Competition assays with MLS 1765 - 92 cells transduced with an RFP ‑ labeled NTC shRNA or an RFP - labeled shRNA against the YAP1 3 ' UTR following transduction with EV or the YAP1 coding sequence . Flow cytometric quantification of RFP ‑ positive cells on day 17 relative to day 3 showed that cell viability was rescued by expression of the shRNA ‑ resistant YAP1 cDNA . Error bars represent the mean ± SD of two independent experiments .

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

Competition assays with MLS 1765-92 cells transduced with an RFP‑labeled NTC shRNA or an RFP-labeled shRNA against the YAP1 3' UTR following transduction with EV or the YAP1 coding sequence. Flow cytometric quantification of RFP‑positive cells on day 17 relative to day 3 showed that cell viability was rescued by expression of the shRNA‑resistant YAP1 cDNA. Error bars represent the mean ± SD of two independent experiments.


Expression of total and exogenous V5 ‑ tagged YAP1 in MLS 1765 ‑ 92 cells shown in ( C ) . One of at least two independent experiments with similar results is 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

Expression of total and exogenous V5‑tagged YAP1 in MLS 1765‑92 cells shown in (C). One of at least two independent experiments with similar results is shown.


Cell viability and expression of FOXM1 and PLK1 in MLS cell lines following siRNA - mediated YAP1 knockdown . Error bars represent the mean ± SD of three independent experiments , unpaired t - test . The blots represent one of at least three independent experiments with similar results .

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

Cell viability and expression of FOXM1 and PLK1 in MLS cell lines following siRNA-mediated YAP1 knockdown. Error bars represent the mean ± SD of three independent experiments, unpaired t-test. The blots represent one of at least three independent experiments with similar results.


Flow cytometric cell cycle analysis of MLS cell lines following shRNA ‑ mediated YAP1 knockdown . Error bars represent the mean ± SD of three independent experiments , two - way ANOVA ; ns , not significant .

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Flow cytometric cell cycle analysis of MLS cell lines following shRNA‑mediated YAP1 knockdown. Error bars represent the mean ± SD of three independent experiments, two-way ANOVA; ns, not significant.


Senescence ‑ associated β ‑ galactosidase ( SABG ) staining intensity in MLS cell lines following shRNA - mediated YAP1 knockdown . Error bars represent the mean ± SD of ten random microscopic fields , two - way ANOVA ) .

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Senescence‑associated β‑galactosidase (SABG) staining intensity in MLS cell lines following shRNA-mediated YAP1 knockdown. Error bars represent the mean ± SD of ten random microscopic fields, two-way ANOVA).


Expression of CDKN1A , CDKN2A , total and phosphorylated RB1 , and TP53 in MLS cell lines following shRNA ‑ mediated YAP1 knockdown . One of at least two independent experiments with similar results is 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

Expression of CDKN1A, CDKN2A, total and phosphorylated RB1, and TP53 in MLS cell lines following shRNA‑mediated YAP1 knockdown. One of at least two independent experiments with similar results is shown.


Expression of cleaved PARP and cleaved caspase 3 / 8 in MLS cell lines following shRNA - mediated YAP1 knockdown . One of at least two independent experiments with similar results is shown .

Expression of cleaved PARP and cleaved caspase 3/8 in MLS cell lines following shRNA-mediated YAP1 knockdown. One of at least two independent experiments with similar results is shown.


Expression of YAP1 and downstream effectors in SCP - 1 cells transduced with FUS ‑ DDIT3 or EV . One of at least three independent experiments with similar results is shown .

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Expression of YAP1 and downstream effectors in SCP-1 cells transduced with FUS‑DDIT3 or EV. One of at least three independent experiments with similar results is shown.


Expression of YAP1 and downstream effectors in cytoplasmic ( yellow ) and nuclear ( blue ) fractions from SCP - 1 cells transduced with FUS ‑ DDIT3 or EV . One of at least two independent experiments with similar results is shown .

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Expression of YAP1 and downstream effectors in cytoplasmic (yellow) and nuclear (blue) fractions from SCP-1 cells transduced with FUS‑DDIT3 or EV. One of at least two independent experiments with similar results is shown.


YAP1 - responsive luciferase activity in SCP - 1 cells transduced with FUS ‑ DDIT3 . Relative luciferase activity is displayed as fold change relative to control . Error bars represent the mean ± SD of three independent experiments , unpaired t - test .

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

YAP1-responsive luciferase activity in SCP-1 cells transduced with FUS‑DDIT3. Relative luciferase activity is displayed as fold change relative to control. Error bars represent the mean ± SD of three independent experiments, unpaired t-test.


Localization of FUS - DDIT3 and YAP1 in MLS 1765 - 92 . Nuclei were counterstained with DAPI . The original magnification was x63 , and images were zoomed in four times for co - localization analysis . The correlation between red and green fluorescence was determined by Pearson coefficient analysis ( square , area for signal acquisition ) .

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

Localization of FUS-DDIT3 and YAP1 in MLS 1765-92. Nuclei were counterstained with DAPI. The original magnification was x63, and images were zoomed in four times for co-localization analysis. The correlation between red and green fluorescence was determined by Pearson coefficient analysis (square, area for signal acquisition).


Co ‑ IP of transiently expressed FUS ‑ DDIT3 and YAP1 from HEK293T cells . V5 - tagged YAP1 was pulled down using an anti - V5 antibody , and interacting proteins were detected by immunoblotting . One of at least two independent experiments with similar results is shown .

Co‑IP of transiently expressed FUS‑DDIT3 and YAP1 from HEK293T cells. V5-tagged YAP1 was pulled down using an anti-V5 antibody, and interacting proteins were detected by immunoblotting. One of at least two independent experiments with similar results is shown.


Co - IP of endogenous FUS - DDIT3 and YAP1 from MLS 1765 - 92 cells . FUS - DDIT3 was pulled down using an anti - DDIT3 antibody , and interacting proteins were detected by immunoblotting . One of at least two independent experiments with similar results is 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

Co-IP of endogenous FUS-DDIT3 and YAP1 from MLS 1765-92 cells. FUS-DDIT3 was pulled down using an anti-DDIT3 antibody, and interacting proteins were detected by immunoblotting. One of at least two independent experiments with similar results is shown.


Viability and proliferation of MLS 402 ‑ 91 , MLS 1765 ‑ 92 , and MLS 2645 - 94 cells cultured in the presence of verteporfin . Error bars represent the mean ± SEM of one representative experiment performed in quintuplicate .

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Viability and proliferation of MLS 402‑91, MLS 1765‑92, and MLS 2645-94 cells cultured in the presence of verteporfin. Error bars represent the mean ± SEM of one representative experiment performed in quintuplicate.


Flow cytometric analysis of apoptosis ( cleaved PARP ) and mitotic fraction ( phosphorylated histone H3S10 ) in MLS cell lines cultured in the presence of 0 . 25 µM Verteporfin . One of two independent experiments with similar results is 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 B-CONTROLLED_VAR O O O O O O O O O O O O

Flow cytometric analysis of apoptosis (cleaved PARP) and mitotic fraction (phosphorylated histone H3S10) in MLS cell lines cultured in the presence of 0.25 µM Verteporfin. One of two independent experiments with similar results is shown.


Expression of total YAP1 and downstream effectors ( FOXM1 and PLK1 ) in MLS cell lines treated with 0 . 5 or 1 µM verteporfin for 15 hours . One of at least three independent experiments with similar results is 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

Expression of total YAP1 and downstream effectors (FOXM1 and PLK1) in MLS cell lines treated with 0.5 or 1 µM verteporfin for 15 hours. One of at least three independent experiments with similar results is shown.


YAP1 - responsive luciferase activity in MLS cell lines transfected with a constitutively active YAP1S127A mutant and treated with 1 µM verteporfin . Relative luciferase activity is displayed relative to control . Error bars represent the mean ± SD of three independent experiments , unpaired t - test .

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

YAP1-responsive luciferase activity in MLS cell lines transfected with a constitutively active YAP1S127A mutant and treated with 1 µM verteporfin. Relative luciferase activity is displayed relative to control. Error bars represent the mean ± SD of three independent experiments, unpaired t-test.


Tumor formation on chicken CAM of MLS cell lines following shRNA - mediated YAP1 knockdown . The number of tumors is given above each bar , Fisher exact test ; ns , not significant .

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Tumor formation on chicken CAM of MLS cell lines following shRNA-mediated YAP1 knockdown. The number of tumors is given above each bar, Fisher exact test; ns, not significant.


Tumor growth on chicken CAM of MLS cell lines following treatment with 1 µM verteporfin . Shown are tumor volumes and representative photographs of tumors .

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Tumor growth on chicken CAM of MLS cell lines following treatment with 1 µM verteporfin. Shown are tumor volumes and representative photographs of tumors.


( A ) Ventricular weight to tibia length ratio ( VW / TL ) of littermate control ( LC ) or cardiomyocyte - specific NCoR1 knockout ( CMNKO ) mice . n = 10 : 9 : 12 : 7 .

(A) Ventricular weight to tibia length ratio (VW/TL) of littermate control (LC) or cardiomyocyte-specific NCoR1 knockout (CMNKO) mice. n=10:9:12:7.


( B ) Ventricular weight to body weight ratio ( VW / BW ) of LC or CMNKO mice . n = 10 : 8 : 12 : 7 .

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(B) Ventricular weight to body weight ratio (VW/BW) of LC or CMNKO mice. n=10:8:12:7.


( C ) Representative H & E staining of cross sections of left ventricles . Scale bar : 50μm .

(C) Representative H&E staining of cross sections of left ventricles. Scale bar: 50μm.


( D ) Representative wheat germ agglutinin ( WGA ) staining of cross section of left ventricles . Scale bar : 10μm .

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(D) Representative wheat germ agglutinin (WGA) staining of cross section of left ventricles. Scale bar: 10μm.


( E ) Quantification of cardiomyocyte size . n = 11 : 10 : 8 : 8 .

(E) Quantification of cardiomyocyte size. n=11:10:8:8.


( F ) qRT - PCR analysis of hypertrophy - related genes in left ventricles . n = 9 : 9 : 11 : 8 .

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(F) qRT-PCR analysis of hypertrophy-related genes in left ventricles. n=9:9:11:8.


( G ) Ejection fraction measured by echocardiography . n = 9 : 7 : 14 : 10 . Student ' s t test was used for statistical analysis .

(G) Ejection fraction measured by echocardiography. n=9:7:14:10. Student's t test was used for statistical analysis.


( H ) Fractional shortening measured by echocardiography . n = 9 : 7 : 14 : 10 . Student ' s t test was used for statistical analysis .

(H) Fractional shortening measured by echocardiography. n=9:7:14:10. Student's t test was used for statistical analysis.


