( J ) Representative picture of oil red O - stained BAs differentiated from EP3γ - expressed EP3 - / - brown pre - adipocytes with WTAP siRNA treatment . Scale bar : 50 μm .

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(J) Representative picture of oil red O-stained BAs differentiated from EP3γ-expressed EP3-/- brown pre-adipocytes with WTAP siRNA treatment. Scale bar: 50 μm.


( K - Q ) qRT - PCR analysis of the relative mRNA levels of brown marker genes in BAs differentiated from EP3γ - expressed EP3 - / - brown pre - adipocytes after transfection with WTAP siRNA ( n = 6 - 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two - way ANOVA followed by Tukey ' s test for post - hoc comparisons K - Q ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(K-Q) qRT-PCR analysis of the relative mRNA levels of brown marker genes in BAs differentiated from EP3γ-expressed EP3-/- brown pre-adipocytes after transfection with WTAP siRNA (n = 6-8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two-way ANOVA followed by Tukey's test for post-hoc comparisons K-Q). *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( A ) Western blotting analysis of ubiquitination of WTAP in the control and EP3 - deficient brown pre - adipocytes with or without sulprostone treatment . Data information : Data are representative of two independent experiments with biological

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(A) Western blotting analysis of ubiquitination of WTAP in the control and EP3-deficient brown pre-adipocytes with or without sulprostone treatment. Data information: Data are representative of two independent experiments with biological replicates


( B ) Western blotting analysis of ERK1 / 2 phosphorylation in the control and EP3 - deficient brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological

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(B) Western blotting analysis of ERK1/2 phosphorylation in the control and EP3-deficient brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates


( C ) Effect of ERK1 / 2 inhibitor , ravoxertinib , on ubiquitin - dependent degradation of WTAP in sulprostone - treated brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological

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(C) Effect of ERK1/2 inhibitor, ravoxertinib, on ubiquitin-dependent degradation of WTAP in sulprostone-treated brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates


( D ) Effect of wortmannin ( Wor ) , pertussis toxin ( PTX ) , U73122 , barbadin , and melittin on ubiquitin - dependent degradation of WTAP in sulprostone - treated brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological

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(D) Effect of wortmannin (Wor), pertussis toxin (PTX), U73122, barbadin, and melittin on ubiquitin-dependent degradation of WTAP in sulprostone-treated brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates


( E ) Analysis of PKA activity in the control and EP3 - deficient brown pre - adipocytes ( n = 4 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological

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(E) Analysis of PKA activity in the control and EP3-deficient brown pre-adipocytes (n = 4). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates


( F ) Effect of PKA inhibitor , H89 , on ubiquitin - dependent degradation of WTAP in EP3 - / - brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological

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(F) Effect of PKA inhibitor, H89, on ubiquitin-dependent degradation of WTAP in EP3-/- brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates


( G ) Effect of ERK1 / 2 phosphorylation site mutation ( HA - WTAP - 2A ) on EP3 - mediated ubiquitin - dependent degradation of WTAP in brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological

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

(G) Effect of ERK1/2 phosphorylation site mutation (HA-WTAP-2A) on EP3-mediated ubiquitin-dependent degradation of WTAP in brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates


( H ) Representative picture of oil red O - stained BAs differentiated from sulprostone - treated brown pre - adipocyte with treatment of PTX , dbcAMP , or ravoxertinib . Scale bar : 50 μm . Data information : Data are representative of two independent experiments with biological

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(H) Representative picture of oil red O-stained BAs differentiated from sulprostone-treated brown pre-adipocyte with treatment of PTX, dbcAMP, or ravoxertinib. Scale bar: 50 μm. Data information: Data are representative of two independent experiments with biological replicates


( I ) Western blotting assay of ERK1 / 2 , WTAP , ZFP410 , PRDM16 , and UCP1 in BAs differentiated from sulprostone - treated brown pre - adipocyte with treatment of PTX , dbcAMP , or ravoxertinib . Data information : Data are representative of two independent experiments with biological

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

(I) Western blotting assay of ERK1/2, WTAP, ZFP410, PRDM16, and UCP1 in BAs differentiated from sulprostone-treated brown pre-adipocyte with treatment of PTX, dbcAMP, or ravoxertinib. Data information: Data are representative of two independent experiments with biological replicates


( B ) Representative pictures of nile red - stained human BAs differentiated from hESC - derived brown progenitors . Scale bar : 50 μm .

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(B)Representative pictures of nile red-stained human BAs differentiated from hESC-derived brown progenitors. Scale bar: 50 μm.


( C ) Time course analysis of the relative mRNA levels of EP3 receptors in hESC - derived BAs during BA differentiation detected by RT - qPCR ( n = 7 - 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates ( C , Statistical significance was evaluated by Welch ANOVA test followed by Tamhane ' s T2 test for post - hoc comparisons ( C ) * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(C) Time course analysis of the relative mRNA levels of EP3 receptors in hESC-derived BAs during BA differentiation detected by RT-qPCR (n = 7-8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates (C, Statistical significance was evaluated by Welch ANOVA test followed by Tamhane's T2 test for post-hoc comparisons (C) *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( D ) Representative pictures of hESC - derived BAs treated with L798106 alone or combined with H89 . Scale bar : 50 μm . Data information : Data are representative of two independent experiments with biological

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(D) Representative pictures of hESC-derived BAs treated with L798106 alone or combined with H89. Scale bar: 50 μm. Data information: Data are representative of two independent experiments with biological replicates


( E ) Effect of H89 on the relative mRNA level of brown marker gene in hESC - derived BAs treated with L798106 detected by RT - qPCR ( n = 6 - 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two - way ANOVA followed by Tukey ' s test for multiple comparisons ( E , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(E) Effect of H89 on the relative mRNA level of brown marker gene in hESC-derived BAs treated with L798106 detected by RT-qPCR (n = 6-8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two-way ANOVA followed by Tukey's test for multiple comparisons (E, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( F ) Effect of H89 on WTAP mRNA expression in hESC - derived BAs treated with L798106 ( n = 6 - 7 ) . ( G ) Effect of H89 on ZNF410 mRNA expression in hESC - derived BAs treated with L798106 ( n = 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by , two - way ANOVA followed by Tukey ' s test for multiple comparisons F , G ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(F) Effect of H89 on WTAP mRNA expression in hESC-derived BAs treated with L798106 (n = 6-7). (G) Effect of H89 on ZNF410 mRNA expression in hESC-derived BAs treated with L798106 (n = 8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by , two-way ANOVA followed by Tukey's test for multiple comparisons F, G). *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( H ) Western blotting assay of ERK1 / 2 , WTAP , ZNF410 , PRDM16 , and UCP1 in hESC - derived BAs treated with L798106 alone or combined with H89 .

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(H) Western blotting assay of ERK1/2, WTAP, ZNF410, PRDM16, and UCP1 in hESC-derived BAs treated with L798106 alone or combined with H89.


( A ) Comparison of migration and invasion abilities between T - and B - lines . PC3 T - and B - line cells were incubated for migration ( 16h ) and invasion ( 24h ) assay . Data shown represent the means ± SD ( n = 3 biological replicates ) . Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ) .

