3 . Spectrum heatmap shows fluorescence intensity . ( C ) DIV 16 cortical neuron transfected GFP and immunolabelled for PIP3 . Spectrum heatmap shows fluorescence intensity . ( D ) Somatic PIP3 quantification in the soma at increasing days in vitro . Data are shown as the mean + / - SEM . P values are significance measured by ANOVA with Tukey ' s post - hoc analysis . N = 3 experiments , 60 neurons . ( E ) Growth cone PIP3 quantification at increasing days in vitro . Data are shown as the mean + / - SEM . P values indicate significance measured by ANOVA with Tukey ' s post - hoc analysis . N = 3 experiments , 60 growth cones .

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Figure 2(A) DIV 3 cortical neurons immunolabelled for PIP3. Spectrum heatmap shows fluorescence intensity. (B) DIV 8 cortical neuron transfected with GFP and immunolabelled for PIP3. Spectrum heatmap shows fluorescence intensity. (C) DIV 16 cortical neuron transfected GFP and immunolabelled for PIP3. Spectrum heatmap shows fluorescence intensity. (D) Somatic PIP3 quantification in the soma at increasing days in vitro. Data are shown as the mean +/- SEM. P values are significance measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 neurons. (E) Growth cone PIP3 quantification at increasing days in vitro. Data are shown as the mean +/- SEM. P values indicate significance measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 growth cones.


Figure 3 ( A ) PIP3 immunofluorescence in the soma of DIV 16 cortical neurons expressing p110 isoforms and GFP . Spectrum heatmap shows fluorescence intensity . ( B ) PIP3 immunofluorescence at the axonal growth cone of DIV 16 neurons expressing p110 isoforms and GFP . Spectrum heatmap shows fluorescence intensity . ( C ) Quantification of PIP3 immunofluorescence in the soma . Data are shown as the mean + / - SEM . P values indicate significance as measured by ANOVA with Tukey ' s post - hoc analysis . N = 3 experiments , 60 neurons . ( D ) Graph showing PIP3 quantification of immunofluorescence at the axon growth cone . Data are shown as the mean + / - SEM . P values indicate significance measured by ANOVA with Tukey ' s post - hoc analysis . N = 3 experiments , 60 growth cones .

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Figure 3(A) PIP3 immunofluorescence in the soma of DIV 16 cortical neurons expressing p110 isoforms and GFP. Spectrum heatmap shows fluorescence intensity. (B) PIP3 immunofluorescence at the axonal growth cone of DIV 16 neurons expressing p110 isoforms and GFP. Spectrum heatmap shows fluorescence intensity. (C) Quantification of PIP3 immunofluorescence in the soma. Data are shown as the mean +/- SEM. P values indicate significance as measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 neurons. (D) Graph showing PIP3 quantification of immunofluorescence at the axon growth cone. Data are shown as the mean +/- SEM. P values indicate significance measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 growth cones.


Figure 4 ( A ) DIV 4 cortical neurons expressing p110 isoforms and GFP . Arrow marks the axon tip . ( B ) Quantification of axon length . n = 3 experiments , 45 neurons per group . ( C ) Quantification of dendrite length . n = 3 experiments , 45 neurons per group . ( D ) Quantification of the axon : dendrite length ratio . n = 3 experiments , 45 neurons per group . ( E ) DIV 14 cortical neurons expressing p110 isoforms and GFP , immunolabelled for pS6 . ( F ) Sholl analysis of branches . n = 60 neurons per group . Data are shown as the mean + / - SEM . P values indicate significance measured by repeated measure ANOVA with Bonferonni ' s post - hoc test . ( G ) Quantification of the total neurite length . n = 3 experiments , 60 neurons per group . ( H ) Quantification of soma area . n = 3 experiments , 60 neurons per group . ( I ) Quantification of the pS6 immunofluorescence . n = 3 experiments , 60 neurons per group .

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Figure 4(A) DIV 4 cortical neurons expressing p110 isoforms and GFP. Arrow marks the axon tip. (B) Quantification of axon length. n = 3 experiments, 45 neurons per group. (C) Quantification of dendrite length. n = 3 experiments, 45 neurons per group. (D) Quantification of the axon:dendrite length ratio. n = 3 experiments, 45 neurons per group. (E) DIV 14 cortical neurons expressing p110 isoforms and GFP, immunolabelled for pS6. (F) Sholl analysis of branches. n = 60 neurons per group. Data are shown as the mean +/- SEM. P values indicate significance measured by repeated measure ANOVA with Bonferonni's post-hoc test. (G) Quantification of the total neurite length. n = 3 experiments, 60 neurons per group. (H) Quantification of soma area. n = 3 experiments, 60 neurons per group. (I) Quantification of the pS6 immunofluorescence. n = 3 experiments, 60 neurons per group.