( A ) Representative images of hearts from LC or CMNKO mice subjected to sham operation or AAC for 2 weeks . Scale bar : 0 . 5cm .

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(A) Representative images of hearts from LC or CMNKO mice subjected to sham operation or AAC for 2 weeks. Scale bar: 0.5cm.


( B ) VW / BW of LC or CMNKO mice 2 weeks after sham operation or AAC . n = 9 : 9 : 16 : 15 .

(B) VW/BW of LC or CMNKO mice 2 weeks after sham operation or AAC. n=9:9:16:15.


( C ) Representative H & E staining of cross sections of left ventricles . Scale bar : 25μm .

(C) Representative H&E staining of cross sections of left ventricles. Scale bar: 25μm.


( D ) Representative WGA staining . Scale bar : 10μm .

(D) Representative WGA staining. Scale bar: 10μm.


( E ) Quantification of cardiomyocyte size . n = 10 : 10 : 8 : 9 .

(E) Quantification of cardiomyocyte size. n=10:10:8:9.


( F ) qRT - PCR analysis of hypertrophy - related genes in left ventricles . n = 8 : 7 : 15 : 15 .

(F) qRT-PCR analysis of hypertrophy-related genes in left ventricles. n=8:7:15:15.


( G ) Representative picrosirius red staining of cross sections of left ventricles . Scale bar : 50μm .

(G) Representative picrosirius red staining of cross sections of left ventricles. Scale bar: 50μm.


( H ) Quantification of fibrotic areas . n = 11 : 10 : 10 : 9 .

(H) Quantification of fibrotic areas. n=11:10:10:9.


( I ) qRT - PCR analysis of fibrosis - related genes in left ventricles . n = 8 : 7 : 15 : 15 .

(I) qRT-PCR analysis of fibrosis-related genes in left ventricles. n=8:7:15:15.


( J ) Ejection fraction measured by echocardiography . n = 9 : 7 : 14 : 12 .

(J) Ejection fraction measured by echocardiography. n=9:7:14:12.


( K ) Fractional shortening measured by echocardiography . n = 9 : 7 : 14 : 12 .

(K) Fractional shortening measured by echocardiography. n=9:7:14:12.


( L ) Lung weight to body weight ratio ( LW / BW ) of LC or CMNKO mice 2 weeks after sham operation or AAC . n = 8 : 7 : 16 : 15 .

(L) Lung weight to body weight ratio (LW/BW) of LC or CMNKO mice 2 weeks after sham operation or AAC. n=8:7:16:15.


( M ) Cumulative survival rate of LC or CMNKO mice subjected to AAC . n = 12 : 10 . Two - way ANOVA followed by Bonferoni post - tests was used for statistical analysis .

(M) Cumulative survival rate of LC or CMNKO mice subjected to AAC. n=12:10. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis.


( A ) Western blotting analysis of NCoR1 in neonatal rat ventricular myocytes ( NRVMs ) transfected with siControl or siNCoR1 for 3 days . siControl indicates control siRNA ; siNCoR1 , NCoR1 siRNA .

(A) Western blotting analysis of NCoR1 in neonatal rat ventricular myocytes (NRVMs) transfected with siControl or siNCoR1 for 3 days. siControl indicates control siRNA; siNCoR1, NCoR1 siRNA.


( B ) Representative immunofluorescence staining of α - Actinin in NRVMs transfected with siRNA for 48h and then treated with vehicle ( H2O ) or phenylephrine ( PE ) for another 48h . Scale bar : 50μm .

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(B) Representative immunofluorescence staining of α-Actinin in NRVMs transfected with siRNA for 48h and then treated with vehicle (H2O) or phenylephrine (PE) for another 48h. Scale bar: 50μm.


( C ) Quantification of the surface area of α - Actinin - positive NRVMs with or without NCoR1 knockdown . A total of 40 NRVMs was randomly chosen from 4 replicate coverslips for each group and used for statistical analysis .

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(C) Quantification of the surface area of α-Actinin-positive NRVMs with or without NCoR1 knockdown. A total of 40 NRVMs was randomly chosen from 4 replicate coverslips for each group and used for statistical analysis.


( D ) qRT - PCR analysis of Acta1 and Nppa in NRVMs with or without NCoR1 knockdown . n = 4 : 4 .

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(D) qRT-PCR analysis of Acta1 and Nppa in NRVMs with or without NCoR1 knockdown. n=4:4.


( E ) Western blotting analysis of NCoR1 - flag in NRVMs infected by control lentivirus ( Control ) or NCoR1 - flag lentivirus ( NCoR1 - OV ) for 4 days .

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(E) Western blotting analysis of NCoR1-flag in NRVMs infected by control lentivirus (Control) or NCoR1-flag lentivirus (NCoR1-OV) for 4 days.


( F ) Representative immunofluorescence staining of α - Actinin in NRVMs infected with lentivirus for 48h and then treated with vehicle or PE for another 48h . Scale bar : 50μm .

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

(F) Representative immunofluorescence staining of α-Actinin in NRVMs infected with lentivirus for 48h and then treated with vehicle or PE for another 48h. Scale bar: 50μm.


( G ) Quantification of the surface area of α - Actinin - positive NRVMs with or without NCoR1 overexpression . A total of 30 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis .

(G) Quantification of the surface area of α-Actinin-positive NRVMs with or without NCoR1 overexpression. A total of 30 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis.


( H ) qRT - PCR analysis of Acta1 and Nppa in NRVMs with or without NCoR1 overexpression . n = 4 : 3 . Data represent three independent experiments . Two - way ANOVA followed by Bonferoni post - tests was used for statistical analysis .

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

(H) qRT-PCR analysis of Acta1 and Nppa in NRVMs with or without NCoR1 overexpression. n=4:3. Data represent three independent experiments. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis.


( A ) Representative immunofluorescence staining of α - Actinin in NRVMs transfected with siRNA for 48h and then treated with vehicle ( H2O ) or PE for another 48h . Scale bar : 50μm . siMEF2a indicates MEF2a siRNA .

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

(A) Representative immunofluorescence staining of α-Actinin in NRVMs transfected with siRNA for 48h and then treated with vehicle (H2O) or PE for another 48h. Scale bar: 50μm. siMEF2a indicates MEF2a siRNA.


( B ) Quantification of the surface area of α - Actinin - positive NRVMs with or without knockdown of NCoR1 and / or MEF2a . A total of 15 - 20 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis .

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(B) Quantification of the surface area of α-Actinin-positive NRVMs with or without knockdown of NCoR1 and/or MEF2a. A total of 15-20 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis.


( C ) Representative immunofluorescence staining of α - Actinin in NRVMs transfected with siRNA for 48h and then treated with vehicle or PE for another 48h . Scale bar : 50μm . siMEF2d indicates MEF2d siRNA .

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

(C) Representative immunofluorescence staining of α-Actinin in NRVMs transfected with siRNA for 48h and then treated with vehicle or PE for another 48h. Scale bar: 50μm. siMEF2d indicates MEF2d siRNA.


( D ) Quantification of the surface area of α - Actinin - positive NRVMs with or without knockdown of NCoR1 and / or MEF2d . A total of 15 - 19 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis .

(D) Quantification of the surface area of α-Actinin-positive NRVMs with or without knockdown of NCoR1 and/or MEF2d. A total of 15-19 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis.


( E ) qRT - PCR analysis of Acta1 and Nppa in NRVMs . n = 4 . Data represent three independent experiments . Student ' s t test was used for statistical analysis .

(E) qRT-PCR analysis of Acta1 and Nppa in NRVMs. n=4. Data represent three independent experiments. Student's t test was used for statistical analysis.


( A ) Luciferase assays of Acta1 and Nppa promoters in HEK293FT cells transfected with NCoR1 - Flag and / or MEF2a - HA or with empty plasmids . Student ' s t test was used for statistical

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(A) Luciferase assays of Acta1 and Nppa promoters in HEK293FT cells transfected with NCoR1-Flag and/or MEF2a-HA or with empty plasmids. Student's t test was used for statistical analysis


( B ) Co - immunoprecipitation ( Co - IP ) analysis of NCoR1 and MEF2a in HEK293FT cells transfected with full - length NCoR1 - Flag and MEF2a - HA .

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(B) Co-immunoprecipitation (Co-IP) analysis of NCoR1 and MEF2a in HEK293FT cells transfected with full-length NCoR1-Flag and MEF2a-HA.


( C ) Co - IP analysis of NCoR1 and truncated MEF2a in HEK293FT cells . Schematic illustration of MEF2a - HA construct is shown above the Co - IP results .

(C) Co-IP analysis of NCoR1 and truncated MEF2a in HEK293FT cells. Schematic illustration of MEF2a-HA construct is shown above the Co-IP results.


( D ) Co - IP analysis of MEF2a and truncated NCoR1 in HEK293FT cells . Schematic illustration of NCoR1 - Flag construct is shown above the Co - IP results .

(D) Co-IP analysis of MEF2a and truncated NCoR1 in HEK293FT cells. Schematic illustration of NCoR1-Flag construct is shown above the Co-IP results.


( E ) Luciferase assays of Acta1 and Nppa promoters in HEK293FT cells transfected with full - length MEF2a and domain - specific NCoR1 . Student ' s t test was used for statistical

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(E) Luciferase assays of Acta1 and Nppa promoters in HEK293FT cells transfected with full-length MEF2a and domain-specific NCoR1. Student's t test was used for statistical analysis


( F ) Western blotting analysis of NCoR1 - flag in NRVMs infected by control lentivirus ( Control ) or NCoR1 ( 1939 - 2453 ) - flag lentivirus [ NCoR1 ( 1939 - 2453 ) - OV ] for 4 days .

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(F) Western blotting analysis of NCoR1-flag in NRVMs infected by control lentivirus (Control) or NCoR1 (1939-2453)-flag lentivirus [NCoR1(1939-2453)-OV] for 4 days.


( G ) Representative immunofluorescence staining of α - Actinin in NRVMs infected with lentivirus for 48h and then treated with vehicle ( DMSO ) or PE for another 48h . Scale bar : 50μm .

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

(G) Representative immunofluorescence staining of α-Actinin in NRVMs infected with lentivirus for 48h and then treated with vehicle (DMSO) or PE for another 48h. Scale bar: 50μm.


( H ) Quantification of the surface area of α - Actinin - positive NRVMs . A total of 30 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis . Two - way ANOVA followed by Bonferoni post - tests was used for statistical

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

(H) Quantification of the surface area of α-Actinin-positive NRVMs. A total of 30 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis


( I ) qRT - PCR analysis of Acta1 and Nppa in NRVMs . n = 4 : 4 . Data represent three independent experiments . Two - way ANOVA followed by Bonferoni post - tests was used for statistical

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

(I) qRT-PCR analysis of Acta1 and Nppa in NRVMs. n=4:4. Data represent three independent experiments. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis


( A ) ChIP analysis showing enrichment of NCoR1 in MEF2 binding regions on the promoters of Acta1 and Nppa in ventricular samples .