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(A) Comparison of migration and invasion abilities between T- and B- lines. PC3 T- and B-line cells were incubated for migration (16h) and invasion (24h) assay. Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01).


( B ) Comparison of cell proliferation rate between T - and B - lines . Cell proliferation assay was performed by MTS reagents . Data are the means ± SD ( n = 3 biological replicates ) .

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(B) Comparison of cell proliferation rate between T- and B- lines. Cell proliferation assay was performed by MTS reagents. Data are the means ± SD (n=3 biological replicates).


( C ) Western blot analysis of the endogenous CTH in PC3 orthotopically implanted primary tumor - derived ( T2 and T3 - lines ) and bone - metastases - derived cancer cells ( B2 and B3 - lines ) .

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(C) Western blot analysis of the endogenous CTH in PC3 orthotopically implanted primary tumor-derived (T2 and T3-lines) and bone-metastases-derived cancer cells (B2 and B3-lines).


( D ) Top : H2S catalyzing activity of cell lysates from PC3 T2 , B2 , T3 , and B3 lines . Lead acetate - soaked paper strips show a PbS brown stain as a result of reaction with H2S . Bottom : The level of H2S production was quantified by densitometry , and the histograms represent the means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc test was used for the statistical analysis ( * P < 0 . 05 ) .

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(D) Top: H2S catalyzing activity of cell lysates from PC3 T2, B2, T3, and B3 lines. Lead acetate-soaked paper strips show a PbS brown stain as a result of reaction with H2S. Bottom: The level of H2S production was quantified by densitometry, and the histograms represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis (*P<0.05).


( E , F ) 1x106 PC3 - T2 , B2 , T3 , and B3 cells were orthotopically injected into mouse prostate for 15 - 20 days . Incidences of paraaortic lymph nodes metastasis ( E ) and bone metastasis ( F ) are shown ( n = 6 - 8 mice per group ) . Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ) .

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(E, F) 1x106 PC3-T2, B2, T3, and B3 cells were orthotopically injected into mouse prostate for 15-20 days. Incidences of paraaortic lymph nodes metastasis (E) and bone metastasis (F) are shown (n=6-8 mice per group). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01).


( G ) Top : expression of CTH in prostate tumors by immunocytochemistry analysis of commercial tissue arrays . Samples were sub - grouped by TNM stages . The representative images from different stages are shown . S : Stroma ; Scale bars : 250μm . Bottom : The statistical significance was determined using the χ2

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(G) Top: expression of CTH in prostate tumors by immunocytochemistry analysis of commercial tissue arrays. Samples were sub-grouped by TNM stages. The representative images from different stages are shown. S: Stroma; Scale bars: 250μm. Bottom: The statistical significance was determined using the χ2 test


The RNA - seq data of CTH was downloaded from the TCGA database ( H ) The mRNA expression of CTH in PC specimens and normal tissues . Data are presented as means ± SD ( n = 50 cases for adjancent non - tumor parts and 469 cases for PC tumors ) . Student ' s t - test was used for the statistical analysis ( * * P < 0 . 01 ) .

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The RNA-seq data of CTH was downloaded from the TCGA database (H) The mRNA expression of CTH in PC specimens and normal tissues. Data are presented as means ± SD (n=50 cases for adjancent non-tumor parts and 469 cases for PC tumors). Student's t-test was used for the statistical analysis (**P<0.01).


( I ) Kaplan - Meier survival analysis of PC patients with high or low CTH expression . The statistical significance was determined using the χ2 test .

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(I) Kaplan-Meier survival analysis of PC patients with high or low CTH expression. The statistical significance was determined using the χ2 test.


( A ) Western blot analysis of the CTH level in PC3 - T2 cells with CTH overexpression by pCMV - CTH - HA or PC3 - B2 cells with CTH knockdown by siCTH - 1 .

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(A) Western blot analysis of the CTH level in PC3-T2 cells with CTH overexpression by pCMV-CTH-HA or PC3-B2 cells with CTH knockdown by siCTH-1.


( B ) Top : The H2S production capacity of cell lysates from PC3 - T2 cells with CTH overexpression by pCMV - CTH - HA or PC3 - B2 cells with CTH knockdown by siCTH - 1 . Lead acetate - soaked paper strips show a PbS brown stain as a result of reaction with H2S . Bottom : The level of H2S production was quantified by densitometry , and the histograms represent the means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc test was used for the statistical analysis ( * P < 0 . 05 ) .

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(B) Top: The H2S production capacity of cell lysates from PC3-T2 cells with CTH overexpression by pCMV-CTH-HA or PC3-B2 cells with CTH knockdown by siCTH-1. Lead acetate-soaked paper strips show a PbS brown stain as a result of reaction with H2S. Bottom: The level of H2S production was quantified by densitometry, and the histograms represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis (*P<0.05).


( C ) PC3 - B2 cells were transfected with control or siCTH - 1 and then were subjected to migration ( 16h ) and invasion ( 24h ) assays . Data information : Data shown represent the means ± SD ( n = 3 biological replicates ) . Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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(C) PC3-B2 cells were transfected with control or siCTH-1 and then were subjected to migration (16h) and invasion (24h) assays. Data information: Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001).


( D ) Cell proliferation rates of PC3 - B2 cells with CTH knockdown . Cell proliferation assay was performed by MTS reagent . Data information : Data shown represent the means ± SD ( n = 3 biological replicates ) . Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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(D) Cell proliferation rates of PC3-B2 cells with CTH knockdown. Cell proliferation assay was performed by MTS reagent. Data information: Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001).


( E ) Western blot analysis of the CTH levels in PC3 cells transfected with HA - CTH or pCMV - HA vector control .

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(E) Western blot analysis of the CTH levels in PC3 cells transfected with HA-CTH or pCMV-HA vector control.


( F ) PC3 cells were transfected with HA - CTH or pCMV - HA vector control and then incubated for migration ( 16h ) and invasion assay ( 24h ) . Data information : Data shown represent the means ± SD ( n = 3 biological replicates ) . Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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(F) PC3 cells were transfected with HA-CTH or pCMV-HA vector control and then incubated for migration (16h) and invasion assay (24h). Data information: Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001).


( G ) PC3 cells were pre - treated with 10μM PAG for 16 hrs , and incubated for migration ( 16h ) and invasion assay ( 24h ) in the presence of 10μM PAG . Data information : Data shown represent the means ± SD ( n = 3 biological replicates ) . Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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(G) PC3 cells were pre-treated with 10μM PAG for 16 hrs, and incubated for migration (16h) and invasion assay (24h) in the presence of 10μM PAG. Data information: Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001).


( A ) Real - Time RT - PCR analysis for IL - 1β mRNA levels in PC3 cells with control or CTH knockdown . Recombinant IL - 1β ( 10ng / ml ) was added for 24h in serum - free medium . Data information : All data shown represent the means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc test was used for the statistical analysis . ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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(A) Real-Time RT-PCR analysis for IL-1β mRNA levels in PC3 cells with control or CTH knockdown. Recombinant IL-1β (10ng/ml) was added for 24h in serum-free medium. Data information: All data shown represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001).