Figure 5 ( A ) Axotomised DIV 14 - 16 rat cortical neurons expressing p110δ and GFP or GFP alone . Axons were cut > 1000 μm from the soma imaged for 14 h . Black arrows mark the cut site , white arrows marks the axon tip . See also Movies EV7 and EV8 . ( B ) Percentage of regenerating axons 14 h after laser axotomy . Numbers on bars are injured axons per group , from 4 experiments . ( C ) Quantification of axon regeneration length 14 h post - axotomy . n = 9 axons for GFP , 9 for p110α , 25 for p110αH1047R and 27 for p110δ from 4 experiments . ( D ) Quantification of time to start of regeneration . n = 9 axons for GFP , 9 for p110α , 25 for p110αH1047R and 27 for p110δ from 4 experiments . ( E ) Axotomised human embryonic stem cell neurons p110δ and GFP or GFP alone . Black arrow marks the cut site , white arrows mark the axon tip . ( F ) Percentage of regenerating axons of human hESC neurons , Numbers on bars are injured axons per group , from 5 experiments . ( G ) Quantification of axon regeneration length . n = 22 axons for GFP , 44 for p110δ , from 5 experiments . Data are shown as the mean + / - SEM . Data were analysed by two tailed Student ' s T - test . ( H ) Quantification of time to start of regeneration . n = 22 axons for GFP , 44 for p110δ , from 5 experiments . Data are shown as the mean + / - SEM . Data were analysed by two tailed Student ' s T - test .

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Figure 5(A) Axotomised DIV 14-16 rat cortical neurons expressing p110δ and GFP or GFP alone. Axons were cut >1000 μm from the soma imaged for 14 h. Black arrows mark the cut site, white arrows marks the axon tip. See also Movies EV7 and EV8. (B) Percentage of regenerating axons 14 h after laser axotomy. Numbers on bars are injured axons per group, from 4 experiments. (C) Quantification of axon regeneration length 14 h post-axotomy. n=9 axons for GFP, 9 for p110α, 25 for p110αH1047R and 27 for p110δ from 4 experiments. (D) Quantification of time to start of regeneration. n=9 axons for GFP, 9 for p110α, 25 for p110αH1047R and 27 for p110δ from 4 experiments. (E) Axotomised human embryonic stem cell neurons p110δ and GFP or GFP alone. Black arrow marks the cut site, white arrows mark the axon tip. (F) Percentage of regenerating axons of human hESC neurons, Numbers on bars are injured axons per group, from 5 experiments. (G) Quantification of axon regeneration length. n=22 axons for GFP, 44 for p110δ, from 5 experiments. Data are shown as the mean +/- SEM. Data were analysed by two tailed Student's T-test. (H) Quantification of time to start of regeneration. n=22 axons for GFP, 44 for p110δ, from 5 experiments. Data are shown as the mean +/- SEM. Data were analysed by two tailed Student's T-test.


Figure 6 ( A ) Percentage of regenerating axons after laser axotomy of DIV 14 - 17 rat cortical neurons expressing either GFP ( control ) or GFP and p110δ in the presence of the indicated inhibitors , 14h after laser injury . Numbers on bars are injured axons per group . ( B ) Single confocal section through a DIV 16 neuron expressing p110δ and GFP ( upper panels ) . Green colour is GFP , red is overexpressed p110δ , detected with anti - p110δ . Lower panels show p110δ and GFP in a distal axon section , and at the growth cone . Arrow indicates the axon . ( C ) Active ARF and total ARF6 ( red ) in axons of DIV 16 rat cortical neurons expressing GFP or GFP and p110δ , as indicated . Arrows indicate axons . ( D ) Quantification of mean axonal ARF activity and total ( mean ) ARF6 in DIV 16 neurons expressing GFP or GFP and p110δ n = 60 axons for each condition . ( E ) Kymographs showing dynamics of α9 integrin - GFP in the distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . ( F ) Quantification of α9 integrin - GFP dynamics in the distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . n numbers are axon sections analysed for each condition . ( G ) Quantification of total α9 integrin - GFP number in the distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . n = 43 for control , 47 for co - transfected with p110δ . n numbers are axon sections analysed for each condition . ( H ) Kymographs showing dynamics of Rab11 - GFP in the distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . ( I ) Quantification of Rab11 - GFP dynamics in the distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . n numbers are axon sections analysed for each condition . ( J ) Quantification of total Rab11 - GFP in the distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . n = 43 for control , 53 for co - transfected with p110δ . n numbers are axon sections analysed for each condition .

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

Figure 6(A) Percentage of regenerating axons after laser axotomy of DIV 14-17 rat cortical neurons expressing either GFP (control) or GFP and p110δ in the presence of the indicated inhibitors, 14h after laser injury. Numbers on bars are injured axons per group.(B) Single confocal section through a DIV 16 neuron expressing p110δ and GFP (upper panels). Green colour is GFP, red is overexpressed p110δ, detected with anti-p110δ. Lower panels show p110δ and GFP in a distal axon section, and at the growth cone. Arrow indicates the axon.(C) Active ARF and total ARF6 (red) in axons of DIV 16 rat cortical neurons expressing GFP or GFP and p110δ, as indicated. Arrows indicate axons. (D) Quantification of mean axonal ARF activity and total (mean) ARF6 in DIV 16 neurons expressing GFP or GFP and p110δ n=60 axons for each condition. (E) Kymographs showing dynamics of α9 integrin-GFP in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. (F) Quantification of α9 integrin-GFP dynamics in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. n numbers are axon sections analysed for each condition. (G) Quantification of total α9 integrin-GFP number in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. n=43 for control, 47 for co-transfected with p110δ. n numbers are axon sections analysed for each condition.(H) Kymographs showing dynamics of Rab11-GFP in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. (I) Quantification of Rab11-GFP dynamics in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. n numbers are axon sections analysed for each condition. (J) Quantification of total Rab11-GFP in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. n=43 for control, 53 for co-transfected with p110δ. n numbers are axon sections analysed for each condition.


