( D ) Intraperitoneal glucose tolerance ( IPGTT ) was assessed in normal mice , islet transplant mice receiving vehicle ( on day when grafts were considered rejected ) , islet transplant mice treated with IL - 33 ( at day 30 day 80 post - islet transplantation ) . Data shown are mean ± ( n = 6 - 9 per group ) . ( E ) under curve ( AUC ) for IPGTT was assessed . Data shown are mean ± ( n = 6 - 9 per group ) a one - way ANOVA was performed , * * * P < 0 . 001 .

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

(D) Intraperitoneal glucose tolerance test (IPGTT) was assessed in normal mice, islet transplant mice receiving vehicle (on the day when grafts were considered rejected), and islet transplant mice treated with IL-33 (at day 30 and day 80 post-islet transplantation). Data shown are the mean ± SEM (n=6-9 per group). (E) Area under the curve (AUC) for IPGTT was assessed. Data shown are the mean ± SEM (n=6-9 per group) and a one-way ANOVA was performed, ***P<0.001.


( F ) Representative immunohistochemical for insulin in graft samples from mice receiving vehicle or IL - 33 . Scale bar = 100 µm .

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(F) Representative immunohistochemical staining for insulin in graft samples from mice receiving vehicle or IL-33. Scale bar = 100 µm.


C57BL / 6 mice were treated with mouse recombinant IL - 33 or PBS daily for 5 consecutive days . ( A , B ) Representative FACS showing proportion of Tregs ( CD4 + Foxp3 + ) in CD4 + T compartment from spleens ( A ) kidneys ( B ) at day 3 after treatment in C57BL / 6 mice receiving PBS ( n = 4 ) or IL - 33 ( n = 6 ) .

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

C57BL/6 mice were treated with mouse recombinant IL-33 or PBS daily for 5 consecutive days. (A, B) Representative FACS analysis showing the proportion of Tregs (CD4+Foxp3+) in the CD4+ T cell compartment from the spleens (A) and kidneys (B) at day 3 after treatment in C57BL/6 mice receiving PBS (n=4) or IL-33 (n=6).


C57BL / 6 mice were treated with mouse recombinant IL - 33 or PBS daily for 5 consecutive days . ( C , D ) Representative FACS showing proportion of ILC2s ( Lin - GATA - 3 + ) in CD45 + leukocyte compartment from spleens ( C ) kidneys ( D ) at day 3 after treatment in C57BL / 6 mice receiving PBS ( n = 4 ) or IL - 33 ( n = 6 ) .

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

C57BL/6 mice were treated with mouse recombinant IL-33 or PBS daily for 5 consecutive days. (C, D) Representative FACS analysis showing the proportion of ILC2s (Lin-GATA-3+) in the CD45+ leukocyte compartment from the spleens (C) and kidneys (D) at day 3 after treatment in C57BL/6 mice receiving PBS (n=4) or IL-33 (n=6).


( E , F ) proportion of Tregs ( E ) ILC2s ( F ) in spleen kidney in PBS - injected controls ( n = 4 ) at 1 - 12 after IL - 33 treatment ( n = 4 - 6 per IL - 33 - treated group ) . Data information : Data shown are mean ± ; Statistical was performed with an unpaired t - , * * P < 0 . 01 , * * * P < 0 . 001 .

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

(E, F) proportion of Tregs (E) and ILC2s (F) in the spleen and kidney in PBS-injected controls (n=4) and at weeks 1-12 after IL-33 treatment (n=4-6 per IL-33-treated group). Data information: Data shown are the mean ± SEM; Statistical analysis was performed with an unpaired t-test, **P<0.01, ***P<0.001.


( B - D ) Proportion or numbers of CD4 + Foxp3 + Tregs in spleens , kidneys islet grafts of normal , islet transplant mice receiving vehicle islet transplant mice with IL - 33 treatment ( at day 7 , 30 80 post - islet transplantation ) . Data shown are mean ± ( n = 4 - 6 per group ) a one - way ANOVA was performed ; * * * P < 0 . 001 .

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

(B-D) Proportion or numbers of CD4+Foxp3+ Tregs in the spleens, kidneys and islet grafts of normal, islet transplant mice receiving vehicle and islet transplant mice with IL-33 treatment (at day 7, 30 and 80 post-islet transplantation). Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; ***P<0.001.


( E ) Representative confocal microscopy of immunostaining for CD4 , Foxp3 insulin in islet grafts . Scale bar = 50 µm .

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(E) Representative confocal microscopy images of immunostaining for CD4, Foxp3 and insulin in islet grafts. Scale bar = 50 µm.


( F - H ) Proportion or numbers of ILC2s in spleens , kidneys islet graft of mice with or without IL - 33 treatment . Data shown are mean ± ( n = 4 - 6 per group ) a one - way ANOVA was performed ; * * P < 0 . 01 , * * * P < 0 . 001 .

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

(F-H) Proportion or numbers of ILC2s in the spleens, kidneys and islet graft of mice with or without IL-33 treatment. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; **P<0.01, ***P<0.001.


( I ) Representative confocal microscopy of immunostaining for CD127 , ST2 , CD3 insulin in islet grafts . Scale bar = 50 µm .

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

(I) Representative confocal microscopy images of immunostaining for CD127, ST2, CD3 and insulin in islet grafts. Scale bar = 50 µm.


( J ) mRNA expression of IL - 25 , IL - 33 TSLP in islet grafts of mice with or without IL - 33 treatment was examined by qPCR , expressed relative to control of each experiment . Data shown are mean ± ( n = 4 - 6 per group ) a one - way ANOVA was performed ; * * P < 0 . 01 , * * * P < 0 . 001 .

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

(J) The mRNA expression of IL-25, IL-33 and TSLP in islet grafts of mice with or without IL-33 treatment was examined by qPCR, and expressed relative to the control of each experiment. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; **P<0.01, ***P<0.001.


Proportion of CD4 + Foxp3 + Tregs ( B ) from spleens kidneys of islet transplant mice receiving vehicle , IL - 33 , IL - 33 / DT , IL - 33 / PC61 , or IL - 33 / DT / PC61 at day 5 post - islet transplantation . Data shown are mean ± ( n = 4 - 5 per group ) a one - way ANOVA was performed ; NS : non significant , * * * P < 0 . 001 .

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

Proportion of CD4+Foxp3+ Tregs (B) from the spleens and kidneys of islet transplant mice receiving vehicle, IL-33, IL-33/DT, IL-33/PC61, or IL-33/DT/PC61 at day 5 post-islet transplantation. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed; NS: non significant, ***P<0.001.


Proportion of Lin - GATA - 3 + ILC2s ( C ) from spleens kidneys of islet transplant mice receiving vehicle , IL - 33 , IL - 33 / DT , IL - 33 / PC61 , or IL - 33 / DT / PC61 at day 5 post - islet transplantation . Data shown are mean ± ( n = 4 - 5 per group ) a one - way ANOVA was performed ; NS : non significant , * * * P < 0 . 001 .

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

Proportion of Lin-GATA-3+ ILC2s (C) from the spleens and kidneys of islet transplant mice receiving vehicle, IL-33, IL-33/DT, IL-33/PC61, or IL-33/DT/PC61 at day 5 post-islet transplantation. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed; NS: non significant, ***P<0.001.


( D ) Islet graft survival of five groups of mice was assessed by monitoring blood glucose calculated using Kaplan - Meier method . Cumulative data from two independent experiments are shown . Statistical was performed with a log - rank . * P < 0 . 05 , * * P < 0 . 01 .

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

(D) Islet graft survival of five groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. *P<0.05, **P<0.01.