( B ) ELISA assay for IL - 1β protein level in the culture medium of PC3 cells with control or CTH knockdown . Data information All data shown represent the means ± SD ( n = 3 biological replicates ) . Student ' s t - test ( B ) was used for the statistical analysis . ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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(B) ELISA assay for IL-1β protein level in the culture medium of PC3 cells with control or CTH knockdown. Data information All data shown represent the means ± SD (n=3 biological replicates). Student's t-test (B) was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001).


( C ) PC3 cells transfected with control or CTH knockdown were incubated for invasion ( 24h ) assay with recombinant IL - 1β ( 10ng / ml ) added to the medium of the lower chamber . Data information : All data shown represent the means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc test was used for the statistical analysis . ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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(C) PC3 cells transfected with control or CTH knockdown were incubated for invasion (24h) assay with recombinant IL-1β (10ng/ml) added to the medium of the lower chamber. Data information: All data shown represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001).


( D ) PC3 cells transfected with control or CTH siRNA were exposed to IL - 1β ( 20ng / ml ) for 1hr . Subcellular localization of p65 was detected by immunocytochemistry . Nuclei were counterstained with DAPI . The representative images are shown . Scale bars : 25μm .

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(D) PC3 cells transfected with control or CTH siRNA were exposed to IL-1β (20ng/ml) for 1hr. Subcellular localization of p65 was detected by immunocytochemistry. Nuclei were counterstained with DAPI. The representative images are shown. Scale bars: 25μm.


( E ) The percent of nuclear translocation for p65 was scored by counting the number of nuclear positive stained cells of p65 to the total number of cells in random microscopic fields . Data information : All data shown represent the means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc test was used for the statistical analysis . ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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(E) The percent of nuclear translocation for p65 was scored by counting the number of nuclear positive stained cells of p65 to the total number of cells in random microscopic fields. Data information: All data shown represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001).


Real - Time RT - PCR analysis for VEGF ( F ) mRNA level in PC3 cells with control or CTH knockdown . Recombinant IL - 1β ( 10ng / ml ) was added for 24h in serum - free medium . Data information : All data shown represent the means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc test was used for the statistical analysis . ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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Real-Time RT-PCR analysis for VEGF (F) mRNA level in PC3 cells with control or CTH knockdown. Recombinant IL-1β (10ng/ml) was added for 24h in serum-free medium. Data information: All data shown represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001).


Real - Time RT - PCR analysis for MMP - 13 ( G ) mRNA level in PC3 cells with control or CTH knockdown . Recombinant IL - 1β ( 10ng / ml ) was added for 24h in serum - free medium . Data information : All data shown represent the means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc test was used for the statistical analysis . ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

Real-Time RT-PCR analysis for MMP-13 (G) mRNA level in PC3 cells with control or CTH knockdown. Recombinant IL-1β (10ng/ml) was added for 24h in serum-free medium. Data information: All data shown represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001).


( A ) PC3 cells were incubated for migration ( 16h ) and invasion ( 24h ) assay . DMEM / 10 % FBS , together with various concentration of NaHS ( 10nM - 1mM ) acting as a chemoattractant Data information Data shown represent the means ± SD ( n = 3 biological replicates ) ANOVA followed by Tukey ' s post - hoc tes was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 )

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

(A) PC3 cells were incubated for migration (16h) and invasion (24h) assay. DMEM/10% FBS, together with various concentration of NaHS (10nM-1mM) acting as a chemoattractant Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)


( B ) PC3 cells transfected with control or CTH siRNA were incubated for invasion ( 24h ) assay . DMEM / 10 % FBS , with or without NaHS ( 1μM ) , acting as a chemoattractant Data information Data shown represent the means ± SD ( n = 3 biological replicates ) ANOVA followed by Tukey ' s post - hoc tes was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 )

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

(B) PC3 cells transfected with control or CTH siRNA were incubated for invasion (24h) assay. DMEM/10% FBS, with or without NaHS (1μM), acting as a chemoattractant Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)


( C ) Top : PC3 cells treated with 100 μM NaHS for 30 min at 37 ° C . Cells were lyzed and treated with or without DTT for 10min at 4 ° C before subjecting to the modified biotin switch assay with the antibody against p65 to detect S - sulfhydration . Down : quantitative analysis of SSH - p65 protein level , and normalized with total p65 level Data information Data shown represent the means ± SD ( n = 3 biological replicates ) ANOVA followed by Tukey ' s post - hoc tes was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 )

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

(C) Top: PC3 cells treated with 100 μM NaHS for 30 min at 37°C. Cells were lyzed and treated with or without DTT for 10min at 4°C before subjecting to the modified biotin switch assay with the antibody against p65 to detect S-sulfhydration. Down: quantitative analysis of SSH-p65 protein level, and normalized with total p65 level Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)


( D ) PC3 cells were incubate for invasion assay for 24 hrs . DMEM / 10 % FBS , together with 1μM NaHS and an NF - κB inhibitor ( 50μg / ml SN50 , or 100nM QNZ ) , acting as a chemoattractant Data information Data shown represent the means ± SD ( n = 3 biological replicates ) ANOVA followed by Tukey ' s post - hoc tes was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 )

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

(D) PC3 cells were incubate for invasion assay for 24 hrs. DMEM/10% FBS, together with 1μM NaHS and an NF-κB inhibitor (50μg/ml SN50, or 100nM QNZ), acting as a chemoattractant Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)


( E ) Real - Time RT - PCR analysis for IL - 1β , VEGF , and MMP - 13 mRNA levels in PC3 cells treated with 1μM NaHS for 24h Data information Data shown represent the means ± SD ( n = 3 biological replicates ) Student ' s t - tes was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 )

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

(E) Real-Time RT-PCR analysis for IL-1β, VEGF, and MMP-13 mRNA levels in PC3 cells treated with 1μM NaHS for 24h Data information Data shown represent the means ± SD (n=3 biological replicates) Student's t-tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)


( F ) ELISA assay for IL - 1β protein level in the culture medium of PC3 cells with various concentrations of NaHS ( 10nM - 10μM ) added for 24h Data information Data shown represent the means ± SD ( n = 3 biological replicates ) ANOVA followed by Tukey ' s post - hoc tes was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 )

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

(F) ELISA assay for IL-1β protein level in the culture medium of PC3 cells with various concentrations of NaHS (10nM-10μM) added for 24h Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)


( G ) Western blot analysis of the p65 expression in PC3 cells with p65 knock - out and then transfected with p65 wild - type or C38S mutant Data information Data shown represent the means ± SD ( n = 3 biological replicates )

O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of the p65 expression in PC3 cells with p65 knock-out and then transfected with p65 wild-type or C38S mutant Data information Data shown represent the means ± SD (n=3 biological replicates)


( H ) PC3 cells with p65 knock - out with or without overexpression of p65 wild - type or C38S mutant were incubated for invasion assay for 24 hrs . DMEM / 10 % FBS was used as a chemoattractant Data information Data shown represent the means ± SD ( n = 3 biological replicates ) ANOVA followed by Tukey ' s post - hoc tes was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 )