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(A) ChIP analysis showing enrichment of NCoR1 in MEF2 binding regions on the promoters of Acta1 and Nppa in ventricular samples.


( B ) ChIP analysis of enrichment of MEF2a on promoters of Acta1 and Nppa in ventricular samples . n = 3 : 3 .

(B) ChIP analysis of enrichment of MEF2a on promoters of Acta1 and Nppa in ventricular samples. n=3:3.


ChIP analysis of enrichment of HDAC4 ( C ) on promoters of Acta1 and Nppa in ventricular samples . n = 4 : 4 for ( C )

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ChIP analysis of enrichment of HDAC4 (C) on promoters of Acta1 and Nppa in ventricular samples. n=4:4 for (C)


ChIP analysis of enrichment of HDAC5 ( D ) on promoters of Acta1 and Nppa in ventricular samples . n = 3 : 3 for (

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ChIP analysis of enrichment of HDAC5 (D) on promoters of Acta1 and Nppa in ventricular samples. n=3:3 for (D


ChIP analysis of enrichment of , acetylated - Histone 4 ( ac - H4 ) ( E ) on promoters of Acta1 and Nppa in ventricular samples . , n = 3 :

ChIP analysis of enrichment of , acetylated-Histone 4 (ac-H4) (E) on promoters of Acta1 and Nppa in ventricular samples. , n=3:3


ChIP analysis of enrichment of ac - H3 ( F ) on promoters of Acta1 and Nppa in ventricular samples . n = 3 :

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ChIP analysis of enrichment of ac-H3 (F) on promoters of Acta1 and Nppa in ventricular samples. n=3:3


( G ) ChIP analysis of RNA Polymerase II enrichment on the transcriptional regions of Acta1 and Nppa in ventricular samples . n = 3 : 3 .

(G) ChIP analysis of RNA Polymerase II enrichment on the transcriptional regions of Acta1 and Nppa in ventricular samples. n=3:3.


( A ) Western blotting analysis of RIDs of NCoR1 in left ventricular samples from mice infected with AAV9 GFP or AAV9 NCoR1 - RIDs ( 1939 - 2453 ) - Flag .

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(A) Western blotting analysis of RIDs of NCoR1 in left ventricular samples from mice infected with AAV9 GFP or AAV9 NCoR1-RIDs (1939-2453)-Flag.


( B ) VW / BW of mice 2 weeks after sham operation or AAC . n = 7 : 7 : 12 : 12 .

(B) VW/BW of mice 2 weeks after sham operation or AAC. n=7:7:12:12.


( C ) Representative H & E staining of cross sections of left ventricles . Scale bar : 10μm .

(C) Representative H&E staining of cross sections of left ventricles. Scale bar: 10μm.


( D ) Representative WGA staining . Scale bar : 10μm .

(D) Representative WGA staining. Scale bar: 10μm.


( E ) Quantification of cardiomyocyte size . n = 6 : 6 : 7 : 8 .

(E) Quantification of cardiomyocyte size. n=6:6:7:8.


( F ) qRT - PCR analysis of hypertrophy - related genes in left ventricles . n = 8 : 8 : 11 : 11 .

(F) qRT-PCR analysis of hypertrophy-related genes in left ventricles. n=8:8:11:11.


( G ) Representative picrosirius red staining of cross sections of left ventricles . Scale bar : 20μm .

(G) Representative picrosirius red staining of cross sections of left ventricles. Scale bar: 20μm.


( H ) Quantification of fibrotic areas . n = 5 : 5 : 7 : 7 .

(H) Quantification of fibrotic areas. n=5:5:7:7.


( I ) qRT - PCR analysis of fibrosis - related genes in left ventricles . n = 8 : 8 : 11 : 11 .

(I) qRT-PCR analysis of fibrosis-related genes in left ventricles. n=8:8:11:11.


( J ) Ejection fraction measured before , 2 weeks and 4 weeks after AAC . n = 6 : 6 . Two - way ANOVA followed by Bonferoni post - tests was used for statistical analysis .

(J) Ejection fraction measured before, 2 weeks and 4 weeks after AAC. n=6:6. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis.


( K ) Fractional shortening measured before , 2 weeks and 4 weeks after AAC . n = 6 : 6 . Two - way ANOVA followed by Bonferoni post - tests was used for statistical analysis .

(K) Fractional shortening measured before, 2 weeks and 4 weeks after AAC. n=6:6. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis.


( A ) Representative immunofluorescence images of Sec31A marked ERES in HeLa cells cultured on crossbow shaped micropatterned surface of small ( upper panel ) or large ( lower panel ) size for 4 hours . Cells were treated with DMSO ( Ctrl ) or 50 µM NSC23766 ( Rac1 inhibitor ) for 4 hours prior to fixation and immunostaining . The number of ERES per cell for each pattern size in HeLa ( B ) The number of ERES was quantified from at least 74 cells per condition for HeLa ( B ) Each dot represents an individual cell .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative immunofluorescence images of Sec31A marked ERES in HeLa cells cultured on crossbow shaped micropatterned surface of small (upper panel) or large (lower panel) size for 4 hours. Cells were treated with DMSO (Ctrl) or 50 µM NSC23766 (Rac1 inhibitor) for 4 hours prior to fixation and immunostaining. The number of ERES per cell for each pattern size in HeLa (B) The number of ERES was quantified from at least 74 cells per condition for HeLa (B) Each dot represents an individual cell.


( A ) Representative immunofluorescence images of HeLa cells cultured on micropatterned surface of multiple geometries . Cells were transfected with 10 nM non - targeting control ( siCtrl ) or co - transfected with siRNAs targeting Sar1A and Sar1B . After 48 h , cells were trypsinized and 80 , 000 cells were seeded on micropatterned chips . Cells were allowed to attach for 3 h , stained with CellMask for 5 min and processed for microscopy . Scale bars : 5 µm ( B ) Quantification graph showing the percentage of cells failing to entirely cover the different geometries ( described in A ) . Data derived from at least 100 cells from a set of three independent experiments . Error bars represent standard deviation . Asterisks ( * ) denote statistical significance ( p - value < 0 . 05 ; chi - squared test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative immunofluorescence images of HeLa cells cultured on micropatterned surface of multiple geometries. Cells were transfected with 10 nM non-targeting control (siCtrl) or co-transfected with siRNAs targeting Sar1A and Sar1B. After 48 h, cells were trypsinized and 80,000 cells were seeded on micropatterned chips. Cells were allowed to attach for 3 h, stained with CellMask for 5 min and processed for microscopy. Scale bars: 5 µm (B) Quantification graph showing the percentage of cells failing to entirely cover the different geometries (described in A). Data derived from at least 100 cells from a set of three independent experiments. Error bars represent standard deviation. Asterisks (*) denote statistical significance (p-value < 0.05; chi-squared test).


( D ) Representative immunofluorescence images of HeLa cells cultured on micropatterned surface of multiple geometries . Cells were treated with 50 µM of the Rac1 inhibitor NSC23766 for 4 hours prior to staining with CellMask and processing for immunofluorescence microscopy . ( E ) Quantification of ( D ) . Percentage of cells failing to cover entire micropatterned surface is shown for control ( ctrl ) and the Rac1 inhibitor NSC23766 . Between 113 - 117 cells were counted in total from a set of three independent experiments .

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

(D) Representative immunofluorescence images of HeLa cells cultured on micropatterned surface of multiple geometries. Cells were treated with 50 µM of the Rac1 inhibitor NSC23766 for 4 hours prior to staining with CellMask and processing for immunofluorescence microscopy. (E) Quantification of (D). Percentage of cells failing to cover entire micropatterned surface is shown for control (ctrl) and the Rac1 inhibitor NSC23766. Between 113 - 117 cells were counted in total from a set of three independent experiments.


( A ) HeLa cells were transfected with 10 nM non - targeting control siRNA ( siCtrl ) or siRNAs targeting Rac1 ( siRac1 ) . After 72 h , cells were fixed and processed for immunostaining against Sec31A to label ERES . Representative confocal microscopy images are shown . ( B ) Quantification graph shows the number of ERES / cell in cells transfected with control siRNA ( siCtrl ) or siRNA against Rac1 ( two siRNAs # 1 and # 2 ) . Values are expressed as % of siCtrl . From three independent experiments , with > 30 cells per condition were counted . ( C ) Representative immunofluorescence images showing ERES in HeLa cells treated with DMSO ( Ctrl ) , Rac1 inhibitor NSC23766 ( 50 µM , 4 h ) , or NSC23766 washout ( 50 µM for 4h , then washout for 2h ) . ( D ) Graph shows the number of ERES per cell ( displayed as % of Ctrl ) derived from at least 30 cells per condition from three experiments .

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(A) HeLa cells were transfected with 10 nM non-targeting control siRNA (siCtrl) or siRNAs targeting Rac1 (siRac1). After 72 h, cells were fixed and processed for immunostaining against Sec31A to label ERES. Representative confocal microscopy images are shown. (B) Quantification graph shows the number of ERES/cell in cells transfected with control siRNA (siCtrl) or siRNA against Rac1 (two siRNAs #1 and #2). Values are expressed as % of siCtrl. From three independent experiments, with >30 cells per condition were counted. (C) Representative immunofluorescence images showing ERES in HeLa cells treated with DMSO (Ctrl), Rac1 inhibitor NSC23766 (50 µM, 4 h), or NSC23766 washout (50 µM for 4h, then washout for 2h). (D) Graph shows the number of ERES per cell (displayed as % of Ctrl) derived from at least 30 cells per condition from three experiments.


( E ) Fluorescence recovery after photobleaching ( FRAP ) of GFP - Sec16A marked ERES in HeLa cells . Images show an individual ERES before ( - 1 ) , immediately after ( 0 ) and 28 sec after photobleaching . The graph illustrates FRAP analysis of individual ERES from 23 ( for control ) and 24 ( for NSC23766 ) regions in 3 experiments . Line connects individual time points . Data are ± SD . ( F ) Quantification of the mobile fraction of GFP - Sec16A in control and Rac1 inhibited cells . Mobile fraction is derived from a total of 23 ( Ctrl ) and 24 ( NSC23766 ) individual ERES from three independent experiments .