Figure 7C ) Retinal sections from AAV - injected mice , immunolabelled for phospho - S6 . Blue colour is DAPI to indicate nuclei . Inset images show individual colours at the same scale as the full image . ( D ) Percentage of phospho - S6 positive cells in the RGC layer 2 weeks after delivery of AAV - Cre - GFP or AAV - GFP . ( E ) Retinal whole mounts of AAV - injected mice immunolabelled for the RGC marker Brn3A to indicate cell survival . ( F ) Analysis of RGC survival 28 days after optic nerve crush ( counts of Brn3A positive RGC neurons ) . G ) Representative images of CTB - labelled RGC axons 4 weeks after optic nerve crush in wild type ( c57bl / 6 ) , Rosa26 - p110αH1047R , and Rosa26 - p110δ transgenic mice injected with AAV2 . Cre . GFP . Dashed line indicates crush site . ( H ) Regenerating axons at different distances distal to lesion sites .

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Figure 7C) Retinal sections from AAV-injected mice, immunolabelled for phospho-S6. Blue colour is DAPI to indicate nuclei. Inset images show individual colours at the same scale as the full image.(D) Percentage of phospho-S6 positive cells in the RGC layer 2 weeks after delivery of AAV-Cre-GFP or AAV-GFP.(E) Retinal whole mounts of AAV-injected mice immunolabelled for the RGC marker Brn3A to indicate cell survival.(F) Analysis of RGC survival 28 days after optic nerve crush (counts of Brn3A positive RGC neurons).G) Representative images of CTB-labelled RGC axons 4 weeks after optic nerve crush in wild type (c57bl/6), Rosa26-p110αH1047R, and Rosa26-p110δ transgenic mice injected with AAV2.Cre.GFP. Dashed line indicates crush site.(H) Regenerating axons at different distances distal to lesion sites.


Figure 8 ( A ) Retinal sections from mice injected with AAV2 viruses as indicated , immunolabeled for phospho - S6 . Blue colour is DAPI to indicate nuclei . Inset images show individual colours at the same scale as the full image . ( B ) Percentage of phospho S6 positive cells in the retinal ganglion layer of mice injected with AAV2 . shScramble . GFP or AAV2 . shPTEN . GFP . ( C ) Number of pS6 - postive RGCs of mice injected with AAV2 . GFP or AAV2 . p110δ . ( D ) Representative images of retinal whole mounts from AAV - injected mice stained for the RGC marker Brn3A to indicate RGC survival . ( E ) RGC survival 28 days after optic nerve crush . ( F ) CTB - labelled RGC axons 4 weeks after optic nerve crush . Boxes i . to iv . show regenerating axons . Dashed line indicates crush site . White arrows mark the end of axons . Graph shows quantification of regenerating axons at different distances distal to the lesion sites .

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Figure 8(A) Retinal sections from mice injected with AAV2 viruses as indicated, immunolabeled for phospho-S6. Blue colour is DAPI to indicate nuclei. Inset images show individual colours at the same scale as the full image. (B) Percentage of phospho S6 positive cells in the retinal ganglion layer of mice injected with AAV2.shScramble.GFP or AAV2.shPTEN.GFP. (C) Number of pS6-postive RGCs of mice injected with AAV2.GFP or AAV2.p110δ.(D) Representative images of retinal whole mounts from AAV-injected mice stained for the RGC marker Brn3A to indicate RGC survival. (E) RGC survival 28 days after optic nerve crush. (F) CTB-labelled RGC axons 4 weeks after optic nerve crush. Boxes i. to iv. show regenerating axons. Dashed line indicates crush site. White arrows mark the end of axons. Graph shows quantification of regenerating axons at different distances distal to the lesion sites.


Figure 1 ( C ) NAD + / NADH ratio was measured in mBA cell lines and 17 . 5 days embryo liver . Data represented as mean ± SEMs , and obtained from three different cell line and embryo liver . Significance determined by Student ' s t test , two - tailed , * P < 0 . 05 , * * P < 0 . 01 . ( D ) Immunostaining of 17 . 5 days embryo tissues ( mid - brain , heart , liver , and skin ) with anti - PAR antibody . Insets show higher magnification . Scale bar indicates 100 μm ( insert ) . ( E ) Wild - type ( WT ) and Ews - / - embryos mid - brain at E17 . 5 days were subjected to TUNEL assay . Scale bar indicates 100 μm ( lnsert ) .

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Figure 1(C) NAD+/NADH ratio was measured in mBA cell lines and 17.5 days embryo liver. Data represented as mean ±SEMs, and obtained from three different cell line and embryo liver. Significance determined by Student's t test, two-tailed, *P < 0.05, **P < 0.01.(D) Immunostaining of 17.5 days embryo tissues (mid-brain, heart, liver, and skin) with anti-PAR antibody. Insets show higher magnification. Scale bar indicates 100 μm (insert).(E) Wild-type (WT) and Ews-/- embryos mid-brain at E17.5 days were subjected to TUNEL assay. Scale bar indicates 100 μm (lnsert).