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(H) PC3 cells with p65 knock-out with or without overexpression of p65 wild-type or C38S mutant were incubated for invasion assay for 24 hrs. DMEM/10% FBS was used as a chemoattractant Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)


1x106 PC3 cells with p65 knock - out were transiently re - expressed p65 wild - type or C38S mutant and inoculated into the bloodstream via tail - vein injection . After 7 days of inoculation , mouse lung was collected to examine lung metastasis . ( I ) Images show H & E staining of the lungs from mice injected with p65 knockout PC3 cells re - expressing p65 wild - type or C38S mutant . Red arrows indicate metastatic nodules . Scale bar :

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

1x106 PC3 cells with p65 knock-out were transiently re-expressed p65 wild-type or C38S mutant and inoculated into the bloodstream via tail-vein injection. After 7 days of inoculation, mouse lung was collected to examine lung metastasis. (I) Images show H&E staining of the lungs from mice injected with p65 knockout PC3 cells re-expressing p65 wild-type or C38S mutant. Red arrows indicate metastatic nodules. Scale bar: 1mm


1x106 PC3 cells with p65 knock - out were transiently re - expressed p65 wild - type or C38S mutant and inoculated into the bloodstream via tail - vein injection . After 7 days of inoculation , mouse lung was collected to examine lung metastasis The total metastatic nodules per lung ( J were quantified . Data are presented as means ± SEM ( n = 7 mice per group ) . Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 )

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

1x106 PC3 cells with p65 knock-out were transiently re-expressed p65 wild-type or C38S mutant and inoculated into the bloodstream via tail-vein injection. After 7 days of inoculation, mouse lung was collected to examine lung metastasis The total metastatic nodules per lung (J were quantified. Data are presented as means ± SEM (n=7 mice per group). Student's t-test was used for the statistical analysis (*P<0.05)


1x106 PC3 cells with p65 knock - out were transiently re - expressed p65 wild - type or C38S mutant and inoculated into the bloodstream via tail - vein injection . After 7 days of inoculation , mouse lung was collected to examine lung metastasis percentage of the metastatic area per lung ( K ) were quantified . Data are presented as means ± SEM ( n = 7 mice per group ) . Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 )

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

1x106 PC3 cells with p65 knock-out were transiently re-expressed p65 wild-type or C38S mutant and inoculated into the bloodstream via tail-vein injection. After 7 days of inoculation, mouse lung was collected to examine lung metastasis percentage of the metastatic area per lung (K) were quantified. Data are presented as means ± SEM (n=7 mice per group). Student's t-test was used for the statistical analysis (*P<0.05)


( A ) Western blot analysis of the CTH expression in PC3 cells stably expressing control or CTH shRNA .

(A) Western blot analysis of the CTH expression in PC3 cells stably expressing control or CTH shRNA.


1x106 PC3 cells with control or CTH knockdown were orthotopically injected into the mouse prostate for 60 days . The tumor weight ( B ) , and tumor volume ( C ) were compared . Data are presented as means ± SEM ( n = 10 mice per group ) . Data information : Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ) .

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1x106 PC3 cells with control or CTH knockdown were orthotopically injected into the mouse prostate for 60 days. The tumor weight (B), and tumor volume (C) were compared. Data are presented as means ± SEM (n = 10 mice per group). Data information: Student's t-test was used for the statistical analysis (*P<0.05).


1x106 PC3 cells with control or CTH knockdown were orthotopically injected into the mouse prostate for 60 days . Incidences of paraaortic lymph nodes metastasis ( D ) and bone metastasis ( E ) are shown . Data are presented as means ± SEM ( n = 10 mice per group ) . Data information : Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ) .

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1x106 PC3 cells with control or CTH knockdown were orthotopically injected into the mouse prostate for 60 days. Incidences of paraaortic lymph nodes metastasis (D) and bone metastasis (E) are shown. Data are presented as means ± SEM (n = 10 mice per group). Data information: Student's t-test was used for the statistical analysis (*P<0.05).


( F ) Real - Time RT - PCR analysis for CTH mRNA levels in PC3 orthotopic tumors from shCon or shCTH group . Data shown represent normalized means ± SEM ( n = 10 mice per group ) . Data information : Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ) .

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(F) Real-Time RT-PCR analysis for CTH mRNA levels in PC3 orthotopic tumors from shCon or shCTH group. Data shown represent normalized means ± SEM (n=10 mice per group). Data information: Student's t-test was used for the statistical analysis (*P<0.05).


Immunohistochemical analysis of paraffin - embedded xenograft prostate tumor using CD31 ( G ) antibody . The total vessels number per mm2 was quantified . Scale bars : 0 . 1 mm . Data are presented as means ± SEM ( n = 4 - 5 mice per group ) . Data information : Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ) .

Immunohistochemical analysis of paraffin-embedded xenograft prostate tumor using CD31 (G) antibody. The total vessels number per mm2 was quantified. Scale bars: 0.1 mm. Data are presented as means ± SEM (n=4-5 mice per group). Data information: Student's t-test was used for the statistical analysis (*P<0.05).


Immunohistochemical analysis of paraffin - embedded xenograft prostate tumor using LYVE - 1 ( H ) antibody . The total vessels number per mm2 was quantified . Scale bars : 0 . 1 mm . Data are presented as means ± SEM ( n = 4 - 5 mice per group ) . Data information : Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ) .

Immunohistochemical analysis of paraffin-embedded xenograft prostate tumor using LYVE-1 (H) antibody. The total vessels number per mm2 was quantified. Scale bars: 0.1 mm. Data are presented as means ± SEM (n=4-5 mice per group). Data information: Student's t-test was used for the statistical analysis (*P<0.05).


Fluorescence staining for the apical marker CD13 on liver tissue sections from untreated mice or animals at indicated time points post PH . Shown are maximum projections of 50 µm z - stacks covering an entire CV - PV axis ( CV , left ; PV , right ) . Indicated regions of the CV ( orange ) and PV ( blue ) areas are shown as magnifications on the left and right , respectively .

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Fluorescence staining for the apical marker CD13 on liver tissue sections from untreated mice or animals at indicated time points post PH. Shown are maximum projections of 50 µm z-stacks covering an entire CV-PV axis (CV, left; PV, right). Indicated regions of the CV (orange) and PV (blue) areas are shown as magnifications on the left and right, respectively.


Quantification of BC diameter within 18 zones along the CV - PV axis ( zone 1 , peri - central ; zone 18 , peri - portal ) in livers from untreated animals or mice at indicated timepoints post PH . The diameter was measured from 3D BC network reconstructions of IF image stacks of CD13 as shown in ( a ) . The zones directly adjacent to the CV and PV were excluded from the analysis ( ~ 1 cell layer ) . Mean ± s . e . m , n = 3 - 6 mice per timepoint . BC diameter of untreated mice vs . 0 . 8 d , p = 1 . 62 * 10 - 11 ; untreated mice vs . 1 . 5 d , p = 1 . 18 * 10 - 12 ; untreated mice vs . 2 . 0 d , p = 5 . 69 * 10 - 11 ; untreated mice vs . 3 . 0 d , p = 6 . 92 * 10 - 12 ; untreated mice vs . 5 . 0 d , p = 1 . 65 * 10 - 7 .