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

(E) Fluorescence recovery after photobleaching (FRAP) of GFP-Sec16A marked ERES in HeLa cells. Images show an individual ERES before (-1), immediately after (0) and 28 sec after photobleaching. The graph illustrates FRAP analysis of individual ERES from 23 (for control) and 24 (for NSC23766) regions in 3 experiments. Line connects individual time points. Data are ± SD. (F) Quantification of the mobile fraction of GFP-Sec16A in control and Rac1 inhibited cells. Mobile fraction is derived from a total of 23 (Ctrl) and 24 (NSC23766) individual ERES from three independent experiments.


( G ) The rate of ER - export was monitored using GFP - ManII - RUSH in HeLa cells after perturbation of Rac1 with siRNA ( siRac1 ) , or Rac1 inhibitor NSC23766 , or a combination of both . Representative images show GFP - ManII - RUSH distribution in HeLa cells at indicated time points . ( H ) Quantification shows ratio of ManII fluorescence intensity within Golgi to outside Golgi region after addition of biotin at indicated time points . Between 76 - 104 cells were used for measurement of ManII intensity in 3 experiments .

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(G) The rate of ER-export was monitored using GFP-ManII-RUSH in HeLa cells after perturbation of Rac1 with siRNA (siRac1), or Rac1 inhibitor NSC23766, or a combination of both. Representative images show GFP-ManII-RUSH distribution in HeLa cells at indicated time points. (H) Quantification shows ratio of ManII fluorescence intensity within Golgi to outside Golgi region after addition of biotin at indicated time points. Between 76 - 104 cells were used for measurement of ManII intensity in 3 experiments.


( A ) Co - occurrence of mCherry - Rac1 and GFP - Sec61β at the endoplasmic reticulum ( ER ) in HeLa cells stably expressing mCherry - Rac1 and transiently expressing GFP - Sec61β as monitored by confocal microscopy . SRRF imaging mode was used in Andor DragonFly spinning disk confocal microscope for obtaining the image . Scale bar : 5 µm and 1 µm ( inset ) . See also movieEV3 . ( B ) Line profile of two marked spots from ( A ) showing the overlap between mCherry - Rac1 and GFP - Sec61β signal at the ER .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-occurrence of mCherry-Rac1 and GFP-Sec61β at the endoplasmic reticulum (ER) in HeLa cells stably expressing mCherry-Rac1 and transiently expressing GFP-Sec61β as monitored by confocal microscopy. SRRF imaging mode was used in Andor DragonFly spinning disk confocal microscope for obtaining the image. Scale bar: 5 µm and 1 µm (inset). See also movieEV3. (B) Line profile of two marked spots from (A) showing the overlap between mCherry-Rac1 and GFP-Sec61β signal at the ER.


( E ) Representative images showing Sec31A marked ERES in HeLa cells transfected with indicated plasmids . HeLa cells were transfected with the plasmids 16 h before immunolabelling with Sec31A . Scale bar : 5 µm . ( F ) Quantification of ERES number in HeLa cells from the experiment described in C . Between 90 - 100 cells were quantified from 4 independent experiments . Average ERES per cell ± standard deviation is shown . Each dot represents average ERES / cell from an experiment .

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(E) Representative images showing Sec31A marked ERES in HeLa cells transfected with indicated plasmids. HeLa cells were transfected with the plasmids 16 h before immunolabelling with Sec31A. Scale bar: 5 µm. (F) Quantification of ERES number in HeLa cells from the experiment described in C. Between 90-100 cells were quantified from 4 independent experiments. Average ERES per cell ± standard deviation is shown. Each dot represents average ERES/cell from an experiment.


( G ) Images represent ERES in HeLa cells that were transfected with the indicated plasmids and cultured on the crossbow micropattern for 4 hours . Cells were transfected with the plasmids 16 h before they were trypsinized and seeded on the micropatterns for ERES staining . ( H ) The dot plot shows ERES count in individual cells from ( G ) . Mean ± standard deviation is shown for each condition . At least 58 cells were used for counting ERES number . Independent experiments were performed 3 times .

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(G) Images represent ERES in HeLa cells that were transfected with the indicated plasmids and cultured on the crossbow micropattern for 4 hours. Cells were transfected with the plasmids 16 h before they were trypsinized and seeded on the micropatterns for ERES staining. (H) The dot plot shows ERES count in individual cells from (G). Mean ± standard deviation is shown for each condition. At least 58 cells were used for counting ERES number. Independent experiments were performed 3 times.


( I ) Merged confocal images show distribution of Rac1 specific GEF TIAM ( magenta ) in HeLa cells before ( 0 ) , and after optogenetic recruitment at indicated time points . CIBN - Sec61β ( green ) was used to recruit Cry2 - TIAM to the ER with 488 nm laser . ( J ) Graph shows the number of SNAP - Sec16A marked ERES in HeLa cells before ( 0 ) and after ( 30 , 60 and 90 seconds ) recruitment of TIAM to the ER from 2 independent experiments . Whiskers indicate min - max range and central band indicates the median of all data . Number of cells = 9 .

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(I) Merged confocal images show distribution of Rac1 specific GEF TIAM (magenta) in HeLa cells before (0), and after optogenetic recruitment at indicated time points. CIBN-Sec61β (green) was used to recruit Cry2-TIAM to the ER with 488 nm laser. (J) Graph shows the number of SNAP-Sec16A marked ERES in HeLa cells before (0) and after (30, 60 and 90 seconds) recruitment of TIAM to the ER from 2 independent experiments. Whiskers indicate min-max range and central band indicates the median of all data. Number of cells = 9.


( A and B ) Representative immunofluorescence images showing Sec31A labelled ERES in HeLa cells treated with DMSO ( Ctrl ) , 0 . 5 µM cytochalasin D ( CytoD ) and 0 . 5 µM latrunculin A ( LatA ) for 15 and 30 min ( A ) . Scale bar : 5 µm . The number of ERES per cell in HeLa cells treated with different doses of CytoD and LatA for 30 min are displayed as % of control ± standard deviation ( 90 - 100 cells from 3 experiments ) ( B ) .

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

(A and B) Representative immunofluorescence images showing Sec31A labelled ERES in HeLa cells treated with DMSO (Ctrl), 0.5 µM cytochalasin D (CytoD) and 0.5 µM latrunculin A (LatA) for 15 and 30 min (A). Scale bar: 5 µm. The number of ERES per cell in HeLa cells treated with different doses of CytoD and LatA for 30 min are displayed as % of control ± standard deviation (90 -100 cells from 3 experiments) (B).


( C ) Distribution of ManII - RUSH in HeLa cells . Cells were pre - treated with DMSO ( Ctrl ) or 1 µM LatA for 15 min prior to addition of biotin to initiate ManII - trafficking . Golgi arrival for ManII - RUSH was monitored for 30 min .

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

(C) Distribution of ManII-RUSH in HeLa cells. Cells were pre-treated with DMSO (Ctrl) or 1 µM LatA for 15 min prior to addition of biotin to initiate ManII-trafficking. Golgi arrival for ManII-RUSH was monitored for 30 min.


( D - E ) Graph showing ratio of ManII within Golgi to outside Golgi . Values were derived from HeLa cells treated with LatA or CytoD as described in C . At least 30 cells per condition were used for quantification in each experiment ( n = 3 ) .

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(D-E) Graph showing ratio of ManII within Golgi to outside Golgi. Values were derived from HeLa cells treated with LatA or CytoD as described in C. At least 30 cells per condition were used for quantification in each experiment (n = 3).


( F ) HeLa cells cultured on PDMS membranes were treated with DMSO or 1 µM cytochalasin D ( CytoD ) and ERES were labelled with anti - Sec31A antibody before ( no strain ) or after 15 % equibiaxial strain ( 15 % strain ) . Anti - phalloidin staining was performed to visualize actin in cells . Representative images are shown . ( G ) Quantification of experiment described in ( F ) . The graph shows average ERES / Cell . Data were derived from a total of 90 - 115 cells from 3 experiments . Each dot represents several cells and error bars show standard deviation .

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

(F) HeLa cells cultured on PDMS membranes were treated with DMSO or 1 µM cytochalasin D (CytoD) and ERES were labelled with anti-Sec31A antibody before (no strain) or after 15% equibiaxial strain (15% strain). Anti-phalloidin staining was performed to visualize actin in cells. Representative images are shown. (G) Quantification of experiment described in (F). The graph shows average ERES/Cell. Data were derived from a total of 90 - 115 cells from 3 experiments. Each dot represents several cells and error bars show standard deviation.


( H and I ) siRNA mediated depletion of INF2 does not phenocopy Rac1 effect . HeLa cells were transfected with non - targeting control siRNA ( siCtrl ) or siRNA against INF2 ( siINF2 ) for 72 h before processing cells immunostaining with Sec31A to quantify ERES . Representative images showing Sec31A - labelled ERES in HeLa cells ( H ) , and quantification of ERES per cell presented as % of control ( I ) . In total 137 cells ( siCtrl ) and 113 cells ( siINF2 ) were used for ERES quantification from three independent experiments .

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

(H and I) siRNA mediated depletion of INF2 does not phenocopy Rac1 effect. HeLa cells were transfected with non-targeting control siRNA (siCtrl) or siRNA against INF2 (siINF2) for 72 h before processing cells immunostaining with Sec31A to quantify ERES. Representative images showing Sec31A-labelled ERES in HeLa cells (H), and quantification of ERES per cell presented as % of control (I). In total 137 cells (siCtrl) and 113 cells (siINF2) were used for ERES quantification from three independent experiments.


( J and K ) Knockdown of INF2 does not alter the kinetics of ER - to - Golgi transport . HeLa cells stably expressing ManII - RUSH were transfected with siRNAs as described in E , and the ER - to - Golgi transport of ManII - RUSH was followed by confocal microscopy ( J ) . Representative images for indicated time points are shown . Scale bars , 5 µm . The ratio of ManII - RUSH within the Golgi to outside the Golgi was calculated for the indicated time points ( K ) . Three independent experiments were performed and a total of 82 ( siCtrl , 0 min ) , 72 ( siINF2 , 0 min ) and 93 ( siCtrl and siINF2 , 20 min ) were used for quantification .

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(J and K) Knockdown of INF2 does not alter the kinetics of ER-to-Golgi transport. HeLa cells stably expressing ManII-RUSH were transfected with siRNAs as described in E, and the ER-to-Golgi transport of ManII-RUSH was followed by confocal microscopy (J). Representative images for indicated time points are shown. Scale bars, 5 µm. The ratio of ManII-RUSH within the Golgi to outside the Golgi was calculated for the indicated time points (K). Three independent experiments were performed and a total of 82 (siCtrl, 0 min), 72 (siINF2, 0 min) and 93 (siCtrl and siINF2, 20 min) were used for quantification.


( A ) Co - localization of mCherry - Rac1 and GFP - Sec16A in HeLa cells as monitored by SRRF live cell imaging . See also movieS4 .