Figure 2 ( A ) Western blot analysis of Parp1 and DNA damage markers in Wild - type ( WT ) and Ews - / - mBA cells . Cells were treated with MMS ( 0 . 02 % ) in a time dependent manner . Proteins were fractionated into two groups , chromatin - bound ( Chro ) proteins and soluble ( Solu ) proteins . ( B ) After treatment with MMS ( 0 . 02 % , 1hour ) , the media was replaced to release the DNA damage . The proteins were fractionated and the specific protein kinetics at chromatin was analyzed by western blot . ( C ) GFP - PARP1 U2OS cells were transfected with siControl and siEWS . Local DNA damage was induced by micro - irradiation using a 405nm laser . Data represented as mean ± SEMs and more than six cells were analyzed from six independent experiments . The statistical significance determined by One - way ANOVA , * * * P < 0 . 001 . Scale bar indicates 5 μm . ( D ) Fluorescence correlation spectroscopy ( FCS ) was measured after 15 minutes later following micro - irradiation ( Upper ) or under normal condition in GFP - PARP1 U2OS cells with either siControl or siEWS . Data represented as mean ± SEMs , more than 50 cells were analyzed , and significance determined by One - way ANOVA , * * * P < 0 . 001 . Scale bar indicate 5 μm . ( E ) AsiSI endonuclease - integrated U2OS cells were transfected with siControl and siEWS . Cells were incubated with doxycycline for 4 hours to induce DSBs . With or without changing the Dox - added media to fresh media for 2 hours ( for release samples ) , the amount of chromatin - associated PARP1 was measured using ChIP assay . ( N . T : Non - treat , T : AsiSI treat , Release : Damage released samples ) . Data represented as mean ± SEMs , and technical repeats ( n = 3 ) , significance determined by two - way ANOVA , * * * P < 0 . 001 .

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

Figure 2(A) Western blot analysis of Parp1 and DNA damage markers in Wild-type (WT) and Ews-/- mBA cells. Cells were treated with MMS (0.02%) in a time dependent manner. Proteins were fractionated into two groups, chromatin-bound (Chro) proteins and soluble (Solu) proteins. (B) After treatment with MMS (0.02%, 1hour), the media was replaced to release the DNA damage. The proteins were fractionated and the specific protein kinetics at chromatin was analyzed by western blot.(C) GFP-PARP1 U2OS cells were transfected with siControl and siEWS. Local DNA damage was induced by micro-irradiation using a 405nm laser. Data represented as mean ±SEMs and more than six cells were analyzed from six independent experiments. The statistical significance determined by One-way ANOVA, ***P< 0.001. Scale bar indicates 5 μm.(D) Fluorescence correlation spectroscopy (FCS) was measured after 15 minutes later following micro-irradiation (Upper) or under normal condition in GFP-PARP1 U2OS cells with either siControl or siEWS. Data represented as mean ±SEMs, more than 50 cells were analyzed, and significance determined by One-way ANOVA, ***P< 0.001. Scale bar indicate 5 μm.(E) AsiSI endonuclease-integrated U2OS cells were transfected with siControl and siEWS. Cells were incubated with doxycycline for 4 hours to induce DSBs. With or without changing the Dox-added media to fresh media for 2 hours (for release samples), the amount of chromatin-associated PARP1 was measured using ChIP assay. (N.T: Non-treat, T: AsiSI treat, Release: Damage released samples). Data represented as mean ±SEMs, and technical repeats (n=3), significance determined by two-way ANOVA, ***P< 0.001.


Figure 3 ( A ) Total levels of PAR in whole cell lysate were measured using western blot analysis . Wild - type ( WT ) and Ews - / - mBA cells were treated with H2O2 , ( 1 mM , 20 minutes ) with or without Olaparib ( 5 μM , 7 hours ) . ( B ) The kinetics of PAR accumulation was analyzed by western blotting . WT and Ews - / - cells were treated with H2O2 ( 1 mM , 20 minutes ) , followed by incubation in fresh media for the indicated times to allow release of DNA damage ( C ) Immunohistochemistry detection of PAR by ADP - ribose antibody in WT and Ews - / - cells after H2O2 treatment ( 1 mM , 20 minutes ) and recovery from DNA damage . Right graph displays mean of intensities measured from 500 cells . Data represented as mean ± SEMs , significance determined by One - way ANOVA , * * * P < 0 . 001 . Scale bar indicate 20 μm . ( D , E ) Whole cell level of PAR was measured in WT and Ews - / - mBA cells upon treatment with ( D ) MMS ( 0 . 02 % , 60 minutes ) ( E ) with or without recovery from DNA damage . ( F ) NAD + / NADH ratio was measured in mBA cell lines following treatment of H2O2 ( 1 mM , 10 minutes ) . Data represented as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Student ' s t test , two - tailed , * * P < 0 . 01 , * * * P < 0 . 001 .