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Quantification of BC diameter within 18 zones along the CV-PV axis (zone 1, peri-central; zone 18, peri-portal) in livers from untreated animals or mice at indicated timepoints post PH. The diameter was measured from 3D BC network reconstructions of IF image stacks of CD13 as shown in (a). The zones directly adjacent to the CV and PV were excluded from the analysis (~ 1 cell layer). Mean ± s.e.m, n=3-6 mice per timepoint. BC diameter of untreated mice vs. 0.8 d, p=1.62*10-11; untreated mice vs. 1.5 d, p=1.18*10-12; untreated mice vs. 2.0 d, p=5.69*10-11; untreated mice vs. 3.0 d, p=6.92*10-12; untreated mice vs. 5.0 d, p=1.65*10-7.


EM images of BC on liver tissue sections of untreated mice ( left ) and 1 . 8 d post sham OP ( middle ) or PH ( right ) .

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EM images of BC on liver tissue sections of untreated mice (left) and 1.8 d post sham OP (middle) or PH (right).


Quantification of BC perimeter ( d ) and total BC membrane length ( e ) from EM images as representatively shown in ( c ) . Box - whisker plot with median , 25 - 75 quartiles and minimum / maximum error bars , n = 5 - 6 mice per condition . In ( d ) , BC perimeter of untreated vs . sham condition , p = 0 . 94 ( n . s . ) ; Untreated vs . PH condition , p = 0 . 01 . In ( e ) , BC membrane length of untreated vs . sham condition , p = 0 . 86 ( n . s . ) ; Untreated vs . PH condition , p = 0 . 03 .

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Quantification of BC perimeter (d) and total BC membrane length (e) from EM images as representatively shown in (c). Box-whisker plot with median, 25-75 quartiles and minimum/maximum error bars, n=5-6 mice per condition. In (d), BC perimeter of untreated vs. sham condition, p=0.94 (n.s.); Untreated vs. PH condition, p=0.01. In (e), BC membrane length of untreated vs. sham condition, p=0.86 (n.s.); Untreated vs. PH condition, p=0.03.


IF stainings of the apical marker CD13 on liver tissue sections from livers at 1 d post sham OP or BDL . Shown are maximum projections of 50 µm z - stacks in the CV ( left , orange ) and PV ( right , blue ) region . Images in ( a ) and ( f ) are background - subtracted . Scale bars , 50 µm ( a ) , 1 µm ( c ) , 5 µm ( f ) .

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IF stainings of the apical marker CD13 on liver tissue sections from livers at 1 d post sham OP or BDL. Shown are maximum projections of 50 µm z-stacks in the CV (left, orange) and PV (right, blue) region. Images in (a) and (f) are background-subtracted. Scale bars, 50 µm (a), 1 µm (c), 5 µm (f).


Fluorescence stainings for F - actin and the apical marker CD13 in the PV area on liver tissue sections from untreated mice or animals at indicated time points post PH . Arrows indicate BC . Scale bar , 5 µm Quantification of apical F - actin intensity from images as representatively shown in ( a , as well as Appendix Fig . S2A , B , at indicated time points post PH ( solid line ) or sham OP ( dashed line ) . Data is normalized to untreated animals ( timepoint 0 ) . Mean ± s . e . m , n = 3 - 5 ( b ) mice per timepoint .

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Fluorescence stainings for F-actin and the apical marker CD13 in the PV area on liver tissue sections from untreated mice or animals at indicated time points post PH. Arrows indicate BC. Scale bar, 5 µm Quantification of apical F-actin intensity from images as representatively shown in (a, as well as Appendix Fig. S2A,B, at indicated time points post PH (solid line) or sham OP (dashed line). Data is normalized to untreated animals (timepoint 0). Mean ± s.e.m, n=3-5 (b) mice per timepoint.


Fluorescence stainings for pMLC and the apical marker CD13 in the PV area on liver tissue sections from untreated mice or animals at indicated time points post PH . Arrows indicate BC . Scale bar , 5 µm Quantification of pMLC ( d ) intensity from images as representatively shown in ( a , c ) as well as Appendix Fig . S2A , B , at indicated time points post PH ( solid line ) or sham OP ( dashed line ) . Data is normalized to untreated animals ( timepoint 0 ) . Mean ± s . e . m , n = 2 - 5 ( d ) mice per timepoint .

Fluorescence stainings for pMLC and the apical marker CD13 in the PV area on liver tissue sections from untreated mice or animals at indicated time points post PH. Arrows indicate BC. Scale bar, 5 µm Quantification of pMLC (d) intensity from images as representatively shown in (a,c) as well as Appendix Fig. S2A,B, at indicated time points post PH (solid line) or sham OP (dashed line). Data is normalized to untreated animals (timepoint 0). Mean ± s.e.m, n=2-5 (d) mice per timepoint.


Fluorescence stainings for YAP , PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice and animals at indicated time points post PH . Images show an entire CV - PV axis ( CV , left ; PV , right ) . Note , the bright YAP fluorescence in the PV area stem from cholangiocytes of the bile duct .

Fluorescence stainings for YAP, PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice and animals at indicated time points post PH. Images show an entire CV-PV axis (CV, left; PV, right). Note, the bright YAP fluorescence in the PV area stem from cholangiocytes of the bile duct.


Fluorescence stainings for YAP , PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice and animals at indicated time points post PH . Images show an entire CV - PV axis ( CV , left ; PV , right ) . Indicated regions ( orange squares ) are shown as magnifications in ( b ) . Note , the bright YAP fluorescence in the PV area stem from cholangiocytes of the bile duct .

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Fluorescence stainings for YAP, PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice and animals at indicated time points post PH. Images show an entire CV-PV axis (CV, left; PV, right). Indicated regions (orange squares) are shown as magnifications in (b). Note, the bright YAP fluorescence in the PV area stem from cholangiocytes of the bile duct.


Quantification of the mean nuclear YAP ( green ) and PCNA ( magenta ) intensity from images of liver tissue sections at indicated timepoints post PH ( solid line ) and sham OP ( dashed line ) as representatively shown in ( a ) and Appendix Fig . S4A . Data was normalized to untreated animals ( timepoint 0 ) . Mean ± s . e . m , n = 3 - 5 mice per timepoint . Nuclear YAP intensity of PH vs . sham time course , p = 2 . 97 * 10 - 5 . Nuclear PCNA intensity of PH vs . sham time course , p = 7 . 51 * 10 - 6 .

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

Quantification of the mean nuclear YAP (green) and PCNA (magenta) intensity from images of liver tissue sections at indicated timepoints post PH (solid line) and sham OP (dashed line) as representatively shown in (a) and Appendix Fig. S4A. Data was normalized to untreated animals (timepoint 0). Mean ± s.e.m, n=3-5 mice per timepoint. Nuclear YAP intensity of PH vs. sham time course, p=2.97*10-5. Nuclear PCNA intensity of PH vs. sham time course, p=7.51*10-6.