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(A) Co-localization of mCherry-Rac1 and GFP-Sec16A in HeLa cells as monitored by SRRF live cell imaging. See also movieS4.


( B ) Representative immunofluorescence images of HeLa cells co - transfected with YFP ( N ) - Rac1 and Sar1 - YFP ( C ) and immunostained with Sec31A . Rac1 and Sar1 form complexes at ERES in HeLa cells , which partially overlap with Sec31A . ( C ) Box plot showing the fraction of ERES that overlap with the Rac1 - Sar1 complex ( number of cells = 24 , number of experiments = 4 ) . Individual data points shown in the graph represent number of images . Whiskers indicate min - max range and central band indicates the median of all data .

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(B) Representative immunofluorescence images of HeLa cells co-transfected with YFP(N)-Rac1 and Sar1-YFP(C) and immunostained with Sec31A. Rac1 and Sar1 form complexes at ERES in HeLa cells, which partially overlap with Sec31A. (C) Box plot showing the fraction of ERES that overlap with the Rac1-Sar1 complex (number of cells = 24, number of experiments = 4). Individual data points shown in the graph represent number of images. Whiskers indicate min-max range and central band indicates the median of all data.


( D ) GFP - tagged Rac1 was immunoprecipitated by GFP - trap and the fraction was immunoblotted for endogenous Sar1 . Representative immunoblot showing co - immunoprecipitation of Sar1 by Rac1 .

O O O O O O O O O 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) GFP-tagged Rac1 was immunoprecipitated by GFP-trap and the fraction was immunoblotted for endogenous Sar1. Representative immunoblot showing co-immunoprecipitation of Sar1 by Rac1.


( E ) Representative immunofluorescence images of HeLa cells co - transfected with YFP ( N ) - Rac1 and Sar1 - YFP ( C ) and cultured on crossbow micropatterns of indicated size . Cells were co - transfected with the indicated DNA constructs and incubated overnight prior to trypsinization and seeding on the micropatterned coverslip . Cells were allowed to grow for 3h , fixed and processed for microscopy . ( F ) Quantification of Rac1 - Sar1 puncta described in ( E ) ( number of experiments = 3 , number of cells = 28 ( small ) and 27 ( large ) ) . In the quantification graph , error bars represent standard deviation and asterisk ( * ) denotes statistical significance at p - value < 0 . 05 .

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(E) Representative immunofluorescence images of HeLa cells co-transfected with YFP(N)-Rac1 and Sar1-YFP(C) and cultured on crossbow micropatterns of indicated size. Cells were co-transfected with the indicated DNA constructs and incubated overnight prior to trypsinization and seeding on the micropatterned coverslip. Cells were allowed to grow for 3h, fixed and processed for microscopy. (F) Quantification of Rac1-Sar1 puncta described in (E) (number of experiments = 3, number of cells = 28 (small) and 27 (large)). In the quantification graph, error bars represent standard deviation and asterisk (*) denotes statistical significance at p-value < 0.05.


( E ) Cells expressing GFP - Rac1 were lysed followed by immunoprecipitation against GFP and immunoblotting against endogenous Sar1 and Sec12 . Blotting against GFP was performed to determine efficiency of the IP .

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(E) Cells expressing GFP-Rac1 were lysed followed by immunoprecipitation against GFP and immunoblotting against endogenous Sar1 and Sec12. Blotting against GFP was performed to determine efficiency of the IP.


( F ) HeLa cells were co - transfected with the indicated plasmids and cells were incubated overnight prior to cell lysis and immunoprecipitation against GFP . Immunoblotting against anti - mCherry was performed to detect Sar1A - mCherry . The IP efficiency was determined using anti - GFP . Different exposure times are shown for anti - mCherry blot .

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(F) HeLa cells were co-transfected with the indicated plasmids and cells were incubated overnight prior to cell lysis and immunoprecipitation against GFP. Immunoblotting against anti-mCherry was performed to detect Sar1A-mCherry. The IP efficiency was determined using anti-GFP. Different exposure times are shown for anti-mCherry blot.


( A ) Microsomes isolated from HeLa cells were incubated as indicated . Active Sar1 pulldown was then carried out , followed by immunoblotting against Sar1 .

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(A) Microsomes isolated from HeLa cells were incubated as indicated. Active Sar1 pulldown was then carried out, followed by immunoblotting against Sar1.


( B ) Microsomes ( M ) from HeLa cells stably expressing mCherry - ManII - RUSH were incubated with ATP and an ATP - regenerating system , GTP , biotin and cytosol in the presence or absence of recombinant Rac1 for 30 min at 25 ° C . COPII vesicles ( V ) were pelleted by ultracentrifugation ( 100 , 000 xg ) and immunoblotted for anti - mCherry to detect ManII - RUSH and anti - calnexin ( ER marker ) . ( C ) Quantification data of two independent vesicle budding assays . All values were normalized to 4 ℃ Ctrl condition .

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(B) Microsomes (M) from HeLa cells stably expressing mCherry-ManII-RUSH were incubated with ATP and an ATP-regenerating system, GTP, biotin and cytosol in the presence or absence of recombinant Rac1 for 30 min at 25°C. COPII vesicles (V) were pelleted by ultracentrifugation (100,000 xg) and immunoblotted for anti-mCherry to detect ManII-RUSH and anti-calnexin (ER marker). (C) Quantification data of two independent vesicle budding assays. All values were normalized to 4 ℃ Ctrl condition.


Entry of MLV pseudotyped with SARS - CoV - 2 S , SARS - CoV - 2 Sfur / mut and SARS - CoV S in VeroE6 cells .

Entry of MLV pseudotyped with SARS-CoV-2 S, SARS-CoV-2 Sfur/mut and SARS-CoV S in VeroE6 cells.


Entry of MLV pseudotyped with SARS - CoV - 2 S or SARS - CoV - 2 Sfur / mut in BHK cells transiently transfected with hACE2 . The experiments were carried out in triplicate with two independent pseudovirus preparations and a representative experiment is shown .

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Entry of MLV pseudotyped with SARS-CoV-2 S or SARS-CoV-2 Sfur/mut in BHK cells transiently transfected with hACE2. The experiments were carried out in triplicate with two independent pseudovirus preparations and a representative experiment is shown.


. Western blot analysis of SARS - CoV - 2 S - MLV , SARS - CoV - 2 Sfur / mut - MLV and SARS - CoV S - MLV pseudovirions using an anti - SARS - CoV S2 antibody .

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. Western blot analysis of SARS-CoV-2 S-MLV, SARS-CoV-2 Sfur/mut-MLV and SARS-CoV S-MLV pseudovirions using an anti-SARS-CoV S2 antibody.


Biolayer interferometry binding analysis of the hACE2 ectodomain to immobilized SARS - CoV - 2 SB ( B ) or SARS - CoV

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Biolayer interferometry binding analysis of the hACE2 ectodomain to immobilized SARS-CoV-2 SB (B) or SARS-CoV SB


Two orthogonal views of the closed SARS - CoV - 2 S trimer cryoEM map . C . Atomic model of the closed SARS - CoV - 2 S trimer in the same orientation as in panel A .

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Two orthogonal views of the closed SARS-CoV-2 S trimer cryoEM map. C. Atomic model of the closed SARS-CoV-2 S trimer in the same orientation as in panel A.


D - E . Two orthogonal views of the partially open SARS - CoV - 2 S trimer cryoEM map ( one SB domain is open ) . F . Atomic model of the closed SARS - CoV - 2 S trimer in the same orientation as in panel D . The glycans were omitted for clarity .

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D-E. Two orthogonal views of the partially open SARS-CoV-2 S trimer cryoEM map (one SB domain is open). F. Atomic model of the closed SARS-CoV-2 S trimer in the same orientation as in panel D. The glycans were omitted for clarity.


Entry of SARS - CoV - 2 and SARS - CoV S pseudotyped viruses is potently inhibited by four SARS - CoV S mouse polyclonal immune plasma .

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Entry of SARS-CoV-2 and SARS-CoV S pseudotyped viruses is potently inhibited by four SARS-CoV S mouse polyclonal immune plasma.


A Western blots of acid - extracted histones from activated CD4 + T cells showing global histone lactylation and H3K18 lactylation levels in the presence and absence of glucose at 24 hours after stimulation of cells . One of three similar experiments is shown .

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A Western blots of acid-extracted histones from activated CD4+ T cells showing global histone lactylation and H3K18 lactylation levels in the presence and absence of glucose at 24 hours after stimulation of cells. One of three similar experiments is shown.


C , D Western blots of acid - extracted histones from activated CD4 + T cells in the presence of glycolysis inhibitor 2 - DG and PDK inhibitor DCA , respectively . Cells were analysed for global lactylation and H3K18 lactylation 24 hours after activation of TCR with anti - CD3 ( 5 µg / ml ) .

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C, D Western blots of acid-extracted histones from activated CD4+ T cells in the presence of glycolysis inhibitor 2-DG and PDK inhibitor DCA, respectively. Cells were analysed for global lactylation and H3K18 lactylation 24 hours after activation of TCR with anti-CD3 (5 µg/ml).


E Immunoblotting of acid - extracted lactylated histones from anti - CD3 - activated CD4 + T cells in the presence of extracellular lactate ( 25 mM ) at 24 hours of cell culture . One representative out of three experiments is shown . For all immunoblotting experiments , ex vivo purified , non - activated CD4 + T cells were used as control lymphocytes .

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E Immunoblotting of acid-extracted lactylated histones from anti-CD3-activated CD4+ T cells in the presence of extracellular lactate (25 mM) at 24 hours of cell culture. One representative out of three experiments is shown. For all immunoblotting experiments, ex vivo purified, non-activated CD4+ T cells were used as control lymphocytes.


F ChIP - sequncing showing H3K18 lactylation signals in control Th17 cells and lactate - treated Th17 lymphocytes ( 25 mM ) . Biological duplicates are shown .

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F ChIP-sequncing showing H3K18 lactylation signals in control Th17 cells and lactate-treated Th17 lymphocytes (25 mM). Biological duplicates are shown.


A , Schematic representation of signaling events . Left : time course of changes in cAMP ( dotted line ) and cGMP ( solid line ) ; right : time course of changes in Vm ( green ) , pHi ( red ) ; [ Na + ] i ( orange ) , and [ Ca2 + ] i ( pink ) . Vertical dotted lines indicate time of stimulation .

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A, Schematic representation of signaling events. Left: time course of changes in cAMP (dotted line) and cGMP (solid line); right: time course of changes in Vm (green), pHi (red); [Na+]i (orange), and [Ca2+]i (pink). Vertical dotted lines indicate time of stimulation.