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Figure 3(A) Total levels of PAR in whole cell lysate were measured using western blot analysis. Wild-type (WT) and Ews-/- mBA cells were treated with H2O2, (1 mM, 20 minutes) with or without Olaparib (5 μM, 7 hours).(B) The kinetics of PAR accumulation was analyzed by western blotting. WT and Ews-/- cells were treated with H2O2 (1 mM, 20 minutes), followed by incubation in fresh media for the indicated times to allow release of DNA damage(C) Immunohistochemistry detection of PAR by ADP-ribose antibody in WT and Ews-/- cells after H2O2 treatment (1 mM, 20 minutes) and recovery from DNA damage. Right graph displays mean of intensities measured from 500 cells. Data represented as mean ±SEMs, significance determined by One-way ANOVA, ***P < 0.001. Scale bar indicate 20 μm.(D, E) Whole cell level of PAR was measured in WT and Ews-/- mBA cells upon treatment with (D) MMS (0.02%, 60 minutes) (E) with or without recovery from DNA damage.(F) NAD+/NADH ratio was measured in mBA cell lines following treatment of H2O2 (1 mM, 10 minutes). Data represented as mean ±SEMs, and technical repeats (n=3). Significance determined by Student's t test, two-tailed, **P < 0.01, ***P< 0.001.


Figure 4 ( A ) PARP1 and EWS interaction was visualized in cells using the CUPID system . After transfection of mRCD - EWS and GC3 - PARP1 ( EGFP - PARP1 ) plasmid , the cells were treated with either H2O2 ( 1 mM , 10 minutes ) or PMA . Scale bar indicates 5 μm . ( B ) Endogenous interaction between EWS and PARP1 was measured using immunoprecipitation western blot analysis . Cells were treated with H2O2 ( 1 mM , 10 minutes ) with or without Olaparib ( 5 μM , 7 hours ) . ( C ) MMS - treated cells , with or without Olaparib , were immunoprecipitated using an anti - EWS antibody and immunoblotted by anti - PAR antibody ( upper ) . Pre - Olaparib group was treated with Olaparib for 3 hours before MMS treatment ( 0 . 02 % 30 minutes ) and after - Olaparib group treated Olaparib after 10 minutes following treatment of MMS . Lower blot indicates input of anti - EWS antibody . ( D ) In vitro PAR binding assay . THRAP3 binding to PAR was used as a positive control and BSA binding to PAR was used as a negative control . ( E ) Schematic map of nine EWS mutants ( left ) and IP - western blot analysis ( right ) . GFP - PARP1 and siPARG were transfected into HEK - 293 cells with each EWS mutants plasmid . After H2O2 treatment , the cells were conducted to IP with anti - Flag antibody . ( F ) Western blot analysis of PARP1 accumulation on chromatin . EWS - WT and M4 mutants were transfected into EWS depleted HEK - 293 cells . After treatment of MMS ( 0 . 02 % , 1 hour ) , the proteins were fractionated to either chromatin - bound or soluble fraction . ( G ) Quantification of relative amount of PARP1 and γH2AX on chromatin , divided by chromatin H3 , from three independent western experiments are presented . Data represented as mean ± SEMs , were measured from 3 independent experiments . Significance determined by t - test . * P < 0 . 05 , * * P < 0 . 01 . n . s indicate non - significance ( H ) WT and Ews - / - cells were transfected with EWS - WT and - M4 mutant and cell viability was measured 24 hours after MMS treatment ( indicated concentration ) . Error bars represent as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * P < 0 . 01 . n . s indicate non - significance ,

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 4(A) PARP1 and EWS interaction was visualized in cells using the CUPID system. After transfection of mRCD-EWS and GC3-PARP1 (EGFP-PARP1) plasmid, the cells were treated with either H2O2 (1 mM, 10 minutes) or PMA. Scale bar indicates 5 μm.(B) Endogenous interaction between EWS and PARP1 was measured using immunoprecipitation western blot analysis. Cells were treated with H2O2 (1 mM, 10 minutes) with or without Olaparib (5 μM, 7 hours).(C) MMS-treated cells, with or without Olaparib, were immunoprecipitated using an anti-EWS antibody and immunoblotted by anti-PAR antibody (upper). Pre-Olaparib group was treated with Olaparib for 3 hours before MMS treatment (0.02% 30 minutes) and after-Olaparib group treated Olaparib after 10 minutes following treatment of MMS. Lower blot indicates input of anti-EWS antibody.(D) In vitro PAR binding assay. THRAP3 binding to PAR was used as a positive control and BSA binding to PAR was used as a negative control.(E) Schematic map of nine EWS mutants (left) and IP-western blot analysis (right). GFP-PARP1 and siPARG were transfected into HEK-293 cells with each EWS mutants plasmid. After H2O2 treatment, the cells were conducted to IP with anti-Flag antibody.(F) Western blot analysis of PARP1 accumulation on chromatin. EWS-WT and M4 mutants were transfected into EWS depleted HEK-293 cells. After treatment of MMS (0.02%, 1 hour), the proteins were fractionated to either chromatin-bound or soluble fraction. (G) Quantification of relative amount of PARP1 and γH2AX on chromatin, divided by chromatin H3, from three independent western experiments are presented. Data represented as mean ±SEMs, were measured from 3 independent experiments. Significance determined by t-test. * P < 0.05, **P < 0.01. n.s indicate non-significance(H) WT and Ews-/- cells were transfected with EWS-WT and -M4 mutant and cell viability was measured 24 hours after MMS treatment (indicated concentration). Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, **P < 0.01. n.s indicate non-significance,