Spatial analysis of the mean nuclear YAP intensity from images as representatively shown in ( a ) and Appendix Fig . S4A , at indicated time points post PH ( solid line ) or sham OP ( dashed line ) in 10 zones within the CV - PV axis ( zone 1 , CV area ; zone 10 , PV area ) . Data is normalized to untreated animals ( not shown ) . Mean ± s . e . m , n = 3 - 5 mice per timepoint . Nuclear YAP intensity of sham vs . PH mice at 0 . 8d , p = 5 . 35 * 10 - 5 ; 1 . 5d , p = 3 . 95 * 10 - 5 ; 2d , p = 5 . 99 * 10 - 5 ; 3d , p = 2 . 16 * 10 - 5 ; 5d , p = 0 . 01 .

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

Spatial analysis of the mean nuclear YAP intensity from images as representatively shown in (a) and Appendix Fig. S4A, at indicated time points post PH (solid line) or sham OP (dashed line) in 10 zones within the CV-PV axis (zone 1, CV area; zone 10, PV area). Data is normalized to untreated animals (not shown). Mean ± s.e.m, n=3-5 mice per timepoint. Nuclear YAP intensity of sham vs. PH mice at 0.8d, p=5.35*10-5; 1.5d, p=3.95*10-5; 2d, p=5.99*10-5; 3d, p=2.16*10-5; 5d, p=0.01.


Fluorescence stainings for YAP , CD13 and with the nuclear marker DAPI on liver tissue sections from an untreated mouse or animals at indicated time points post PH . Arrows indicate BC . Note the enrichment of apical YAP at 0 . 5 and 1 . 5d post PH .

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Fluorescence stainings for YAP, CD13 and with the nuclear marker DAPI on liver tissue sections from an untreated mouse or animals at indicated time points post PH. Arrows indicate BC. Note the enrichment of apical YAP at 0.5 and 1.5d post PH.


Fractionation of liver tissue at 2d post PH or sham ( Sh ) OP and untreated ( UT ) mice for nuclear ( Nuc ) , cytoplasmic ( Cyto ) and membrane ( Mem ) fraction . TRF2 , transferrin receptor2 ( membrane marker ) ; Histone H3 ( nuclear marker ) ; GAPDH , Glyceraldehyde 3 - phosphate dehydrogenase ( cytoplasmic marker ) . Quantification of YAP in the nuclear , cytoplasmic and membrane fraction from immunoblots as representatively shown in ( f ) . YAP signal is normalized to the fraction marker ( GAPDH , cytoplasm ; Histon H3 , nuclei ; TRF2 , membrane ) and to the untreated condition ( not shown ) . n = 3 ( sham ) or 4 ( PH ) ; data is shown as mean + / - s . e . m . Differences between sham and PH conditions for cytoplasmic , nuclear and membrane fractions are not significant .

Fractionation of liver tissue at 2d post PH or sham (Sh) OP and untreated (UT) mice for nuclear (Nuc), cytoplasmic (Cyto) and membrane (Mem) fraction. TRF2, transferrin receptor2 (membrane marker); Histone H3 (nuclear marker); GAPDH, Glyceraldehyde 3-phosphate dehydrogenase (cytoplasmic marker). Quantification of YAP in the nuclear, cytoplasmic and membrane fraction from immunoblots as representatively shown in (f). YAP signal is normalized to the fraction marker (GAPDH, cytoplasm; Histon H3, nuclei; TRF2, membrane) and to the untreated condition (not shown). n = 3 (sham) or 4 (PH); data is shown as mean +/- s.e.m. Differences between sham and PH conditions for cytoplasmic, nuclear and membrane fractions are not significant.


EM image of immunogold - labelled YAP on liver tissue sections at 1 . 8 d post PH . Image ( A ) shows a BC in between two hepatocytes . Indicated regions ( black squares ) of the apical ( a ' ) and the basolateral ( a ' ' ) membrane of the hepatocyte are shown as magnification . Arrows indicate gold particles .

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EM image of immunogold-labelled YAP on liver tissue sections at 1.8 d post PH. Image (A) shows a BC in between two hepatocytes. Indicated regions (black squares) of the apical (a') and the basolateral (a'') membrane of the hepatocyte are shown as magnification. Arrows indicate gold particles.


Correlative light and electron microcopy of F - actin ( B ) and YAP ( C ) on a liver tissue section at 1 . 8 d post PH . YAP was detected by immunogold - labelling , F - actin by fluorescence staining with phalloidin . Indicated BC regions with high ( c ' , c ' ' ) or low ( c ' ' ' ) F - actin levels are shown as magnifications . Arrows indicate gold particles .

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Correlative light and electron microcopy of F-actin (B) and YAP (C) on a liver tissue section at 1.8 d post PH. YAP was detected by immunogold-labelling, F-actin by fluorescence staining with phalloidin. Indicated BC regions with high (c', c'') or low (c''') F-actin levels are shown as magnifications. Arrows indicate gold particles.


Fluorescence stainings for YAP ( green ) , F - actin ( magenta ) and with the nuclear marker DAPI ( blue ) of primary hepatocyte cultures treated with DMSO ( control ) , Y27 , deoxycholic acid ( DCA ) or DCA + Y27 for ~ 18 h . Indicated areas ( dashed rectangle ) are shown as magnifications in insets .

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Fluorescence stainings for YAP (green), F-actin (magenta) and with the nuclear marker DAPI (blue) of primary hepatocyte cultures treated with DMSO (control), Y27, deoxycholic acid (DCA) or DCA+Y27 for ~18 h. Indicated areas (dashed rectangle) are shown as magnifications in insets.


Quantification of the mean nuclear YAP intensity from fluorescence images of primary hepatocytes treated with DMSO ( control ) , Y27 , DCA or DCA + Y27 for ~ 18 h as representatively shown in ( A ) . Data is normalized to untreated cells ( not shown ) . Mean ± s . e . m . , n = 7 ; DCA vs . DMSO p = 8 . 98 * 10 - 5 ; DCA + Y27 vs . DMSO , p = 0 . 05 ; Y27 vs . DMSO , p = 0 . 55 ( n . s . ) ; DCA + Y27 vs . DCA p = 0 .

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

Quantification of the mean nuclear YAP intensity from fluorescence images of primary hepatocytes treated with DMSO (control), Y27, DCA or DCA+Y27 for ~18 h as representatively shown in (A). Data is normalized to untreated cells (not shown). Mean ± s.e.m., n = 7; DCA vs. DMSO p=8.98*10-5; DCA+Y27 vs. DMSO, p=0.05; Y27 vs. DMSO, p=0.55 (n.s.); DCA+Y27 vs. DCA p = 0.05


Western blot of pMLC and GAPDH ( loading control ) of primary hepatocyte culture lysates . Cells were untreated or incubated for 18 h with the indicated

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Western blot of pMLC and GAPDH (loading control) of primary hepatocyte culture lysates. Cells were untreated or incubated for 18 h with the indicated compounds


Fluorescence stainings of primary hepatocytes for YAP ( green ) , F - actin ( magenta ) and with the nuclear marker DAPI ( blue ) . Cells were treated with DMSO ( control ) , Y27 , SMIFH2 or Cytochalasin D ( CytoD ) for 6 h . Indicated areas ( dashed rectangle ) are shown as magnifications in insets . Note the dilation of canaliculi ( asterisks ) and fragmentation of F - actin ( arrows ) upon CytoD treatment .