A - D , Longitudinal ( A and C , proximal view on left ) and cross - sectional ( B and D , proximal - to - distal view ) tomographic slices of representative intact flagella from wild - type Chlamydomonas reinhardtii ( C . r . ) flagella ( A and B ) and sea urchin Strongylocentrotus purpuratus ( S . p . ) sperm ( C and D ) . Note that in Chlamydomonas , the flagellar membrane ( m ) is surrounded by a glycocalyx ( g ) . Other labels : central pair complex ( c ) and doublet microtubule ( d ) . Scale bar , 100 nm .

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A-D, Longitudinal (A and C, proximal view on left) and cross-sectional (B and D, proximal-to-distal view) tomographic slices of representative intact flagella from wild-type Chlamydomonas reinhardtii (C.r.) flagella (A and B) and sea urchin Strongylocentrotus purpuratus (S.p.) sperm (C and D). Note that in Chlamydomonas, the flagellar membrane (m) is surrounded by a glycocalyx (g). Other labels: central pair complex (c) and doublet microtubule (d). Scale bar, 100 nm.


E , Diameter of intact flagella from Chlamydomonas and sea urchin sperm measured on cross - sectional views of cryo - tomographic reconstructions . Numbers indicate average diameters ± s . d . ( n = 84 for sea urchin ( S . p . ) and n = 18 for Chlamydomonas ( C . r . ) )

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E, Diameter of intact flagella from Chlamydomonas and sea urchin sperm measured on cross-sectional views of cryo-tomographic reconstructions. Numbers indicate average diameters ± s.d. (n = 84 for sea urchin (S.p.) and n = 18 for Chlamydomonas (C.r.))


A , Time course of the change in voltage Vm evoked by a 2 - ms light flash in sperm that was loaded with caged cGMP ( 15 μM ) and recorded in 100KASW . Inset : extended timescale showing the rise of Vm . Fit of data ( black ) using two exponentials with time constants τrise = 17 . 4 ms and τrelax = 354 ms . Light flash in all panels is depicted as dashed magenta line .

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A, Time course of the change in voltage Vm evoked by a 2-ms light flash in sperm that was loaded with caged cGMP (15 μM) and recorded in 100KASW. Inset: extended timescale showing the rise of Vm. Fit of data (black) using two exponentials with time constants τrise = 17.4 ms and τrelax = 354 ms. Light flash in all panels is depicted as dashed magenta line.


B , Time course of the changes in Vm evoked by 100 - ms flash in 100KASW using caged cGMP ( blue ) and caged 8 - Br - cGMP ( black ) . The flashes released 260 nM cGMP or 2 , 600 nM 8 - Br - cGMP .

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B, Time course of the changes in Vm evoked by 100-ms flash in 100KASW using caged cGMP (blue) and caged 8-Br-cGMP (black). The flashes released 260 nM cGMP or 2,600 nM 8-Br-cGMP.


C , Time course of changes in Vm in 100KASW evoked by 2 - s flashes at different light energy that correspond to 440 , 220 , 75 , and 8 nM s - 1 of cGMP . Data was fitted using a heuristic model described by equation 3 ( Methods section ) ( black lines ) . D , Amplitude ( red ) and decay time τrelax ( black ) for different light energies and release rates for experiments as shown in panel C . τrelax was determined using a single exponential fit . The linear fit of the Vm amplitude was restricted to low light levels ; at higher levels , the Vm response saturates ( see panel C ) . Points indicate mean ± s . d . from 5 experiments .

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C, Time course of changes in Vm in 100KASW evoked by 2-s flashes at different light energy that correspond to 440, 220, 75, and 8 nM s-1 of cGMP. Data was fitted using a heuristic model described by equation 3 (Methods section) (black lines). D, Amplitude (red) and decay time τrelax (black) for different light energies and release rates for experiments as shown in panel C. τrelax was determined using a single exponential fit. The linear fit of the Vm amplitude was restricted to low light levels; at higher levels, the Vm response saturates (see panel C). Points indicate mean ± s.d. from 5 experiments.


A , Time course of cGMP - evoked changes in Vm in 100KASW ( red , 500 nM cGMP ; pink , 9 nM cGMP ) and normal ASW ( blue , 500 nM cGMP ; navy , 9 nM cGMP ) . The time constants of recovery after cGMP release ( 9 nM ) were 289 ms ( depolarization ) and 303 ms ( hyperpolarization ) . Signals in panels were detected using the potentiometric probe FluoVolt ; dashed magenta lines indicate the timing of the photolysis flash .

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A, Time course of cGMP-evoked changes in Vm in 100KASW (red, 500 nM cGMP; pink, 9 nM cGMP) and normal ASW (blue, 500 nM cGMP; navy, 9 nM cGMP ). The time constants of recovery after cGMP release (9 nM) were 289 ms (depolarization) and 303 ms (hyperpolarization). Signals in panels were detected using the potentiometric probe FluoVolt; dashed magenta lines indicate the timing of the photolysis flash.


, Representative measurement of the dependence of recovery - time constant on the amount of released cGMP . Inset : decay time was constant for low stimulation levels equivalent to ≲ 30 cGMP molecules or ≤ 3 resact molecules .

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, Representative measurement of the dependence of recovery-time constant on the amount of released cGMP. Inset: decay time was constant for low stimulation levels equivalent to ≲ 30 cGMP molecules or ≤ 3 resact molecules.


, Voltage response evoked by photolysis of DEACM - caged cGMP ( blue ) or 8 - Br - cGMP ( red ) . The light energy was adjusted such that Vm response amplitudes became similar ; for 8 - Br - cGMP , about 5 - times more light energy was required . Light pulse 10 ms .

, Voltage response evoked by photolysis of DEACM-caged cGMP (blue) or 8-Br-cGMP (red). The light energy was adjusted such that Vm response amplitudes became similar; for 8-Br-cGMP, about 5-times more light energy was required. Light pulse 10 ms.


, Vm responses evoked by cGMP in the presence ( blue ) and absence ( red ) of the HCN channel blocker ZD7288 ( 50 μM ) .

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, Vm responses evoked by cGMP in the presence (blue) and absence (red) of the HCN channel blocker ZD7288 (50 μM).


E , cGMP - evoked Vm response in ASW ( black ) , in the presence of IBMX ( 1 mM ) ( red ) , and in Ca2 + - free ASW ( [ Ca2 + ] ≤ 500 nM ) in the presence of IBMX ( 1 mM ) ( blue ) . Signals were detected using the potentiometric probe Di - 8 - ANEPPS in the ratio mode . F , Normalized responses from panel E .

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E, cGMP-evoked Vm response in ASW (black), in the presence of IBMX (1 mM) (red), and in Ca2+-free ASW ([Ca2+] ≤ 500 nM) in the presence of IBMX (1 mM) (blue). Signals were detected using the potentiometric probe Di-8-ANEPPS in the ratio mode. F, Normalized responses from panel E.


A , B , Ca2 + signals elicited by cGMP release and recorded with FluoForte . The UV flash released 28 ( A ) and 215 B ) cGMP molecules . Light flashes are indicated by dashed magenta lines .

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A, B, Ca2+ signals elicited by cGMP release and recorded with FluoForte. The UV flash released 28 (A) and 215 B) cGMP molecules. Light flashes are indicated by dashed magenta lines.


C , pHi signals , elicited by the release of approximately 1 , 400 cGMP molecules , were recorded with pHrodo Red . An increase of pHi is indicated by a decrease of ∆ F / F . Exemplary recording from a single cell . D , Average of pHi signals recorded from n = 25 cells . Error bars represent s . d .

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C, pHi signals, elicited by the release of approximately 1,400 cGMP molecules, were recorded with pHrodo Red. An increase of pHi is indicated by a decrease of ∆F/F. Exemplary recording from a single cell. D, Average of pHi signals recorded from n = 25 cells. Error bars represent s.d.


B , Relationship between cGMP synthesis rate and free cGMP concentration . We assume that a single GC molecule can synthesize 10 cGMP molecules during its lifetime of 150 ms Inset : expanded scale .

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B, Relationship between cGMP synthesis rate and free cGMP concentration. We assume that a single GC molecule can synthesize 10 cGMP molecules during its lifetime of 150 ms Inset: expanded scale.


( A ) The AH109 strain was co ‐ transformed with pGBKT7 containing the C ‐ terminal ( CT ) domain of mAtg9 ( bait ) and pGADT7 , or pACT2 , containing the CT domain of p38IP ( prey ) . The interaction was then visualized by growth of co ‐ transformed yeast on SD ‐ Trp / Leu / His plates for 3 days at 30 ° C .

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(A) The AH109 strain was co‐transformed with pGBKT7 containing the C‐terminal (CT) domain of mAtg9 (bait) and pGADT7, or pACT2, containing the CT domain of p38IP (prey). The interaction was then visualized by growth of co‐transformed yeast on SD‐Trp/Leu/His plates for 3 days at 30°C.


( C ) HEK293A cells were transfected with RFP - mAtg9 and HA - p38IP . HA - p38IP was immunoprecipitated with an anti ‐ HA antibody and the co ‐ immunoprecipitated RFP - mAtg9 was detected with an anti ‐ mAtg9 antibody . HA - p38IP was detected with an anti ‐ HA antibody . Asterisk indicates a non ‐ specific band .

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(C) HEK293A cells were transfected with RFP-mAtg9 and HA-p38IP. HA-p38IP was immunoprecipitated with an anti‐HA antibody and the co‐immunoprecipitated RFP-mAtg9 was detected with an anti‐mAtg9 antibody. HA-p38IP was detected with an anti‐HA antibody. Asterisk indicates a non‐specific band.


Indirect immunofluorescence analysis of mAtg9 with ( D ) HA - full ‐ length p38IP ( HA - FL ) , ( E ) HA - C ‐ terminal p38IP ( HA - CT ) , and ( F ) HA - N ‐ terminal p38IP ( HA - NT ) . Bars = 5 μm . The co ‐ localization seen in ( D ) and ( E ) was not because of overlap of randomly distributed vesicles . Cross ‐ correlation function ( CCF ) analysis ( van Steensel et al , 1996 ) showed a peak and maximum Rp value around ΔX = 0 , indicating that co ‐ localization between mAtg9 and p38IP is positively correlated and non random ( Supplementary Figure S1 ) .

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Indirect immunofluorescence analysis of mAtg9 with (D)HA-full‐length p38IP (HA-FL), (E)HA-C‐terminal p38IP (HA-CT), and (F) HA-N‐terminal p38IP (HA-NT). Bars=5 μm. The co‐localization seen in (D) and (E) was not because of overlap of randomly distributed vesicles. Cross‐correlation function (CCF) analysis ( van Steensel et al, 1996) showed a peak and maximum Rp value around ΔX=0, indicating that co‐localization between mAtg9 and p38IP is positively correlated and non random ( Supplementary Figure S1).