( E ) Data are shown as blood glucose measurement in islet transplant mice treated with IL - 33 / DT , IL - 33 / PC61 , or IL - 33 / DT / PC61 . horizontal black indicates a BGL of 16 mmol / liter , threshold for rejection . Each represents one mouse .

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

(E) Data are shown as blood glucose measurement in islet transplant mice treated with IL-33/DT, IL-33/PC61, or IL-33/DT/PC61. The horizontal black line indicates a BGL of 16 mmol/liter, the threshold for rejection. Each line represents one mouse.


( A , B ) ILC210 non - IL - 10 producing ILC2s were assessed in islet graft ( A ) kidney ( B ) by intracellular IL - 10 at day 5 post islet transplantation . Data shown are mean ± ( n = 5 per group ) an unpaired t - was performed ; * * * P < 0 . 001 . ST2 : suppression of tumorigenicity 2 .

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

(A,B) ILC210 and non-IL-10 producing ILC2s were assessed in islet graft (A) and kidney (B) by intracellular IL-10 staining at day 5 post islet transplantation. Data shown are the mean ± SEM (n=5 per group) and an unpaired t-test was performed; ***P<0.001. ST2: suppression of tumorigenicity 2.


( C ) IL - 10 reporter C57BL / 6 mice were treated with PBS , IL - 33 alone or IL - 33 IL - 2C daily for 5 consecutive days . Proportion of lin - CD127 + ST2 + IL - 10 - GFP ILC210 from kidneys at day 3 after treatment in IL - 10 reporter C57BL / 6 mice . Data shown are mean ± ( n = 4 - 6 per group ) a one - way ANOVA was performed ; * * * P < 0 . 001 .

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

(C) IL-10 reporter C57BL/6 mice were treated with PBS, IL-33 alone or IL-33 and IL-2C daily for 5 consecutive days. Proportion of lin-CD127+ST2+IL-10-GFP ILC210 from the kidneys at day 3 after treatment in IL-10 reporter C57BL/6 mice. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; ***P<0.001.


( D ) Kidney ILC2s isolated from normal IL - 10 reporter C57BL / 6 mice were cultured with Medium only or Medium with IL - 33 IL - 2C for six days . Proportion of ILC210 was assessed by flow cytometry . Data shown are mean ± ( n = 6 per group ) an unpaired t - was performed ; * * * P < 0 . 001 . ST2 : suppression of tumorigenicity 2 .

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

(D) Kidney ILC2s isolated from normal IL-10 reporter C57BL/6 mice were cultured with Medium only or Medium with IL-33 and IL-2C for six days. Proportion of ILC210 was assessed by flow cytometry. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed; ***P<0.001. ST2: suppression of tumorigenicity 2.


( E ) secreted cytokine IL - 10 was analyzed via ELISA . Data shown are mean ± ( n = 6 per group ) an unpaired t - was performed ; * * * P < 0 . 001 .

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

(E) The secreted cytokine IL-10 was analyzed via ELISA. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed; ***P<0.001.


( F ) Kidney ILC2s isolated from C57BL / 6 mice were cultured with Medium only or Medium with IL - 33 IL - 2C for 30 minutes . expression of phosphorylated STAT5 was examined in kidney ILC2s by flow cytometry . p - STAT5 ( red lines ) isotype controls ( gray - filled areas ) are shown . Data represent mean ± of evaluations of MFI from each group ( n = 6 per group ) an unpaired t - was performed ; * * * P < 0 . 001 . MFI , mean .

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

(F) Kidney ILC2s isolated from C57BL/6 mice were cultured with Medium only or Medium with IL-33 and IL-2C for 30 minutes. The expression of phosphorylated STAT5 was examined in kidney ILC2s by flow cytometry. p-STAT5 (red lines) and isotype controls (gray-filled areas) are shown. Data represent the mean ± SEM of evaluations of MFI from each group (n=6 per group) and an unpaired t-test was performed; ***P<0.001. MFI, mean fluorescence intensity.


( G ) kidney ILC2s were cultured with Medium , IL - 33 / IL - 2C , or IL - 33 / IL - 2C STAT5 inhibitor ( STAT5 - IN ) for three days . secreted cytokine IL - 10 was analyzed via ELISA . Data shown are mean ± ( n = 6 per group ) a one - way ANOVA was performed ; * * * P < 0 . 001 .

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

(G) The kidney ILC2s were cultured with Medium, IL-33/IL-2C, or IL-33/IL-2C and STAT5 inhibitor (STAT5-IN) for three days. The secreted cytokine IL-10 was analyzed via ELISA. Data shown are the mean ± SEM (n=6 per group) and a one-way ANOVA was performed; ***P<0.001.


( A ) ILC210 non - ILC210 were isolated from ex vivo - expanded kidney ILC2 by flow sorting . ILC210 were transfected with control ( ILC210 - C ) or IL - 10 CRISPR - Cas9 ( ILC210 - IL - 10 ) . IL - 10 was measured in culture supernatant of ILC210 - C ILC210 - IL - 10 via ELISA . Data shown are mean ± ( n = 6 per group ) an unpaired t - was performed ; * * * P < 0 . 001 .

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

(A) ILC210 and non-ILC210 were isolated from ex vivo-expanded kidney ILC2 by flow sorting. ILC210 were transfected with control (ILC210-C) or IL-10 CRISPR-Cas9 (ILC210-IL-10). IL-10 was measured in culture supernatant of ILC210-C and ILC210-IL-10 via ELISA. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed; ***P<0.001.


( C ) Transfused ILC2s ( CFSE labeled , Green ) were observed in islet graft at day 5 post islet transplantation . Scale bar = 50 µm . numbers of CFSE - labeled ILC2s in islet graft were counted . Data shown are mean ± a one - way ANOVA was performed ( n = 4 per group ) . NS , not significant .

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

(C) Transfused ILC2s (CFSE labeled, Green) were observed in islet graft at day 5 post islet transplantation. Scale bar = 50 µm. The numbers of CFSE-labeled ILC2s in the islet graft were counted. Data shown are the mean ± SEM and a one-way ANOVA was performed (n=4 per group). NS, not significant.


( D ) mRNA expression of IL - 10 in islet grafts at day 5 post islet transplantation was examined by qPCR . Data shown are mean ± ( n = 4 per group ) a one - way ANOVA was performed ; * * P < 0 . 01 , * * * P < 0 . 001 .

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

(D) The mRNA expression of IL-10 in islet grafts at day 5 post islet transplantation was examined by qPCR. Data shown are the mean ± SEM (n=4 per group) and a one-way ANOVA was performed; **P<0.01, ***P<0.001.


( E ) Islet graft survival of four groups of mice was assessed by monitoring blood glucose calculated using Kaplan - Meier method . Cumulative data from two independent experiments are shown . Statistical was performed with a log - rank . * * * P < 0 . 001 .

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

(E) Islet graft survival of four groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001.


( F ) CD4 + T isolated from C57BL / 6 splenocytes were cultured with irradiated splenocytes derived from BALB / c in presence of ILC210 at indicated ratios for 4 days . CD4 + T proliferation was assessed using [ 3H ] - thymidine incorporation assays . Data shown are mean ± ( n = 4 - 6 per group ) .

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

(F) CD4+ T cells isolated from C57BL/6 splenocytes were cultured with irradiated splenocytes derived from BALB/c in the presence of ILC210 at the indicated ratios for 4 days. CD4+ T cells proliferation was assessed using [3H]-thymidine incorporation assays. Data shown are the mean ± SEM (n=4-6 per group).


( G ) Neutralizing anti - IL - 10 antibodies or genetic ablation of IL - 10 were used to block effect of ILC210 on CD4 + T proliferation . Data shown are mean ± ( n = 6 per group ) an unpaired t - was performed . * * * P < 0 . 001 .