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

Fluorescence stainings of primary hepatocytes for YAP (green), F-actin (magenta) and with the nuclear marker DAPI (blue). Cells were treated with DMSO (control), Y27, SMIFH2 or Cytochalasin D (CytoD) for 6 h. Indicated areas (dashed rectangle) are shown as magnifications in insets. Note the dilation of canaliculi (asterisks) and fragmentation of F-actin (arrows) upon CytoD treatment.


Quantification of the mean nuclear YAP intensity from images of primary hepatocytes treated with DMSO , Saline , Y27 , Fasudil , SMIFH2 , CK666 , CytoD or Latrunculin A ( LatA ) for 6 h . Saline serves as control for Fasudil , DMSO serves as control for all other conditions . Inhibitors affecting similar actin processes are displayed in the same grey level . Saline , Fasudil , CK666 and LatA conditions are not shown in ( d ) . Data is normalized to untreated cells ( not shown ) . Mean ± s . e . m . , n = 3 - 5 ; DMSO vs . Y27 , p = 0 . 002 ; DMSO vs . CytoD , p = 5 . 11 * 10 - 7 ; DMSO vs . LatA , p = 0 . 01 ; DMSO vs . CK666 , p = 0 . 15 ( n . s . ) ; DMSO vs . SMIFH2 , p = 0 . 11 ( n . s . ) ; Saline vs . Fasudil , p = 0 . 92 .

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

Quantification of the mean nuclear YAP intensity from images of primary hepatocytes treated with DMSO, Saline, Y27, Fasudil, SMIFH2, CK666, CytoD or Latrunculin A (LatA) for 6 h. Saline serves as control for Fasudil, DMSO serves as control for all other conditions. Inhibitors affecting similar actin processes are displayed in the same grey level. Saline, Fasudil, CK666 and LatA conditions are not shown in (d). Data is normalized to untreated cells (not shown). Mean ± s.e.m., n=3-5; DMSO vs. Y27, p=0.002; DMSO vs. CytoD, p=5.11*10-7; DMSO vs. LatA, p=0.01; DMSO vs. CK666, p=0.15 (n.s.); DMSO vs. SMIFH2, p= 0.11 (n.s.); Saline vs. Fasudil, p= 0.92.


Western blot of pMLC and GAPDH ( loading control ) in untreated or actin inhibitor - treated primary hepatocyte culture lysates . Inhibitor treatments are the same as in ( E ) . Scale bars , 20 µm ( A , D ) .

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Western blot of pMLC and GAPDH (loading control) in untreated or actin inhibitor-treated primary hepatocyte culture lysates. Inhibitor treatments are the same as in (E). Scale bars, 20 µm (A, D).


Fluorescence stainings for the apical marker CD13 on liver tissue sections from mice at 2 d post sham OP or PH , treated with saline ( control ) or the Rho kinase inhibitor Fasudil for 1 h . Shown are maximum projections of 50 µm stacks in the PV area . Quantification of BC diameter within 18 zones along the CV - PV axis ( zone 1 , peri - central ; zone 18 , peri - portal ) from mice at 2 d post sham OP ( green ) or PH ( red ) , treated with saline ( control , solid line ) or Fasudil ( dashed line ) . Diameter was measured from 3D BC network reconstructions of image stacks as representatively shown in ( a ) . The zones directly adjacent to the CV and PV were excluded from the analysis ( ~ 1 cell layer ) . Mean ± s . e . m , n = 7 - 8 mice per condition from 3 independent experiments . BC diameter of sham operated mice treated with saline vs . Fasudil , p = 6 . 59 * 10 - 8 ; BC diameter of PH mice treated with saline vs . Fasudil , p = 2 . 31 * 10 - 14 .

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

Fluorescence stainings for the apical marker CD13 on liver tissue sections from mice at 2 d post sham OP or PH, treated with saline (control) or the Rho kinase inhibitor Fasudil for 1 h. Shown are maximum projections of 50 µm stacks in the PV area. Quantification of BC diameter within 18 zones along the CV-PV axis (zone 1, peri-central; zone 18, peri-portal) from mice at 2 d post sham OP (green) or PH (red), treated with saline (control, solid line) or Fasudil (dashed line). Diameter was measured from 3D BC network reconstructions of image stacks as representatively shown in (a). The zones directly adjacent to the CV and PV were excluded from the analysis (~ 1 cell layer). Mean ± s.e.m, n=7-8 mice per condition from 3 independent experiments. BC diameter of sham operated mice treated with saline vs. Fasudil, p= 6.59*10-8; BC diameter of PH mice treated with saline vs. Fasudil, p=2.31*10-14.


Fluorescence stainings for YAP , PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice or animals at 2 d post sham OP or PH , treated with saline ( control ) or Fasudil for 1 h . Indicated regions ( dashed rectangle ) are shown as magnifications in insets . Quantification of the mean nuclear YAP intensity within 10 zones along the CV - PV axis , ( zone 1 , peri - central ; zone 10 , peri - portal ) from IF images as representatively shown in ( c ) . Data was normalized to untreated animals . Mean ± s . e . m , n = 7 - 8 mice per condition from 3 independent experiments . Nuclear YAP intensity of sham operated mice treated with saline vs . Fasudil , p = 0 . 13 ( n . s . ) ; nuclear YAP intensity of PH mice treated with saline vs . Fasudil , p = 0 . 0004 .

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

Fluorescence stainings for YAP, PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice or animals at 2 d post sham OP or PH, treated with saline (control) or Fasudil for 1 h. Indicated regions (dashed rectangle) are shown as magnifications in insets. Quantification of the mean nuclear YAP intensity within 10 zones along the CV-PV axis, (zone 1, peri-central; zone 10, peri-portal) from IF images as representatively shown in (c). Data was normalized to untreated animals. Mean ± s.e.m, n=7-8 mice per condition from 3 independent experiments. Nuclear YAP intensity of sham operated mice treated with saline vs. Fasudil, p=0.13 (n.s.) ; nuclear YAP intensity of PH mice treated with saline vs. Fasudil, p=0.0004.


b ) Caco - 2 cells were infected with SARS - CoV - 2 as per Figure 1A . Cells were washed , fixed and stained with DAPI ( blue ) to visualise cell nuclei and incubated with anti - N protein ( red ) to visualise SARS - CoV - 2 infected cells . Scale bar denotes 10 µM .

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b) Caco-2 cells were infected with SARS-CoV-2 as per Figure 1A. Cells were washed, fixed and stained with DAPI (blue) to visualise cell nuclei and incubated with anti-N protein (red) to visualise SARS-CoV-2 infected cells. Scale bar denotes 10 µM.


c ) Infection rates were calculated using automated imaging software ( see materials and methods ) calculated from six fields of view per time point per biological replicate . Data point indicates the sample means and the error bars the standard error of the mean ( SEM ) ( n = 3 ) . d ) SARS - CoV - 2 detected proteins plotted as a function of time where each data point denotes a different replicate ( n = 3 ) .