( A ) HEK293A cells were transfected with HA - p38IP alone or co ‐ transfected with HA - p38IP and RFP - mAtg9 , homogenized and subjected to centrifugation , and the resulting post ‐ nuclear supernatant ( PNS ) was fractionated by centrifugation at 100 000 g into membrane pellet and cytosol . Equal protein amounts were resolved by SDS - PAGE and immunoblotted with anti ‐ mannose ‐ 6 ‐ phosphate receptor ( MPR ) antibody as a control for membrane ‐ associated proteins , anti ‐ superoxide dismutase ( SOD ) antibody as a control for cytosolic proteins , anti ‐ mAtg9 antibody , and anti ‐ HA antibody . HA - p38IP levels were quantified using ImageJ software and plotted relative to HA - p38IP in the PNS ( n = 4 ) ( * * P = 0 . 0051 ) .

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(A) HEK293A cells were transfected with HA-p38IP alone or co‐transfected with HA-p38IP and RFP-mAtg9, homogenized and subjected to centrifugation, and the resulting post‐nuclear supernatant (PNS) was fractionated by centrifugation at 100 000 g into membrane pellet and cytosol. Equal protein amounts were resolved by SDS-PAGE and immunoblotted with anti‐mannose‐6‐phosphate receptor (MPR) antibody as a control for membrane‐associated proteins, anti‐superoxide dismutase (SOD) antibody as a control for cytosolic proteins, anti‐mAtg9 antibody, and anti‐HA antibody. HA-p38IP levels were quantified using ImageJ software and plotted relative to HA-p38IP in the PNS (n=4) (**P=0.0051).


( B ) HEK293 cells were depleted of mAtg9 using siRNA , then transfected with HA - p38IP and incubated in full medium or EBSS ( data not shown ) . The asterisk indicates mAtg9 ‐ depleted cells . Bar is 5 μM . The intensity of the nuclear versus cytosolic fluorescence in control and cells depleted of mAtg9 under fed and starvation conditions was analysed using ImageJ software ( * * * P = 0 . 0001 , Students t ‐ test )

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(B) HEK293 cells were depleted of mAtg9 using siRNA, then transfected with HA-p38IP and incubated in full medium or EBSS (data not shown). The asterisk indicates mAtg9‐depleted cells. Bar is 5 μM. The intensity of the nuclear versus cytosolic fluorescence in control and cells depleted of mAtg9 under fed and starvation conditions was analysed using ImageJ software (***P=0.0001, Students t‐test)


( C ) HEK293A cells were transfected with HA - p38IP and processed for indirect immunofluorescence . HA - p38IP was detected with an anti ‐ HA antibody and endogenous mAtg9 was detected with an anti ‐ mAtg9 antibody . HA - p38IP localizes to the nucleus and cytoplasmic puncta in full medium or EBSS . HA - p38IP co ‐ localizes with mAtg9 in the periphery of the cell ( arrowheads ) . Bars ( upper panels ) 5 μM , ( lower panels ) 1 μM .

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(C) HEK293A cells were transfected with HA-p38IP and processed for indirect immunofluorescence. HA-p38IP was detected with an anti‐HA antibody and endogenous mAtg9 was detected with an anti‐mAtg9 antibody. HA-p38IP localizes to the nucleus and cytoplasmic puncta in full medium or EBSS. HA-p38IP co‐localizes with mAtg9 in the periphery of the cell (arrowheads). Bars (upper panels) 5 μM, (lower panels) 1 μM.


( D ) Anti ‐ mAtg9 antibody was used to immunoprecipitate mAtg9 from HEK293 cells transfected with HA - p38IP alone or with RFP - mAtg9 and HA - p38IP . A total of 5 % of lysates or immunoprecipitates were probed with anti ‐ HA or anti ‐ mAtg9 antibodies . The left and right hand bottom panels were obtained from the same immunoblot , but exposed for different times to allow the endogenous mAtg9 to be visualized better .

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(D) Anti‐mAtg9 antibody was used to immunoprecipitate mAtg9 from HEK293 cells transfected with HA-p38IP alone or with RFP-mAtg9 and HA-p38IP. A total of 5% of lysates or immunoprecipitates were probed with anti‐HA or anti‐mAtg9 antibodies. The left and right hand bottom panels were obtained from the same immunoblot, but exposed for different times to allow the endogenous mAtg9 to be visualized better.


( E ) HEK293 cells were transfected with control or p38IP siRNA . At 72 h after transfection , cells were incubated in either full medium or EBSS for 2 h , then fixed and immunostained to detect endogenous mAtg9 localization . mAtg9 localization was quantified by visually scoring cells for either a juxta ‐ nuclear or dispersed phenotype . Bars = 5 μm ( data are represented as mean ± s . e . m . of 200 cells , * P = 0 . 0413 , Student ' s t ‐ test ) .

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(E) HEK293 cells were transfected with control or p38IP siRNA. At 72 h after transfection, cells were incubated in either full medium or EBSS for 2 h, then fixed and immunostained to detect endogenous mAtg9 localization. mAtg9 localization was quantified by visually scoring cells for either a juxta‐nuclear or dispersed phenotype. Bars=5 μm (data are represented as mean±s.e.m. of 200 cells, *P=0.0413, Student's t‐test).


( A ) 293 / GFP - LC3 cells were transfected with control or p38IP siRNA . At 72 h after transfection , cells were incubated in either full medium ( FM ) , full medium with leupeptin ( FM Leu ) , EBSS ( ES ) , or EBSS with leupeptin ( ES Leu ) for 2 h . FM Leu ( data not shown ) is identical to FM alone . Quantification of GFP - LC3 ‐ positive autophagosome number was performed by counting in a blinded experiment . Bars = 5 μm ( data are represented as mean ± s . e . m . of 60 cells , * * * P = 0 . 0002 , Students t ‐ test ) .

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(A) 293/GFP-LC3 cells were transfected with control or p38IP siRNA. At 72 h after transfection, cells were incubated in either full medium (FM), full medium with leupeptin (FM Leu), EBSS (ES), or EBSS with leupeptin (ES Leu) for 2 h. FM Leu (data not shown) is identical to FM alone. Quantification of GFP-LC3‐positive autophagosome number was performed by counting in a blinded experiment. Bars=5 μm (data are represented as mean±s.e.m. of 60 cells, ***P=0.0002, Students t‐test).


( B ) HEK293A cells were transfected with siRNA control , siRNA for p38IP , and siRNA for ULK1 . Cell lysates were analysed by SDS - PAGE for endogenous LC3 lipidation using an anti ‐ LC3 antibody , or immunoblotted with anti ‐ ULK1 antibody .

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(B) HEK293A cells were transfected with siRNA control, siRNA for p38IP, and siRNA for ULK1. Cell lysates were analysed by SDS-PAGE for endogenous LC3 lipidation using an anti‐LC3 antibody, or immunoblotted with anti‐ULK1 antibody.


( C ) HeLa cells were transfected with siRNA control , and siRNA for p38IP . After incubation for 2 h as in ( A ) , samples were immunoblotted with anti ‐ LC3 and anti ‐ actin antibodies . Endogenous LC3II / LC3I levels were quantified and the ratio presented as arbitrary units . In ( B ) , n = 4 , * * * P = 0 . 0001 and * P = 0 . 0324 , Students t ‐ test . In ( C ) , data are representative of two experiments .

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(C) HeLa cells were transfected with siRNA control, and siRNA for p38IP. After incubation for 2 h as in (A), samples were immunoblotted with anti‐LC3 and anti‐actin antibodies. Endogenous LC3II/LC3I levels were quantified and the ratio presented as arbitrary units. In (B), n=4, ***P=0.0001 and *P=0.0324, Students t‐test. In (C), data are representative of two experiments.


( D ) 293 / GFP - LC3 cells were transfected with siRNA control or siRNA p38IP , and incubated in full medium , or for 2 and 4 h in EBSS . Samples were immunoblotted with anti ‐ p62 antibodies , and actin as a loading control .

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(D) 293/GFP-LC3 cells were transfected with siRNA control or siRNA p38IP, and incubated in full medium, or for 2 and 4 h in EBSS. Samples were immunoblotted with anti‐p62 antibodies, and actin as a loading control.


( E ) HEK293A cells were transfected with HA - p38IP or myc - ULK1 C ‐ terminal domain ( CTD ) . After 24 h , cells were labelled with [ 14C ] valine as described in Material and Methods section , and incubated in either full medium or EBSS for 2 h . Cells were then collected and analysed for long ‐ lived protein degradation ( data are represented as mean ± s . e . m . of triplicates , representative of two experiments , EBSS mock versus EBSS HA - p38IP ( * * * P = 0 . 036 ) ; EBSS mock versus ULK1 CTD ( * * * P = 0 . 0001 ) ; Students t ‐ test ) .

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(E) HEK293A cells were transfected with HA-p38IP or myc-ULK1 C‐terminal domain (CTD). After 24 h, cells were labelled with [14C]valine as described in Material and Methods section, and incubated in either full medium or EBSS for 2 h. Cells were then collected and analysed for long‐lived protein degradation (data are represented as mean±s.e.m. of triplicates, representative of two experiments, EBSS mock versus EBSS HA-p38IP (***P=0.036); EBSS mock versus ULK1 CTD (***P=0.0001); Students t‐test).


( A ) 293 / GFP - LC3 cells were treated with 10 μM anisomycin for 30 min , or exposed to UV irradiation for 3 min followed by a 40 ‐ min recovery and incubation in full medium , full medium with leupeptin , EBSS , or EBSS with leupeptin for 2 h . Cells were then fixed and visualized by confocal microscopy . GFP - LC3 ‐ positive structures per cell were quantified as in Figure 3A . Bars = 5 μm . Data are represented as mean ± s . e . m . n = 60 cells , mock versus anisomycin EBSS ( * * * P = 0 . 0001 ) ; mock versus UV EBSS ( * * * P = 0 . 0001 ) ; mock versus anisomycin EBSS with leupeptin ( * * * P = 0 . 0001 ) ; mock versus EBSS with leupeptin UV ( * * * P = 0 . 0001 ) . All analysed using Student ' s t - test .

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(A) 293/GFP-LC3 cells were treated with 10 μM anisomycin for 30 min, or exposed to UV irradiation for 3 min followed by a 40‐min recovery and incubation in full medium, full medium with leupeptin, EBSS, or EBSS with leupeptin for 2 h. Cells were then fixed and visualized by confocal microscopy. GFP-LC3‐positive structures per cell were quantified as in Figure 3A. Bars=5 μm. Data are represented as mean±s.e.m. n=60 cells, mock versus anisomycin EBSS (***P=0.0001); mock versus UV EBSS (***P=0.0001); mock versus anisomycin EBSS with leupeptin (***P=0.0001); mock versus EBSS with leupeptin UV (***P=0.0001). All analysed using Student's t-test.