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

(G) Neutralizing anti-IL-10 antibodies or genetic ablation of IL-10 were used to block the effect of ILC210 on CD4+ T cells proliferation. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed. ***P<0.001.


( B ) Islet graft survival of five groups of mice was assessed by monitoring blood glucose calculated using Kaplan - Meier method . Cumulative data from two independent experiments are shown . Statistical was performed with a log - rank . * * * P < 0 . 001 .

(B) Islet graft survival of five groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001.


( C ) To track ILC210 in vivo , CD45 . 2 + ILC210 were co - transplanted with islets in diabetic CD45 . 1 + C57BL / 6 mice . Representative FACS showing proportion of locally transplanted ILC210 ( CD45 . 2 + ST2 + ) in total CD45 + compartment from islet graft at day 5 80 post - islet transplantation or at day when grafts were considered rejected .

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

(C) To track ILC210 in vivo, CD45.2+ ILC210 were co-transplanted with islets in diabetic CD45.1+ C57BL/6 mice. Representative FACS analysis showing the proportion of locally transplanted ILC210 (CD45.2+ST2+) in the total CD45+ cell compartment from islet graft at day 5 and 80 post-islet transplantation or at the day when grafts were considered rejected.


( D , E ) Proportion numbers of locally transplanted CD45 . 2 + ST2 + ILC210 in islet grafts of islet transplant mice over time . Data shown are mean ± ( n = 4 - 5 per group ) a one - way ANOVA was performed , * * P < 0 . 01 , * * * P < 0 . 001 .

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

(D,E) Proportion and numbers of locally transplanted CD45.2+ST2+ ILC210 in islet grafts of islet transplant mice over time. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed, **P<0.01, ***P<0.001.


( F ) expression of IL - 10 - GFP was examined in locally transplanted CD45 . 2 + ST2 + ILC210 by flow cytometry . IL - 10 - GFP ( red lines ) controls ( gray - filled areas ) are shown . Data represent mean ± of evaluations of MFI from each group ( n = 4 - 5 per group ) a one - way ANOVA was performed ; * * * P < 0 . 001 . MFI , mean .

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

(F) The expression of IL-10-GFP was examined in locally transplanted CD45.2+ST2+ ILC210 by flow cytometry. IL-10-GFP (red lines) and controls (gray-filled areas) are shown. Data represent the mean ± SEM of evaluations of MFI from each group (n=4-5 per group) and a one-way ANOVA was performed; ***P<0.001. MFI, mean fluorescence intensity.


B . ST2 were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized IL - 17RSC was isolated via consecutive Flag Strep immunoprecipitation analyzed by MS . As a control , were first solubilized SF - IL - 17 was added only post lysis . Number of identified peptides ( unique + razor ) iBAQ intensities for each in five independent experiments are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 . stoichiometry of IL - 17RSC calculated as between iBAQ intensities of IL - 17RSC components to iBAQ of IL - 17RC . recruitment of kinases TBK1 IKKε as compared to related kinases IKKα IKKβ is significantly enhanced . Mean from five performed MS experiments is shown , statistical significance was determined using unpaired two ‐ Student ' s t ‐ .

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

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 were stimulated with IL - 17 ( 500 ng / ml ) , TNF ( 50 ng / ml ) , or IL - 1α ( 50 ng / ml ) for indicated time points activation of signaling pathways was analyzed by immunoblotting . A representative of two independent experiments is shown .

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

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.


ST2 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 was analyzed by sequencing . In red are transcripts considered to be significantly changed ( log2 fold change > 1 or < - 1 , - log10 Benjamini - Hochberg adjusted p - value > 2 , on of three independent experiments ; tringle is used for transcripts with - log10 adjusted p - value > 100 ) . Names of several significantly upregulated transcripts are indicated .

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

ST2 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 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 was analyzed by sequencing . In red are transcripts considered to be significantly changed ( log2 fold change > 1 or < - 1 , - log10 Benjamini - Hochberg adjusted p - value > 2 , on of three independent experiments ; tringle is used for transcripts with - log10 adjusted p - value > 100 ) . Names of several significantly upregulated transcripts are indicated .

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

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 presence of TBK1 / IKKε inhibitor as compared to IL - 17 alone .

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

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 pretreated or not with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) were left unstimulated or stimulated with IL - 17 ( 500 ng / ml ) for 2 hours induction of mRNA for selected genes was analyzed by real time PCR . Mean + from five independent experiments is shown , statistical significance was determined using unpaired two ‐ Student ' s t ‐ .

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

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 were pretreated or not with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) , stimulated with IL17 ( 500 ng / ml ) for indicated time points analyzed by immunoblotting .

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

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 lacking both kinases ( DKO ) were stimulated with IL - 17 ( 500 ng / ml ) as indicated analyzed by immunoblotting .

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

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 reconstituted with either wild type or kinase dead mutant ( K38A ) version of both kinases were stimulated with IL - 17 ( 500 ng / ml ) as indicated analyzed by immunoblotting .

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

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.


ST2 wild type or TBK1 / IKKε DKO were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized IL - 17RSC was isolated via consecutive Flag Strep immunoprecipitation analyzed by MS . ( A ) shows row - normalized Z - Score calculated from log2 transformed iBAQ intensities from five independent experiments .

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

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. (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 were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized IL - 17RSC was isolated via consecutive Flag Strep immunoprecipitation analyzed by MS . ( B ) between iBAQ intensities of selected IL - 17RSC components to iBAQ of IL - 17RC . Mean + from five independent experiments is shown , statistical significance was determined by two ‐ Mann - Whitney .

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

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 were stimulated with SF - IL - 17 ( 500 ng / ml ) for 15 minutes or were left unstimulated IL - 17 was added post lysis . Lysates were subjected to anti - Flag immunoprecipitation to isolate IL - 17RSC samples were analyzed by immunoblotting . ACT1 phosphorylation detected as upper band in WT is absent in TBK1 / IKKε DKO .

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

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 . were pretreated with TAK1 inhibitor 7 - oxozeanol ( 2 µM ) / or TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) , stimulated with IL - 17 ( 500 ng / ml ) for indicated time points lysates were analyzed by immunoblotting .

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

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 were pretreated with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) , stimulated with IL - 17 ( 500 ng / ml ) as indicated , solubilized , analyzed by immunoblotting . * indicates unspecific band .

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

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 pretreated or not with TBK1 / IKKε inhibitor MRT67307 were stimulated with SF - IL - 17 for 15 minutes or were left unstimulated IL - 17 was added post lysis . IL - 17RSC was isolated analyzed via immunoblotting .

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

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.


A . ST2 wild type ACT1 - deficient were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized IL - 17RSC was isolated via consecutive Flag Strep immunoprecipitation analyzed by MS . As a control , were first solubilized SF - IL - 17 was added only post lysis . Number of identified peptides ( unique + razor ) iBAQ intensities for each in two independent experiments are shown .

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

A. 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 were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated IL - 17 was added post lysis . Lysates were subjected to anti - Flag immunoprecipitation to isolate IL - 17RSC samples were analyzed by immunoblotting .

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

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 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 IL - 17RSC was isolated via consecutive Flag Strep immunoprecipitation analyzed by MS . Number of identified peptides ( unique + razor ) iBAQ intensities for each in two independent experiments are shown .

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

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 reconstituted with TRAF6 ( WT ) , TRAF6 ( C70A ) or empty vector were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated IL - 17 was added post lysis . IL - 17RSC was isolated from lysates by anti - Flag immunoprecipitation analyzed by immunoblotting . Short long exposure of same stained for Act1 are shown for lysate IP samples , respectively .

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

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 reconstituted with indicated constructs were pretreated or not with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) stimulated with IL - 17 ( 500 ng / ml ) for indicated time . ( E ) activation of signaling pathways analyzed by immunoblotting .