Figure 5 ( A ) Relative cell viability of cells transfected with siCon ( WT ) , siEWS ( EWS KD ) , PARP1 ( PARP - 1 KD ) , and double siRNA ( DKD ) after MMS treatment . Error bars represent as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * * P < 0 . 001 . Analysis ( * * * ) indicates differences between DKD with EWS KD . ( B ) Additional Parp1 knockout in Ews knockout cells ( DKO ) were subjected to viability test after MMS treatment . Error bars represent as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * * P < 0 . 001 . * * * indicated differences between DKO with EWS - KO . C ) After transfection of siCon , siEWS , PARP1 , and double siRNA , the effect of co - depletion of PARP1 and EWS on the γH2AX and phosphorylated CHK1 upon MMS treatment was analyzed by western blot . D ) After treatment of MMS , the effect of additional Parp1 knockout in Ews knockout cells ( Double KO ) to the γH2AX and phosphorylated CHK1 were analyzed using western blot . E ) Whole cell expression of PAR in mBA ( WT : Ews - WT , KO : Ews - KO , DKO : double KO ) . F ) Relative NAD + / NADH ratios were measured in WT , Ews - / - and DKO cells . Error bars represent as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * * P < 0 . 001 . G ) PARGi ( 5 μM , 24 hours ) treated WT and Ews - / - cells were subjected to cellular viability assay . Error bars represent as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA . H ) Relative cellular viability was measured after NMN treatment , with or without MMS pretreatment , in WT and Ews - / - cells . Error bars represent as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * * P < 0 . 001 . I ) Chromatin - bound PARP1 was quantified by western blot in WT and Ews - / - cells treated with Olaparib ( 5 μM , 24 hours ) treatment . J ) Relative viability was measured in WT and Ews - / - cells upon treatment of Olaparib for 24 hours . Error bars represent as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * * P < 0 . 001 .

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

Figure 5(A) Relative cell viability of cells transfected with siCon (WT), siEWS (EWS KD), PARP1 (PARP-1 KD), and double siRNA (DKD) after MMS treatment. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001. Analysis (***) indicates differences between DKD with EWS KD.(B) Additional Parp1 knockout in Ews knockout cells (DKO) were subjected to viability test after MMS treatment. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001. *** indicated differences between DKO with EWS-KO. C) After transfection of siCon, siEWS, PARP1, and double siRNA, the effect of co-depletion of PARP1 and EWS on the γH2AX and phosphorylated CHK1 upon MMS treatment was analyzed by western blot. D) After treatment of MMS, the effect of additional Parp1 knockout in Ews knockout cells (Double KO) to the γH2AX and phosphorylated CHK1 were analyzed using western blot.E) Whole cell expression of PAR in mBA (WT: Ews-WT, KO: Ews-KO, DKO: double KO).F) Relative NAD+/NADH ratios were measured in WT, Ews-/- and DKO cells. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001.G) PARGi (5 μM, 24 hours) treated WT and Ews-/- cells were subjected to cellular viability assay. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA.H) Relative cellular viability was measured after NMN treatment, with or without MMS pretreatment, in WT and Ews-/- cells. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001.I) Chromatin-bound PARP1 was quantified by western blot in WT and Ews-/- cells treated with Olaparib (5 μM, 24 hours) treatment.J) Relative viability was measured in WT and Ews-/- cells upon treatment of Olaparib for 24 hours. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001.


Figure 6 ( A ) Western blot analysis of PARP1 in chromatin . Two Ewing Sarcoma cells ( CHP100 and A4573 ) were treated with MMS ( 0 . 02 % , 1 hour ) with or without release ( 1 hour ) from MMS ( N . T : non treat , T : treat , R : release sample ) . ( B ) U2OS and two Ewing Sarcoma cells were treated MMS ( 0 . 02 % , 30 minutes ) and subjected to immunofluorescence by PARP1 antibody after chromatin bound fraction . Scale bar indicates 20 μm ( left ) . Red line indicates mean and more than 300 cells were analyzed . Significance determined by Two - way ANOVA , * * * P < 0 . 001 . ( C ) Human Ewing Sarcoma histology samples were analyzed by immuno - histochemistry using PAR antibody . Scale bar represents 100 mm .

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Figure 6(A) Western blot analysis of PARP1 in chromatin. Two Ewing Sarcoma cells (CHP100 and A4573) were treated with MMS (0.02%, 1 hour) with or without release (1 hour) from MMS (N.T: non treat, T: treat, R: release sample).(B) U2OS and two Ewing Sarcoma cells were treated MMS (0.02%, 30 minutes) and subjected to immunofluorescence by PARP1 antibody after chromatin bound fraction. Scale bar indicates 20 μm (left). Red line indicates mean and more than 300 cells were analyzed. Significance determined by Two-way ANOVA, ***P < 0.001.(C) Human Ewing Sarcoma histology samples were analyzed by immuno-histochemistry using PAR antibody. Scale bar represents 100 mm.