( B ) HEK293A cells were pretreated with 10 μM anisomycin for 30 min , or UV irradiation for 3 min followed by a 40 ‐ min recovery , before incubation in EBSS for 2 h . Images were analysed as in Figure 2E . Bars = 5 μm . Data are represented as mean ± s . e . m . of 60 cells , EBSS mock versus EBSS anisomycin ( * P = 0 . 0142 ) ; EBSS mock versus EBSS UV ( * P = 0 . 0150 ) ; Student ' s t ‐ test .

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(B) HEK293A cells were pretreated with 10 μM anisomycin for 30 min, or UV irradiation for 3 min followed by a 40‐min recovery, before incubation in EBSS for 2 h. Images were analysed as in Figure 2E. Bars=5 μm. Data are represented as mean±s.e.m. of 60 cells, EBSS mock versus EBSS anisomycin (*P=0.0142); EBSS mock versus EBSS UV (*P=0.0150); Student's t‐test.


( A ) 293 / GFP - LC3 cells were pre ‐ treated with 10 μM anisomycin for 30 min before incubation in either full medium or EBSS . Cell lysates were analysed by SDS - PAGE for GFP - LC3 lipidation using an anti ‐ LC3 antibody . The membrane was also probed with anti ‐ β tubulin . The GFP - LC3 lipidation was quantified as the amount of GFP - LC3II / GFP - LC3I ( data are represented as mean ± s . e . m . of triplicates , P = 0 . 0091 , Student ' s t ‐ test ) .

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(A) 293/GFP-LC3 cells were pre‐treated with 10 μM anisomycin for 30 min before incubation in either full medium or EBSS. Cell lysates were analysed by SDS-PAGE for GFP-LC3 lipidation using an anti‐LC3 antibody. The membrane was also probed with anti‐β tubulin. The GFP-LC3 lipidation was quantified as the amount of GFP-LC3II/GFP-LC3I (data are represented as mean±s.e.m. of triplicates, P=0.0091, Student's t‐test).


( B ) HEK293A cells labelled with [ 14C ] valine , as described in Material and Methods section , were pretreated with 10 μM anisomycin for 30 min before incubation in either full medium , EBSS , or EBSS with leupeptin for 2 h . Cells were then analysed for long ‐ lived protein degradation ( data are represented as mean ± s . e . m . of triplicates , representative of two experiments , EBSS untreated versus anisomycin treated ( P = 0 . 0363 ) ; EBSS untreated versus leupeptin treated ( P = 0 . 0001 ) , Student ' s t ‐ test ) .

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(B) HEK293A cells labelled with [14C]valine, as described in Material and Methods section, were pretreated with 10 μM anisomycin for 30 min before incubation in either full medium, EBSS, or EBSS with leupeptin for 2 h. Cells were then analysed for long‐lived protein degradation (data are represented as mean±s.e.m. of triplicates, representative of two experiments, EBSS untreated versus anisomycin treated (P=0.0363); EBSS untreated versus leupeptin treated (P=0.0001), Student's t‐test).


( C ) HEK293 cells were transfected with control siRNA or siRNA for p38IP . After 24 ‐ h incubation , control or p38IP ‐ depleted cells were pre ‐ treated with anisomycin , followed by a 2 ‐ h incubation with full medium , full medium with leupeptin , EBSS , or EBSS with leupeptin . Cell lysates were analysed with anti ‐ p38α or anti ‐ LC3 antibodies . LC3II / LC3I levels were quantified and presented normalized to control , untreated cells incubated in EBSS . Data are representative of two experiments .

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(C) HEK293 cells were transfected with control siRNA or siRNA for p38IP. After 24‐h incubation, control or p38IP‐depleted cells were pre‐treated with anisomycin, followed by a 2‐h incubation with full medium, full medium with leupeptin, EBSS, or EBSS with leupeptin. Cell lysates were analysed with anti‐p38α or anti‐LC3 antibodies. LC3II/LC3I levels were quantified and presented normalized to control, untreated cells incubated in EBSS. Data are representative of two experiments.


( D ) HEK293A cells transiently transfected with HA ‐ p38IP and RFP ‐ mAtg9 were treated with anisomycin for 30 min and mAtg9 was immunoprecipitated . 5 % of the lysates or the immunoprecipitates were probed with anti ‐ HA and anti ‐ mAtg9 antibodies ( data is representative of 3 experiments , * P = 0 . 0332 ) .

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(D) HEK293A cells transiently transfected with HA‐p38IP and RFP‐mAtg9 were treated with anisomycin for 30 min and mAtg9 was immunoprecipitated. 5% of the lysates or the immunoprecipitates were probed with anti‐HA and anti‐mAtg9 antibodies (data is representative of 3 experiments, *P=0.0332).


( A ) HEK293A cells were transfected with RFP - mAtg9 and Flag - p38 ( lanes 2 and 7 ) , RFP - mAtg9 , Flag - p38 and HA - p38IP ( lanes 3 and 8 ) , or RFP - mAtg9 and HA - p38IP ( lanes 4 and 9 ) . RFP - mAtg9 was immunoprecipitated with an anti ‐ mAtg9 antibody , the co ‐ immunoprecipitated HA - p38IP was detected with an anti ‐ HA antibody , and co ‐ immunoprecipitated Flag - p38 was detected with an anti ‐ Flag antibody . RFP - mAtg9 was detected with an anti ‐ Atg9 antibody .

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(A) HEK293A cells were transfected with RFP-mAtg9 and Flag-p38 (lanes 2 and 7), RFP-mAtg9, Flag-p38 and HA-p38IP (lanes 3 and 8), or RFP-mAtg9 and HA-p38IP (lanes 4 and 9). RFP-mAtg9 was immunoprecipitated with an anti‐mAtg9 antibody, the co‐immunoprecipitated HA-p38IP was detected with an anti‐HA antibody, and co‐immunoprecipitated Flag-p38 was detected with an anti‐Flag antibody. RFP-mAtg9 was detected with an anti‐Atg9 antibody.


( B ) HEK293A cells were transfected with RFP - Atg9 and HA - p38IP ( lanes 1 and 5 ) , Flag - Flag , Flag - p38 , and HA - p38IP ( lanes 2 and 6 ) , or Flag - p38 and HA - p38IP ( lanes 3 and 7 ) . HA - p38IP was immunoprecipitated with an anti ‐ HA antibody , the co ‐ immunoprecipitated RFP - Atg9 was detected with an anti ‐ Atg9 antibody , and co ‐ immunoprecipitated Flag - p38 was detected with an anti ‐ p38α antibody . HA - p38IP was detected with an anti ‐ HA antibody .

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(B) HEK293A cells were transfected with RFP-Atg9 and HA-p38IP (lanes 1 and 5), Flag-Flag, Flag-p38, and HA-p38IP (lanes 2 and 6), or Flag-p38 and HA-p38IP (lanes 3 and 7). HA-p38IP was immunoprecipitated with an anti‐HA antibody, the co‐immunoprecipitated RFP-Atg9 was detected with an anti‐Atg9 antibody, and co‐immunoprecipitated Flag-p38 was detected with an anti‐p38α antibody. HA-p38IP was detected with an anti‐HA antibody.


( C ) HEK293A cells were transfected with Flag - p38α for 24 h . Cells were incubated in either full medium , EBSS , or EBSS plus leupeptin for 2 h , lysed , and analysed for endogenous LC3 lipidation using an anti ‐ LC3 antibody , and immunoblotted with anti ‐ Flag antibody . LC3 lipidation was quantified as the amount of LC3II / LC3I ( data are represented as mean ± s . e . m . of triplicates , n = 2 experiments , EBSS control versus EBSS with Flag ‐ p38α , * * * P = 0 . 0026 ) .

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(C) HEK293A cells were transfected with Flag-p38α for 24 h. Cells were incubated in either full medium, EBSS, or EBSS plus leupeptin for 2 h, lysed, and analysed for endogenous LC3 lipidation using an anti‐LC3 antibody, and immunoblotted with anti‐Flag antibody. LC3 lipidation was quantified as the amount of LC3II/LC3I (data are represented as mean±s.e.m. of triplicates, n=2 experiments, EBSS control versus EBSS with Flag‐p38α, ***P=0.0026).


( A ) 293 / GFP - LC3 cells were transfected with control or p38α siRNA . At 72 h after transfection , cells were incubated in either full medium , EBSS , or EBSS with leupeptin for 2 h , then fixed and visualized by confocal microscopy . Bars = 5 μm ( data are represented as mean ± s . e . m . n = 60 cells , EBSS control versus p38α siRNA ( * * * P = 0 . 0001 ) ; EBSS with leupeptin control versus p38α siRNA ( * * * P = 0 . 0001 ) , Student ' s t ‐ test ) .

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(A) 293/GFP-LC3 cells were transfected with control or p38α siRNA. At 72 h after transfection, cells were incubated in either full medium, EBSS, or EBSS with leupeptin for 2 h, then fixed and visualized by confocal microscopy. Bars=5 μm (data are represented as mean±s.e.m. n=60 cells, EBSS control versus p38α siRNA (***P=0.0001); EBSS with leupeptin control versus p38α siRNA (***P=0.0001), Student's t‐test).


( B ) HEK293A cells were transfected with control or p38α siRNA . At 72 h after transfection , cells were incubated in either full medium , EBSS , or EBSS containing leupeptin for 2 h . Cells lysates were analysed for LC3 lipidation using an anti ‐ LC3 antibody . The membrane was also probed with anti ‐ actin and anti ‐ p38α antibodies . LC3 lipidation was quantified as the amount of LC3II / LC3I ( data are represented as mean ± s . e . m . n = 5 , full medium control versus p38α siRNA ( * * P = 0 . 0056 ) ; EBSS control versus p38α siRNA ( * * P = 0 . 048 ) ; EBSS with Leu control versus p38α siRNA ( * * P = 0 . 04 ) , Student ' s t ‐ test ) .

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(B) HEK293A cells were transfected with control or p38α siRNA. At 72 h after transfection, cells were incubated in either full medium, EBSS, or EBSS containing leupeptin for 2 h. Cells lysates were analysed for LC3 lipidation using an anti‐LC3 antibody. The membrane was also probed with anti‐actin and anti‐p38α antibodies. LC3 lipidation was quantified as the amount of LC3II/LC3I (data are represented as mean ±s.e.m. n=5, full medium control versus p38α siRNA (**P=0.0056); EBSS control versus p38α siRNA (**P=0.048); EBSS with Leu control versus p38α siRNA (**P=0.04), Student's t‐test).