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

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 reconstituted with indicated constructs were pretreated or not with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) stimulated with IL - 17 ( 500 ng / ml ) for indicated time . induction of mRNA for selected genes analyzed by real time PCR . Mean + from four independent experiments is shown , statistical significance was determined using unpaired two ‐ Student ' s t ‐ .

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

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 wild type or NEMO KO were stimulated with IL - 17 ( 500 ng / ml ) for indicated time points , solubilized analyzed by immunoblotting .

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

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 were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated IL - 17 was added post lysis . Lysates were subjected to anti - Flag immunoprecipitation to isolate IL - 17RSC . Samples were analyzed by immunoblotting ( H ) .

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

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 were stimulated with SF - IL - 17 or were left unstimulated IL - 17 was added post lysis . results from four independent experiments were quantified by densitometry mean + from four independent experiments is shown ( I ) .

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

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).


ST2 wild type or NEMO KO were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized IL - 17RSC was isolated via consecutive Flag Strep immunoprecipitation analyzed by MS . ( A ) shows row - normalized Z - Score calculated from log2 transformed iBAQ values from two independent experiments .

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

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. (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 were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized IL - 17RSC was isolated via consecutive Flag Strep immunoprecipitation analyzed by MS . ( B ) between iBAQ intensities of selected IL - 17RSC components to iBAQ of IL - 17RC . Mean + from two independent experiments is shown .

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

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 were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated IL - 17 was added post lysis . Lysates were subjected to anti - Flag immunoprecipitation to isolate IL - 17RSC ( C ) analyzed by immunoblotting .

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

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 were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated IL - 17 was added post lysis . Lysates were tested for activation of signaling pathways ( D ) analyzed by immunoblotting .

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

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 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 analyzed by immunoblotting .

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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 were stimulated with TNF ( 50 ng / ml ) for indicated time points lysates were analyzed by immunoblotting .

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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.


B . of ACT1 structure using three different algorithms predicting disordered regions . Detected phosphorylation sites are in disordered region of .

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B. 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 were reconstituted with either wild type ACT1 or indicated mutants or ACT1 with nine identified phospho - sites mutated to alanines ( 9ST mut ) . were stimulated with IL - 17 ( 500 ng / ml ) as indicated lysates were analyzed by immunoblotting . * indicates nonspecific band .

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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 expressing either ACT1 ( WT ) or ACT1 ( 9ST mut ) were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated IL - 17 was added post lysis . IL - 17RSC was isolated via anti - Flag immunoprecipitation analyzed by immunoblotting .

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

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 were reconstituted with either ACT1 ( WT ) or ACT1 ( Δ20 - 380 ) . were stimulated with IL - 17 ( 500 ng / ml ) as indicated lysates were analyzed by immunoblotting . * indicates nonspecific band .

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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 . of iBAQ of ACT1 normalized to iBAQ of IL - 17RC . Data are composite of 14 MS analyses of IL - 17RSC from wild type ST2 performed in this study . Mean + is shown .

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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.


( B ) ACE2 gene expression summary in human tissues in 61 different tissues in NX . Cut - off for what is regarded as expressed was set to 1 . 0 NX .

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(B) ACE2 gene expression summary in human tissues in 61 different tissues and cells in NX. Cut-off for what is regarded as expressed was set to 1.0 NX.


Representative of 20 tissue types histological structures stained on consecutive sections with immunohistochemistry using two antibodies targeting human ACE2 ( brown ) , counterstained with hematoxylin ( blue ) . Most intense antibody was observed in microvilli of intestinal tract renal proximal tubules , in membranes of gallbladder epithelium , epididymis epithelium , testicular Sertoli Leydig , a subset of glandular in seminal vesicle cytoplasm of cardiomyocytes , with HPA000288 also cardiac muscle , while MAB933 only showed in a few . Distinct ACE2 for both antibodies was also present in cornea conjunctiva of eye , interlobular pancreatic , as well as in placental villi , both in cytotrophoblasts , syncytiotrophoblasts , also in extravillous trophoblasts , while placenta decidua was negative . ACE2 could be observed at base of ciliated fallopian tube epithelium , however only for one of antibodies . Note that ACE2 expression was less prominent in crypts of mucosal intestinal layer . ACE2 also stained positive in endothelial pericytes in several tissues , see Fallopian , thyroid , parathyroid , adrenal , pancreas heart . Scale bar = 50µm . Scale bar in dashed squares = 10 µm . ( Brunner = Brunner ' s , EVT = extravillous trophoblasts , endo = endothelial ) .

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Representative images of 20 tissue types and histological structures stained on consecutive sections with immunohistochemistry using two antibodies targeting human ACE2 protein (brown), and counterstained with hematoxylin (blue). Most intense antibody staining was observed in microvilli of the intestinal tract and renal proximal tubules, in membranes of gallbladder epithelium, epididymis epithelium, testicular Sertoli cells and Leydig cells, a subset of glandular cells in seminal vesicle and cytoplasm of cardiomyocytes, with HPA000288 also staining the cardiac muscle fibers, while MAB933 only showed staining in a few cells. Distinct ACE2 staining for both antibodies was also present in cornea and conjunctiva of the eye, interlobular pancreatic ducts, as well as in placental villi, both in cytotrophoblasts, syncytiotrophoblasts, and also in extravillous trophoblasts, while placenta decidua was negative. ACE2 staining could be observed at the base of ciliated fallopian tube epithelium, however only for one of the antibodies. Note that ACE2 protein expression was less prominent in the crypts of the mucosal intestinal layer. ACE2 also stained positive in endothelial cells and pericytes in several tissues, see Fallopian, thyroid, parathyroid, adrenal gland, pancreas and heart. Scale bar = 50µm. Scale bar in dashed squares = 10 µm. (Brunner = Brunner's gland, EVT = extravillous trophoblasts, endo=endothelial cells).


Representative of human respiratory tissues , stained on consecutive sections , with immunohistochemistry using two antibodies targeting human ACE2 ( brown ) , counterstained with hematoxylin ( blue ) . Respiratory tissues were composed of different structures in nasal mucosa , bronchus , smaller bronchioles lung tissue . ACE2 could be observed at base of ciliated in both nasal mucosa bronchial epithelium ( arrowheads ) . Rare ACE2 was present in a few alveolar ( arrows ) . No was observed in nasal squamous epithelium , bronchioles , or submucosal in either tissue . Gender are shown for individuals . Red green colored squares mark positions in TMA cores shown as magnifications . Scale bar = 50µm . Scale bar for in dashed squares = 10 µm . ( re = respiratory ; sq = squamous ) .

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

Representative images of human respiratory tissues, all stained on consecutive sections, with immunohistochemistry using two antibodies targeting human ACE2 protein (brown), and counterstained with hematoxylin (blue). Respiratory tissues were composed of different structures in nasal mucosa, bronchus, smaller bronchioles and lung tissue. ACE2 staining could be observed at the base of ciliated cells in both nasal mucosa and bronchial epithelium (arrowheads). Rare ACE2 staining was present in a few alveolar cells (arrows). No staining was observed in nasal squamous epithelium, bronchioles, or submucosal glands in either tissue. Gender and age are shown for all individuals. Red and green colored squares mark the positions in the TMA cores shown as magnifications. Scale bar = 50µm. Scale bar for images in dashed squares = 10 µm. (re=respiratory; sq=squamous).


( B ) ACE2 abundance in different human tissues from ProteomicsDB , with median expression presented in log10 ( iBAQ ) .

(B) ACE2 protein abundance in different human tissues from ProteomicsDB, with the median expression presented in log10(iBAQ).


( C ) Western blot of ACE2 using five different human tissue lysates . For lung , two different lysates were used : one ( left ) one ( right ) .