Figure 1 ( A - C ) Atg7 + / + and Atg7 - / - MEFs stably expressing GFP ‐ Gal3 ( A , C ) or GFP ‐ Ub ( B ) were treated with 1000 μM LLOMe or 250 μg / ml silica for 3 h . Cells were subjected to immunocytochemistry using the following antibodies : anti ‐ LC3 and anti ‐ Lamp1 ( A ) , anti ‐ p62 and anti ‐ Lamp1 ( B ) , or anti ‐ FK2 and anti ‐ p62 ( C ) . Bars : 10 μm .

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Figure 1(A-C) Atg7+/+ and Atg7-/- MEFs stably expressing GFP‐Gal3 (A, C) or GFP‐Ub (B) were treated with 1000 μM LLOMe or 250 μg/ml silica for 3 h. Cells were subjected to immunocytochemistry using the following antibodies: anti‐LC3 and anti‐Lamp1 (A), anti‐p62 and anti‐Lamp1 (B), or anti‐FK2 and anti‐p62 (C). Bars: 10 μm.


Figure 2 ( A , B ) NIH3T3 cells stably expressing GFP ‐ Gal3 and either empty vector ( control ) or mStrawberry ‐ Atg4BC74A ( Atg4B mutant ) were treated with 1000 μM LLOMe for 1 h . After LLOMe washout , cells were fixed at the indicated time points and subjected to immunocytochemistry for Lamp1 and DAPI ( blue ) ( A ) . The number of GFP ‐ Gal3 or Lamp1 puncta per cell was quantified using G ‐ Count ( see also Supplementary Figure S4A and B ) . Then , the percent of GFP ‐ Gal3 ‐ positive Lamp1 puncta was determined ( B ) . The data represent means ± s . d . At least 70 cells were counted ( n = 3 ) . Bars : 20 μm . Source data for this figure is available on the online supplementary information page .

Figure 2(A, B) NIH3T3 cells stably expressing GFP‐Gal3 and either empty vector (control) or mStrawberry‐Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were fixed at the indicated time points and subjected to immunocytochemistry for Lamp1 and DAPI (blue) (A). The number of GFP‐Gal3 or Lamp1 puncta per cell was quantified using G‐Count (see also Supplementary Figure S4A and B). Then, the percent of GFP‐Gal3‐positive Lamp1 puncta was determined (B). The data represent means±s.d. At least 70 cells were counted (n=3). Bars: 20 μm.Source data for this figure is available on the online supplementary information page.


Figure 3 ( C - E ) HeLa cells transfected with tfGal3 and either One ‐ STrEP ‐ FLAG ‐ tagged Atg4BC74A ( Atg4B mutant ) or empty vector ( control ) were observed by confocal microscopy after treatment as shown in Figure 2A and B . The number of GFP ± RFP + or GFP + RFP + puncta per cell was quantified using G ‐ Count ( D ) . Then , the percent of GFP + RFP + tfGal3 puncta was calculated ( E ) . The data represent means ± s . d . At least 30 cells were counted ( n = 3 ) . Bar : 10 μm . ( F - H ) NIH3T3 cells stably expressing empty vector ( control ) or mStrawberry ‐ Atg4BC74A ( Atg4B mutant ) were treated with 1000 μM LLOMe for 1 h . After LLOMe washout , cells were cultured in the presence or absence of both 10 μg / ml E64d and Pepstatin A , fixed at the indicated time points , and subjected to immunocytochemistry for Gal3 ( green ) and DAPI ( blue ) ( F ) . The number of endogenous Gal3 puncta per control ( G ) or Atg4B ‐ mutant ( H ) cells was quantified by G ‐ Count . The data represent means ± s . d . At least 50 cells were counted ( n = 3 ) . Bars : 20 μm . Source data for this figure is available on the online supplementary information page .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) HeLa cells transfected with tfGal3 and either One‐STrEP‐FLAG‐tagged Atg4BC74A (Atg4B mutant) or empty vector (control) were observed by confocal microscopy after treatment as shown in Figure 2A and B. The number of GFP±RFP+ or GFP+RFP+ puncta per cell was quantified using G‐Count (D). Then, the percent of GFP+RFP+tfGal3 puncta was calculated (E). The data represent means±s.d. At least 30 cells were counted (n=3). Bar: 10 μm.(F-H) NIH3T3 cells stably expressing empty vector (control) or mStrawberry‐Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were cultured in the presence or absence of both 10 μg/ml E64d and Pepstatin A, fixed at the indicated time points, and subjected to immunocytochemistry for Gal3 (green) and DAPI (blue) (F). The number of endogenous Gal3 puncta per control (G) or Atg4B‐mutant (H) cells was quantified by G‐Count. The data represent means±s.d. At least 50 cells were counted (n=3). Bars: 20 μm.Source data for this figure is available on the online supplementary information page.