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(C) Western blot of ACE2 using five different human tissue lysates. For lung, two different lysates were used: one male (left) and one female (right).


( A ) Schematic overview of FMR1 , FXR1 , FXR2 , FMR2 position of 8 selected single nucleotide polymorphisms ( SNPs ) . represents introns , grey box at beginning end of each gene stands for UTR region red boxes represent exons . Gene structure plots generated using FancyGene ( Rambaldi Ciccarelli , 2009 )

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(A) Schematic overview of FMR1, FXR1, FXR2, FMR2 and position of the 8 selected single nucleotide polymorphisms (SNPs). Line represents introns, grey box at the beginning and end of each gene stands for UTR region and red boxes represent exons. Gene structure plots generated using FancyGene (Rambaldi Ciccarelli, 2009)


( B ) Distribution of repeat polymorphism lengths in FMR1 of GRAS schizophrenia healthy controls . ( C ) Distribution of repeat polymorphism lengths in FMR2 of GRAS schizophrenia healthy controls .

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(B) Distribution of repeat polymorphism lengths in FMR1 of male GRAS schizophrenia patients and healthy controls. (C) Distribution of repeat polymorphism lengths in FMR2 of male GRAS schizophrenia patients and healthy controls.


Figure 2 : SNP overview unbiased selection according to standard operating procedure ( SOP ) developed for phenotype - genetic association study ( PGAS ) approaches . ( A ) Genes from ' broader fragile X family ' their chromosomal position . ( B ) SNP numbers available through direct genotyping in here used semi - custom genotyping array ( Affymetrix , Santa Clara , CA , USA ) . ( C ) SNPs fulfilling some of first round of selection criteria ( ' functional ' = SNPs , i . e . located in promoter region , 3 ' UTR or coding ; MAF = MAF ≥ 0 . 2 ; LD = SNPs that ' survive ' after linkage disequilibrium pruning : r² < 0 . 8 ) . Underlined are 13 SNPs selected for PGAS approach using PAUSS ( selection requirements : fulfilled 2 of above criteria or were functional ) . Not more than 3 SNPs per gene are selected to avoid overrepresentation of one gene . ( D ) SNPs with a tendency in PGAS ( see Fig 3A ) at single SNP basis went into final accumulation model .

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Figure 2: SNP overview and unbiased selection according to the standard operating procedure (SOP) developed for phenotype-based genetic association study (PGAS) approaches. (A) Genes from the 'broader fragile X family' and their chromosomal position. (B) SNP numbers available through direct genotyping in the here used semi-custom genotyping array (Affymetrix, Santa Clara, CA, USA). (C) SNPs fulfilling some of the first round of selection criteria ('functional'=SNPs, i.e. located in promoter region, 3'UTR or coding sequence; MAF=MAF≥0.2; LD=SNPs that 'survive' after linkage disequilibrium pruning: r²<0.8). Underlined are the 13 SNPs selected for the PGAS approach using the PAUSS (selection requirements: fulfilled 2 of the above criteria or were functional). Not more than 3 SNPs per gene are selected to avoid overrepresentation of one gene. (D) SNPs with a tendency in PGAS (see Fig 3A) at single SNP basis went into the final accumulation model.


( A - B ) A total of 13 SNPs pre - selected according to SOP presented in Fig 2 underwent PGAS screening as exemplified here : ( A ) PAUSS association pattern of an exemplary fictitious autosomal ( upper panel ) a sex - chromosomal ( lower panel ) SNP which are eligible for accumulation model . genotype associated with highest average PAUSS ( in this example CC ) is ' proautistic genotype ' ( indicated by black arrow ) is assigned a score of 1 . heterozygous genotype is assigned a score of 0 . 5 homozygous genotype associated with lowest PAUSS receives a score of 0 . Please note that difference between genotypes does not have to be statistically significant . ( B ) PAUSS association pattern of an exemplary fictitious autosomal ( upper panel ) a sex - chromosomal ( lower panel ) SNP which would not be selected for accumulation model because of unclear phenotypical / biological relevance .

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

(A-B) A total of 13 SNPs pre-selected according to the SOP presented in Fig 2 underwent PGAS screening as exemplified here: (A) PAUSS association pattern of an exemplary fictitious autosomal (upper panel) and a sex-chromosomal (lower panel) SNP which are eligible for the accumulation model. The genotype associated with the highest average PAUSS (in this example CC) is the 'proautistic genotype' (indicated by the black arrow) and is assigned a score of 1. The heterozygous genotype is assigned a score of 0.5 and the homozygous genotype associated with the lowest PAUSS receives a score of 0. Please note that the difference between genotypes does not have to be statistically significant. (B) PAUSS association pattern of an exemplary fictitious autosomal (upper panel) and a sex-chromosomal (lower panel) SNP which would not be selected for the accumulation model because of unclear phenotypical/biological relevance.


( C - D ) specificity of association of 8SNP accumulation model with an autistic phenotype ( as determined using PAUSS ; compare Fig 4B ) is controlled by applying an unrelated ( or ' non - sense ' ) phenotype , e . g . delusional - depression : ( C ) Intercorrelation pattern of single items included in delusional - depression composite score , used here as example control phenotype . Cronbach ' s alpha is presented as measure of internal consistency . ( D ) delusional - depression composite score is not associated with number of proautistic genotypes of 8SNP risk model in discovery sample .

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(C-D) The specificity of the association of the 8SNP accumulation model with an autistic phenotype (as determined using PAUSS; compare Fig 4B) is controlled by applying an unrelated (or 'non-sense') phenotype, e.g. delusional-depression: (C) Intercorrelation pattern of single items included in the delusional-depression composite score, used here as example control phenotype. Cronbach's alpha is presented as measure of internal consistency. (D) The delusional-depression composite score is not associated with the number of proautistic genotypes of the 8SNP risk model in the discovery sample.


( A ) PAUSS ( PANSS autism severity score ) composition item intercorrelation pattern in GRAS sample of schizophrenic individuals ( discovery sample ) . Cronbach ' s alpha is presented as measure of internal consistency also provided for replication samples I II .

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(A) PAUSS (PANSS autism severity score) composition and item intercorrelation pattern in the GRAS sample of male schizophrenic individuals (discovery sample). Cronbach's alpha is presented as measure of internal consistency and also provided for the male replication samples I and II.


( B ) Association of PAUSS with number of proautistic genotypes of 8SNP risk model in discovery sample .

(B) Association of PAUSS with the number of proautistic genotypes of the 8SNP risk model in the discovery sample.


( C ) PAUSS comparison of extreme groups with high low numbers of accumulated proautistic genotypes in discovery sample ; binary logistic regression with non - z - standardized PAUSS as dependent variable ; mean ± .

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(C) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in the discovery sample; binary logistic regression analysis with non-z-standardized PAUSS as dependent variable; mean±SEM.


( D ) PAUSS comparison of extreme groups with high low numbers of accumulated proautistic genotypes in replication sample I of schizophrenia ; binary logistic regression ; mean ± .

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(D) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in replication sample I of male schizophrenia patients; binary logistic regression analysis; mean±SEM.


( E ) PAUSS comparison of extreme groups with high low numbers of accumulated proautistic genotypes in replication sample II of disease control ; Mann - Whitney U ; mean ± .

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(E) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in replication sample II of male disease control patients; Mann-Whitney U test; mean±SEM.


F ) highly significant correlation of PAUSS social support underlines validity of social support as an autism proxy phenotype ; mean ± .

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F) The highly significant correlation of PAUSS and social support underlines the validity of social support as an autism proxy phenotype; mean±SEM.


( G ) Comparison of extreme groups with high low numbers of accumulated proautistic genotypes for autism proxy phenotype social support in discovery sample ; binary logistic regression ; mean ± .

(G) Comparison of the extreme groups with high and low numbers of accumulated proautistic genotypes for the autism proxy phenotype social support in the discovery sample; binary logistic regression analysis; mean±SEM.


( H ) Comparison of extreme groups with high low numbers of accumulated proautistic genotypes for autism proxy phenotype social support in replication sample III from general population ; binary logistic regression ; mean ± . For replications p - values for one - sided are shown .

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(H) Comparison of the extreme groups with high and low numbers of accumulated proautistic genotypes for the autism proxy phenotype social support in the male replication sample III from general population; binary logistic regression analysis; mean±SEM. For all replications p-values for one-sided tests are shown.


( A ) FXR1 expression in PBMC of individuals carrying rs2601 GG ( low risk ; N = 16 ) versus AA genotype ( high risk ; N = 27 ) .

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(A) FXR1 expression in PBMC of individuals carrying the rs2601 GG (low risk; N=16) versus AA genotype (high risk; N=27).


( B ) PBMC microRNA expression was normalized data is plotted from 4 miR - 181 - 5p members ( miR - 181a , b , c , d - 5p ) .

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(B) PBMC microRNA expression was normalized and data is plotted from all 4 miR-181-5p members (miR-181a, b, c, d-5p).


( C ) homology of 4 human mature miR - 181 species shown together with sequences in broader fragile X gene family containing binding sites for miR - 181 family ( seed matches identified using Target Scan Human SFOLD ) . red letters specify seed sequences seed matches , respectively . Chromosome positions for each seed seed match are denoted ( human genome assembly GRCh38 / hg38 ) .

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(C) Sequence homology of all 4 human mature miR-181 species shown together with sequences in the broader fragile X gene family containing binding sites for the miR-181 family (seed matches identified using Target Scan Human and SFOLD). The red letters specify seed sequences and seed matches, respectively. Chromosome positions for each seed sequence and seed match are denoted (human genome assembly GRCh38/hg38).


( D ) Denoted are ΔΔG values for binding of each of miR - 181 family members to different 3 ' UTR positions . Positions were identified using Target Scan Human SFOLD then processed using PITA algorithm to yield denoted ΔΔG values . ΔΔG is an energetic score , more negative its value , stronger is expected binding of microRNA to given

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(D) Denoted are ΔΔG values for binding of each of the miR-181 family members to different 3'UTR positions. Positions were identified using Target Scan Human and SFOLD and then processed using PITA algorithm to yield the denoted ΔΔG values. ΔΔG is an energetic score, and the more negative its value, the stronger is the expected binding of the microRNA to the given site


( D ) indicated amounts of recombinant wild - type mutant PPM1H or PPM1J ( with a His - Sumo N - terminal tag , expressed in E . coli ) were incubated in vitro with 2 . 5 µg pThr72 phosphorylated Rab8a ( left ) or pThr73 phosphorylated Rab10 ( right ) for 20 min in presence of 10 mM MgCl2 in 40 mM HEPES pH 7 . 5 buffer . Reactions were terminated by addition of SDS Sample Buffer analyzed by Phos - tag gel electrophoresis that separates phosphorylated ( slow migrating ) dephosphorylated Rabs . gel was stained with Instant Blue Coomassie . D288A is a substrate - trapping ( inactive ) variant of PPM1H was used as a control .

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(D) The indicated amounts of recombinant wild-type and mutant PPM1H or PPM1J (with a His-Sumo N-terminal tag, expressed in E. coli) were incubated in vitro with 2.5 µg pThr72 phosphorylated Rab8a (left) or pThr73 phosphorylated Rab10 (right) for 20 min in the presence of 10 mM MgCl2 in 40 mM HEPES pH 7.5 buffer. Reactions were terminated by addition of SDS Sample Buffer and analyzed by Phos-tag gel electrophoresis that separates phosphorylated (slow migrating) and dephosphorylated Rabs. The gel was stained with Instant Blue Coomassie. D288A is a substrate-trapping (inactive) variant of PPM1H and was used as a control.


( D ) Kinetics of phosphate hydrolysis . 25 nM recombinant wild - type PPM1H was incubated with increasing concentrations of pThr72 phosphorylated Rab8a ( GTPγS or GDP ) as described in Materials Methods . Initial velocity ( V0 ) was calculated by dividing of released phosphatase ( μM ) by time ( min ) plotted against substrate for pThr72 phosphorylated Rab8a [ GTP bound conformation ] ( blue ) pThr72 phosphorylated Rab8a [ GDP bound conformation ] ( red ) . experiments were repeated twice , both data points are shown in curves . fittings for left middle panel were performed using mean of two values . Kinetic constants ( Kcat , Vmax , Km ) were obtained using GraphPad software , their uncertainties ( ± ) correspond to SE of mean .

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(D) Kinetics of phosphate hydrolysis. 25 nM recombinant wild-type PPM1H was incubated with increasing concentrations of pThr72 phosphorylated Rab8a (GTPγS or GDP) as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of released phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72 phosphorylated Rab8a[GTP bound conformation] (blue) and pThr72 phosphorylated Rab8a[GDP bound conformation] (red). The experiments were repeated twice, and both data points are shown in curves. Line fittings for the left and middle panel were performed using the mean of the two values. Kinetic constants (Kcat, Vmax, Km) were obtained using GraphPad software, and their uncertainties (±) correspond to the SE of mean.


( E ) Kinetic of PPM1H as in ( D ) using 50 nM PPM1H phosphopeptide substrates , as described in Materials Methods . Initial velocity ( V0 ) was calculated by dividing of phosphatase ( μM ) by time ( min ) plotted against substrate for pThr72 Rab8a phospho - peptide ( blue ) Rab10 pThr73 phospho - peptide ( red ) . Each experiment was performed twice ( data points shown ) .

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(E) Kinetic analysis of PPM1H as in (D) using 50 nM PPM1H and phosphopeptide substrates, as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72 Rab8a phospho-peptide (blue) and Rab10 pThr73 phospho-peptide (red). Each experiment was performed twice (individual data points shown).


( F ) Side by side comparison of catalytic activity against peptide by in vitro malachite green time course . 50 nM recombinant wild - type PPM1H was incubated with 16 μM Rab8a GTPγS pThr72 phosphorylated ( blue ) or Rab8a pThr72 phosphorylated peptide ( red ) for indicated times analysed as described in Materials Methods . experiments were repeated 3 times . error bars represent SE of mean of technical replicates .

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(F) Side by side comparison of the catalytic activity against protein and peptide by in vitro malachite green time course analysis. 50 nM recombinant wild-type PPM1H was incubated with 16 μM Rab8a GTPγS pThr72 phosphorylated protein (blue) or Rab8a pThr72 phosphorylated peptide (red) for indicated times and analysed as described in Materials and Methods. The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates.


( C ) His6 - SUMO - tagged full - variants of PPM1H were used for catalytic assays ( left panel ) . 3 μg of recombinant wild - type or indicated mutant PPM1H were resolved on 4 - 12 % Bis - Tris gradient gel stained with Instant Blue Coomassie . Right , mass photometry histogram for 40nM His6 - SUMO - PPM1H WT ( blue ) His6 - SUMO - PPM1H 2Glu ( brown ) , where WT is 130kDa ( ± 15 . 8 kDa , with 1004 single molecules counted ) 2Glu is 83kDa ( ± 13 . 9 kDa , with 4117 single molecules counted ) . calculated molecular of fusion is approximately 68 . 5 kDa .