Figure 4 ( A - F ) HeLa cells stably expressing GFP ‐ LC3 were transfected with mStrawberry ‐ Gal3 , and treated with 1000 μM LLOMe for 1 h . Then , cells were fixed and observed by confocal microscopy ( A ) . The same specimens were further examined by transmission electron microscopy ( B - F ) . Green : LC3 ; magenta : Gal3 ; blue : DAPI . ( G - I ) NIH3T3 cells stably expressing CFP ‐ Gal3 and YFP ‐ LC3 , and either empty vector ( control ) ( G , H ) or mStrawberry ‐ Atg4BC74A ( I ) were treated with 1000 μM LLOMe for 2 h , fixed , and observed by confocal microscopy . The electron micrographs were taken in the same sample field as the transmission electron microscope . Green : LC3 ; blue : Gal3 and DAPI ; black arrow : single membrane ; white arrow : autophagosome ; white arrowhead : ER membrane .

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 O O O O O O O O O O O O O O O O O O O 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-F) HeLa cells stably expressing GFP‐LC3 were transfected with mStrawberry‐Gal3, and treated with 1000 μM LLOMe for 1 h. Then, cells were fixed and observed by confocal microscopy (A). The same specimens were further examined by transmission electron microscopy (B-F). Green: LC3; magenta: Gal3; blue: DAPI.(G-I) NIH3T3 cells stably expressing CFP‐Gal3 and YFP‐LC3, and either empty vector (control) (G, H) or mStrawberry‐Atg4BC74A (I) were treated with 1000 μM LLOMe for 2 h, fixed, and observed by confocal microscopy. The electron micrographs were taken in the same sample field as the transmission electron microscope. Green: LC3; blue: Gal3 and DAPI; black arrow: single membrane; white arrow: autophagosome; white arrowhead: ER membrane.


Figure 5 ( A ) Plasmacreatinine and ureanitrogen in Atg5F / F and Atg5F / F ; KAPmice treated with vehicle or UA + OA . The values displayed represent means ± s . e . Statistically significant differences ( * P0 . 05 ) are indicated . F / F : Atg5F / Fmice ; F / F ; KAP : Atg5F / F ; KAPmice . ( B , C ) PAS ‐ stained renal cortical region of Atg5F / F and Atg5F / F ; KAP mice treated with vehicle or UA + OA ( n = 4 - 7 ) . Bars : 40 μm ( B ) . Kidney injury score of Atg5F / F and Atg5F / F ; KAP mice treated with vehicle or UA + OA ( C ) . ( D ) Kidney cortexes from Atg5F / F and Atg5F / F ; KAP mice treated with vehicle or UA + OA were subjected to immunohistocheminal analysis of Lamp1 and LC3 . Green : LC3 ; magenta : Lamp1 ; blue : DAPI . F / F : Atg5F / F mice ; F / F ; KAP : Atg5F / F ; KAP mice . Bar : 5 μm . ( E ) Electron micrographs of kidney proximal tubules in Atg5 F / F and Atg5F / F ; KAP mice treated with UA + OA . Yellow arrowhead : autophagosome ; asterisk : lysosome ; Mt : mitochondrion . Bar : 1 μm .

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 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

Figure 5(A) Plasmacreatinine and ureanitrogen in Atg5F/F and Atg5F/F;KAPmice treated with vehicle or UA+OA. The values displayed represent means±s.e. Statistically significant differences (*P0.05) are indicated. F/F: Atg5F/Fmice; F/F;KAP: Atg5F/F;KAPmice.(B, C) PAS‐stained renal cortical region of Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA (n=4-7). Bars: 40 μm (B). Kidney injury score of Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA (C).(D) Kidney cortexes from Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA were subjected to immunohistocheminal analysis of Lamp1 and LC3. Green: LC3; magenta: Lamp1; blue: DAPI. F/F: Atg5F/F mice; F/F;KAP: Atg5F/F;KAP mice. Bar: 5 μm.(E) Electron micrographs of kidney proximal tubules in Atg5 F/F and Atg5F/F;KAP mice treated with UA+OA. Yellow arrowhead: autophagosome; asterisk: lysosome; Mt: mitochondrion. Bar: 1 μm.


figf1Wild - type , apg12 - 1 mutant and Δapg12 cells were cultured in nitrogen - starvation medium containing 1 mM PMSF . After incubation for 6 h , cells were observed under a phase - contrast microscope . Arrows indicate autophagic bodies . c , Wild - type ( squares ) and Δapg12 ( circles ) were cultured in nitrogen - starvation medium and their viability was determined by phloxine B staining . d , Quantification of autophagic activity of wild - type and Δapg12 cells by alkaline phosphatase ( ALP ) assay before ( black bars ) and after ( white bars ) nitrogen starvation for 4 h . Error bars indicate s . d . of three independent experiments .

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figf1Wild-type, apg12-1 mutant and Δapg12 cells were cultured in nitrogen-starvation medium containing 1 mM PMSF. After incubation for 6 h, cells were observed under a phase-contrast microscope. Arrows indicate autophagic bodies.c, Wild-type (squares) and Δapg12 (circles) were cultured in nitrogen-starvation medium and their viability was determined by phloxine B staining.d, Quantification of autophagic activity of wild-type and Δapg12 cells by alkaline phosphatase (ALP) assay before (black bars) and after (white bars) nitrogen starvation for 4 h. Error bars indicate s.d. of three independent experiments.