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(C) His6-SUMO-tagged full-length variants of PPM1H were used for catalytic assays (left panel). 3 μg of recombinant wild-type or indicated mutant PPM1H proteins were resolved on 4-12% Bis-Tris gradient gel and stained with Instant Blue Coomassie. Right, mass photometry histogram for 40nM His6-SUMO-PPM1H WT (blue) and His6-SUMO-PPM1H 2Glu (brown), where WT is 130kDa (±15.8 kDa, with 1004 single molecules counted) and 2Glu is 83kDa (±13.9 kDa, with 4117 single molecules counted). The calculated molecular weight of the fusion protein is approximately 68.5 kDa.


( D ) In vitro malachite green time course of recombinant His6 - SUMO - tagged PPM1H wild - type ( blue ) , 2Glu ( red ) , or D288A ( grey ) against 16 μM pThr72 phosphorylated Rab8a ( GTPγS , left ) , 32 μM pThr72 phosphorylated Rab8a peptide ( middle ) , or 32 μM pThr73 phosphorylated Rab10 peptide ( right ) . experiments were repeated 4 times . error bars represent SE of mean of technical replicates .

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(D) In vitro malachite green assay time course of recombinant His6-SUMO-tagged PPM1H wild-type (blue), 2Glu (red), or D288A (grey) against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS, left), 32 μM pThr72 phosphorylated Rab8a peptide (middle), or 32 μM pThr73 phosphorylated Rab10 peptide (right). The experiments were repeated 4 times. The error bars represent SE of mean of the technical replicates.


( C ) In vitro malachite green time course to determine enzyme activity of recombinant PPM ( 25 nM for substrate , 50 nM for peptide substrate ) against 16 μM pThr72 phosphorylated Rab8a ( GTPγS ) . experiments were repeated 3 times . error bars represent SE of mean of technical replicates . ( D ) In vitro malachite green against 32 μM pThr72 - Rab8a peptide ( left ) pThr73 - Rab10 peptide mimic ( right ) . experiments were repeated 3 times . error bars represent standard error ( SE ) of mean of technical replicates .

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(C) In vitro malachite green assay time course to determine enzyme activity of recombinant PPM proteins (25 nM for the protein substrate, 50 nM for the peptide substrate) against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS). The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates. (D) In vitro malachite green assay against 32 μM pThr72-Rab8a peptide (left) and pThr73-Rab10 peptide mimic (right). The experiments were repeated 3 times. The error bars represent standard error (SE) of mean of the technical replicates.


( E , F ) ( E ) HEK293 overexpressing indicated constructs were treated with ± 200 nM MLi - 2 for 90 min then lysed . 10 μg whole lysate was subjected to immunoblot with indicated antibodies at 1 μg / ml final membranes were analysed using OdysseyClx Western Blot imaging system . Left , each lane represents extract obtained from a different dish of ( two biological replicates per condition without MLi - 2 treatment , one biological replicate per condition with MLi - 2 treatment ) . Right , of phospho - Rab8a / total Rab8a was quantified using Image Studio software data presented relative to phosphorylation observed in PPM1H wild - type expressing . ( F ) As in ( E ) assessing phospho - Rab10 . For quantitation two replicates from presented blots were used . data points are shown on graphs with bar representing mean value of biological replicates .

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(E, F) (E) HEK293 cells overexpressing indicated constructs were treated with ± 200 nM MLi-2 for 90 min and then lysed. 10 μg whole cell lysate was subjected to immunoblot analysis with the indicated antibodies at 1 μg/ml final concentration and membranes were analysed using the OdysseyClx Western Blot imaging system. Left, each lane represents cell extract obtained from a different dish of cells (two biological replicates per condition without MLi-2 treatment, one biological replicate per condition with MLi-2 treatment). Right, the ratio of phospho-Rab8a/total Rab8a was quantified using Image Studio software and data presented relative to the phosphorylation ratio observed in PPM1H wild-type expressing cells. (F) As in (E) assessing phospho-Rab10 levels. For quantitation the two replicates from the presented blots were used. The data points are shown on the graphs with the bar levels representing the mean value of the biological replicates.


( B ) In vitro malachite green time course of recombinant PPM1H mutants against 16 μM pThr72 phosphorylated Rab8a ( GTPγS , left ) , 32 μM pThr72 phosphorylated Rab8a peptide ( middle ) , or 32 μM pThr73 phosphorylated Rab10 peptide ( right ) . experiments were repeated 4 times . error bars represent SE of mean of technical replicates .

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(B) In vitro malachite green assay time course of recombinant PPM1H mutants against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS, left), 32 μM pThr72 phosphorylated Rab8a peptide (middle), or 32 μM pThr73 phosphorylated Rab10 peptide (right). The experiments were repeated 4 times. The error bars represent SE of mean of the technical replicates.


( C ) 3 μg of recombinant wild - type or indicated mutant PPM1H were resolved on 4 - 12 % Bis - Tris gradient gel stained with Instant Blue Coomassie .

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(C) 3 μg of recombinant wild-type or indicated mutant PPM1H proteins were resolved on 4-12% Bis-Tris gradient gel and stained with Instant Blue Coomassie.


( E ) Activity of 25 nM PPM1H flap domain mutants against 16 μM pThr72 phosphorylated Rab8a ( GTPγS ) using malachite green time course . experiments were repeated 3 times . error bars represent SE of mean of technical replicates . Quality of purified ( right panel ) is shown using protocol in ( C ) .

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(E) Activity of 25 nM PPM1H flap domain mutants against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS) using the malachite green assay time course. The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates. Quality of purified proteins (right panel) is shown using the protocol in (C).


( F ) HEK293 overexpressing indicated constructs were treated analysed as described in ( D ) , with pRab10 analysed in upper panel , pRab8a in lower panel .

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(F) HEK293 cells overexpressing indicated constructs were treated and analysed as described in (D), with pRab10 analysed in the upper panel, and pRab8a in the lower panel.


( B ) Incremental deletions of N - terminus from residues 1 - 37 to 1 - 44 , one residue at a time . Upon deletion of R43 ( 44 - end ) , a reduced level of soluble PPM1H expression has no significant catalytic activity .

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(B) Incremental deletions of the N-terminus from residues 1-37 to 1-44, one residue at a time. Upon deletion of R43 (44-end), a reduced level of soluble PPM1H expression has no significant catalytic activity.


( B ) HEK293T were transfected with SFB control , SFB - tagged PR130 SFB - B56δ , lysates were pulled down using streptavidin beads interaction of endogenous PPM1G was detected by western blot ( WB ) .

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(B) HEK293T cells were transfected with SFB control, SFB-tagged PR130 and SFB-B56δ, and the cell lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot (WB).


( C ) Glutathione Sepharose beads bound with bacterially expressed recombinant GST , GST - B56δ or GST - PPP2R1A were incubated with bacterially purified recombinant MBP - PPM1G interaction of B56δ with PPM1G was detected by immunoblotting with MBP antibody . Interaction of B56δ - PPP2R1A was shown similarly using MBP - B56δ GST - PPP2R1A . Recombinant expression is shown by Coomassie .

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(C) Glutathione Sepharose beads bound with bacterially expressed recombinant GST, GST-B56δ or GST-PPP2R1A proteins were incubated with bacterially purified recombinant MBP-PPM1G and interaction of B56δ with PPM1G was detected by immunoblotting with MBP antibody. Interaction of B56δ-PPP2R1A was shown similarly using MBP- B56δ and GST-PPP2R1A proteins. Recombinant protein expression is shown by Coomassie staining.


( D ) HEK293T were transfected with SFB control , SFB - PPP2CA , SFB - PPP2CB , SFB - PPP2R1A , SFB - PPP2R1B SFB - B56δ , lysates were pulled down using streptavidin beads interaction of endogenous PPM1G was detected by western blot using PPM1G antibody .

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(D) HEK293T cells were transfected with SFB control, SFB- PPP2CA, SFB-PPP2CB, SFB-PPP2R1A, SFB-PPP2R1B and SFB- B56δ, and the lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot using PPM1G antibody.


( E ) lysates from HEK293T expressing SFB vector , SFB - B56α , SFB - B56β , SFB - B56γ , SFB - B56δ SFB - B56ε were pulled down interaction with PPM1G was detected by western blotting using PPM1G antibody .

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(E) Cell lysates from HEK293T cells expressing SFB vector, SFB-B56α, SFB-B56β, SFB-B56γ, SFB-B56δ and SFB-B56ε were pulled down and interaction with PPM1G was detected by western blotting using PPM1G antibody.


( G ) expressing B56δ full ( FL ) , Δ91 - 102 , L183A H282A mutants were pulled down with streptavidin beads interaction with PPM1G was detected by western blotting .

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(G) Cells expressing B56δ full length (FL), Δ91-102, L183A and H282A mutants were pulled down with streptavidin beads and interaction with PPM1G was detected by western blotting.


( H ) Various mutants of B56δ were transfected along with myc - PPP2R1A pulled down using streptavidin beads interaction with myc - PPP2R1A was detected by immunoblotting .

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(H) Various mutants of B56δ were transfected along with myc-PPP2R1A and pulled down using streptavidin beads and interaction with myc-PPP2R1A was detected by immunoblotting.


( I ) Binding affinities of B56δ with PPM1G PPP2R1A subunit were determined by Bio - layer interferometry . 6xHis - tagged PPM1G PPP2R1A were immobilized on Ni - NTA biosensors incubated with GST - B56δ wild type ( WT ) or Δ91 - 102 mutant at various concentrations ( 100 - 400 nm ) . Curves represent experimental trace obtained from BLI experiments . Binding affinities ( Kd ) ( + SD ; n = 3 independent experiments ) for B56δ - PPM1G B56δ - PPP2R1A are shown .

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(I) Binding affinities of the B56δ with PPM1G and PPP2R1A subunit were determined by Bio-layer interferometry. 6xHis-tagged PPM1G and PPP2R1A were immobilized on Ni-NTA biosensors and incubated with the GST-B56δ wild type (WT) or Δ91-102 mutant at various concentrations (100-400 nm). Curves represent experimental trace obtained from the BLI experiments. Binding affinities (Kd) (+SD; n=3 independent experiments) for B56δ-PPM1G and B56δ-PPP2R1A are shown.


( A ) MCF7 MDA - MB - 231 transfected with MYC - B56δ were fixed imaged using a confocal microscope after with antibodies against MYC tag PPM1G to determine PPM1G localization . Representative from three independent experiments are shown , Scale bars 5 µm . ( B ) of to ( Cyto / Nuc ) of PPM1G per each was plotted ( n = 35 for MCF - 7 ; n = 25 for MDA - MB - 231 ) . Error bars indicate SD . * * * p < 0 . 001 ( one - way ANOVA , post hoc : Bonferroni ' s multiple comparison ) .

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(A) MCF7 and MDA-MB-231 cells transfected with MYC-B56δ were fixed and imaged using a confocal microscope after staining with antibodies against MYC tag and PPM1G to determine PPM1G localization. Representative images from three independent experiments are shown, Scale bars 5 µm. (B) Quantification of cytoplasmic to nuclear (Cyto/Nuc) ratio of PPM1G intensity per each cell was plotted (n=35 for MCF-7 cells; n=25 for MDA-MB-231 cells). Error bars indicate SD. ***p < 0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( C ) Western blot showing expression of SFB tagged B56δ along with endogenous B56δ using in dicated antibodies .

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(C) Western blot showing expression levels of SFB tagged B56δ along with endogenous B56δ using in dicated antibodies.


( D ) expressing either vector or SFB - B56δ were fractionated into fractions of indicated were determined using western blotting . Tubulin Histone H3 were used to mark fractions respectively .

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(D) Cells expressing either vector or SFB-B56δ were fractionated into nuclear and cytoplasmic fractions and levels of indicated proteins were determined using western blotting. Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively.


( E ) treated with control or B56δ shRNA were fractionated into fractions of indicated were determined using immunoblotting . Tubulin Histone H3 were used to mark fractions respectively .

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(E) Cells treated with control or B56δ shRNA were fractionated into cytoplasmic and nuclear fractions and levels of indicated proteins were determined using immunoblotting. Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively.


( F ) Localization of PPM1G in expressing either empty vector ( control ) , full B56δ ( FL ) or various indicated mutants was determined by confocal imaging after with Flag Myc antibodies . Representative from two independent experiments are shown , Scale bars 10 µm . ( G ) to ( Cyto / Nuc ) of PPM1G per each expressing either vector , B56δ full ( FL ) or mutant versions was quantified plotted ( n = 10 ) . Error bars represents SD . * * * p < 0 . 001 ( one - way ANOVA , post hoc : Bonferroni ' s multiple comparison ) .

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(F) Localization of PPM1G in cells expressing either empty vector (control), full length B56δ (FL) or various indicated mutants was determined by confocal imaging after staining with Flag and Myc antibodies. Representative images from two independent experiments are shown, Scale bars 10 µm. (G) Cytoplasmic to nuclear (Cyto/Nuc) ratio of PPM1G intensity per each cell expressing either vector, B56δ full length (FL) or mutant versions was quantified and plotted (n=10). Error bars represents SD. ***p < 0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( A ) Endogenous association of PPM1G with α - Catenin was detected by immunoblotting with PPM1G antibody after performing immunoprecipitation with either IgG or α - Catenin antibody .

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(A) Endogenous association of PPM1G with α-Catenin was detected by immunoblotting with PPM1G antibody after performing immunoprecipitation with either IgG or α-Catenin antibody.


( B ) SFB - PPM1G was transfected in both Control shRNA B56δ shRNA pulled down using streptavidin beads . Interaction with α - catenin was determined by western blotting .

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(B) SFB-PPM1G was transfected in both Control shRNA and B56δ shRNA cells and pulled down using streptavidin beads. Interaction with α-catenin was determined by western blotting.


( C ) In vitro phosphorylated MBP - tagged α - Catenin was incubated with equal amounts of bacterially purified recombinant wild type catalytically inactive mutant ( D496A ) of PPM1G amount of released phosphate was assayed colorimetrically using malachite green reagent ( A620 nm ) . Data represents mean absorbance from three independent experiments . Error bars indicate SD , * * p < 0 . 01 ( one - way ANOVA , post hoc : Bonferroni ' s multiple comparison ) .

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(C) In vitro phosphorylated MBP-tagged α-Catenin was incubated with equal amounts of bacterially purified recombinant wild type and catalytically inactive mutant (D496A) of PPM1G and the amount of released phosphate was assayed colorimetrically using the malachite green reagent (A620 nm). Data represents mean absorbance from three independent experiments. Error bars indicate SD, **p<0.01 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( D ) In vitro phosphate release was performed using wild type α - Catenin S641A mutant as substrates PPM1G activity on these is plotted , n = 3 , Error bars indicate SD ; * * p < 0 . 01 ( one - way ANOVA , post hoc : Bonferroni ' s multiple comparison ) .

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(D) In vitro phosphate release assay was performed using wild type α-Catenin and S641A mutant as substrates and PPM1G activity on these proteins is plotted, n=3, Error bars indicate SD; **p<0.01 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( E ) MDA - MB - 231 breast cancer were transfected with vector , wild type PPM1G or catalytically inactive PPM1G D496A ( D / A ) mutant . of phosphorylated α - catenin were detected by using phospho - specific antibodies .

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(E) MDA-MB-231 breast cancer cells were transfected with vector, wild type PPM1G or catalytically inactive PPM1G D496A (D/A) mutant. Levels of phosphorylated α-catenin were detected by using phospho-specific antibodies.