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c) Infection rates were calculated using automated imaging software (see materials and methods) calculated from six fields of view per time point per biological replicate. Data point indicates the sample means and the error bars the standard error of the mean (SEM) (n=3). d) SARS-CoV-2 detected proteins plotted as a function of time where each data point denotes a different replicate (n=3).


a ) Total number of significantly up - or down - regulated proteins ( | z - score | > 1 . 96 , q - value < 0 . 05 ) per time point of SARS - CoV - 2 infected Caco - 2 cells as described in 1A . Proteins that were significantly changed in abundance ( left ) and stability ( right ) are sub - grouped according to either down - regulation / destabilization ( red ) or up - regulation / stabilisation ( turquoise ) .

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a) Total number of significantly up- or down-regulated proteins (|z-score| >1.96, q-value <0.05) per time point of SARS-CoV-2 infected Caco-2 cells as described in 1A. Proteins that were significantly changed in abundance (left) and stability (right) are sub-grouped according to either down-regulation/destabilization (red) or up-regulation/stabilisation (turquoise).


c ) SAFB phosphopeptide abundance changes during SARS - CoV - 2 infection . Vero cell phosphopeptide data from ( Bouhaddou et al , 2020 ) . Data previously acquired from Vero cells infected with SARS - CoV - 2 ( Bouhaddou et al , 2020 ) . S604 phosphosite that temporally coincides with SAFB thermal destabilisation ( Potel et al , 2020 ) . Shown above the line plot are the thermostability and abundance profiles of SAFB in SARS - CoV - 2 infected Caco - 2 cells as described in 1A ( n = 3 ) . Asterisk denotes significant regulation ( see methods ) .

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c) SAFB phosphopeptide abundance changes during SARS-CoV-2 infection. Vero cell phosphopeptide data from(Bouhaddou et al, 2020). Data previously acquired from Vero cells infected with SARS-CoV-2 (Bouhaddou et al, 2020). S604 phosphosite that temporally coincides with SAFB thermal destabilisation (Potel et al, 2020). Shown above the line plot are the thermostability and abundance profiles of SAFB in SARS-CoV-2 infected Caco-2 cells as described in 1A (n=3). Asterisk denotes significant regulation (see methods).


D ) Thermal stability profile for all unmodified SAFB peptides ( green ) and the phosphorylated S604 ( pS604 ) ( purple ) in HeLa cells . Data from ( Potel et al , 2020 ) .

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D) Thermal stability profile for all unmodified SAFB peptides (green) and the phosphorylated S604 (pS604) (purple) in HeLa cells. Data from (Potel et al, 2020).


b ) The corresponding HSP90 inhibitor , Tanespimycin , effectively suppressed SARS - CoV - 2 proliferation ( n = 2 ) . Calu - 3 cells were pre - incubated with the compound , followed by the addition of SARS - CoV - 2 and subsequent co - incubation in the presence of the compound for 20 - 24 hours . Viral load was quantified in parallel using antibody - based detection of double stranded RNA ( orange ) . Compound toxicity in Calu - 3 cells was assessed ( purple ) treated with the compound alone for 20 - 24 hours .

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b) The corresponding HSP90 inhibitor, Tanespimycin, effectively suppressed SARS-CoV-2 proliferation (n=2). Calu-3 cells were pre-incubated with the compound, followed by the addition of SARS-CoV-2 and subsequent co-incubation in the presence of the compound for 20-24 hours. Viral load was quantified in parallel using antibody-based detection of double stranded RNA (orange). Compound toxicity in Calu-3 cells was assessed (purple) treated with the compound alone for 20-24 hours.


d ) The Cyp1a1 inhibitor suppressed SARS - CoV - 2 proliferation ( n = 2 ) . Dose response curves were performed as described in 4A . See Appendix Figure S7 for additional tested drugs . Data displayed as described in 5B .

d) The Cyp1a1 inhibitor suppressed SARS-CoV-2 proliferation (n=2). Dose response curves were performed as described in 4A. See Appendix Figure S7 for additional tested drugs. Data displayed as described in 5B.


A . NS1 dimer with one subunit in grey and the other colored by domain ( green , β - roll ; orange , wing with magenta connector subdomain ; blue , central β - ladder ) . N - linked glycosylation sites are shown in sticks , and the glycans are shown in spheres . B . Topology diagram for the NS1 monomer , colored as the pattern in ( A ) . Glycosylation sites are indicated with red hexagons and disulfide bonds with yellow circles . C . Side views of the NS1 dimer from the wing ( left ) and the end of the β - ladder ( right ) . The β - roll ( green ) , connector subdomain ( orange ) and the intertwined loop ( yellow ) of the wing domain form a discontinuous protrusion on one face of the β - ladder with the spaghetti loop ( cyan ) on the other face . The wing domain is omitted from the left image for clarity .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. NS1 dimer with one subunit in grey and the other colored by domain (green, β-roll; orange, wing with magenta connector subdomain; blue, central β-ladder). N-linked glycosylation sites are shown in sticks, and the glycans are shown in spheres.B. Topology diagram for the NS1 monomer, colored as the pattern in (A). Glycosylation sites are indicated with red hexagons and disulfide bonds with yellow circles.C. Side views of the NS1 dimer from the wing (left) and the end of the β-ladder (right). The β-roll (green), connector subdomain (orange) and the intertwined loop (yellow) of the wing domain form a discontinuous protrusion on one face of the β-ladder with the spaghetti loop (cyan) on the other face. The wing domain is omitted from the left image for clarity.


The ZIKV NS1 hexamer is modeled by using the full - length DENV2 NS1 structure ( PDB code : 4O6B ) ( Akey et al , 2014 ) . Each monomer is colored by domains ( green , β - roll ; orange , wing ; magenta , connector subdomain ; blue , central β - ladder ) . The intertwined loop " spike " of ZIKV NS1 contributing to membrane association is colored in red and indicated by dotted lines .

The ZIKV NS1 hexamer is modeled by using the full-length DENV2 NS1 structure (PDB code: 4O6B) (Akey et al, 2014). Each monomer is colored by domains (green, β-roll; orange, wing; magenta, connector subdomain; blue, central β-ladder). The intertwined loop "spike" of ZIKV NS1 contributing to membrane association is colored in red and indicated by dotted lines.


ABC . Electrostatic surface views of NS1 from DENV2 ( PDB : 4O6B , A ) and WNV ( PDB : 4O6C , B ) ( Akey et al , 2014 ) , as well as ZIKV © , showing diverse characteristics on certain regions . Surfaces are colored by electrostatic potential at neutral pH from - 2 kT ( red ) to + 2 kT ( blue ) using PyMOL ( http : / / pymol . org / ) .

ABC. Electrostatic surface views of NS1 from DENV2 (PDB: 4O6B, A) and WNV (PDB: 4O6C, B) (Akey et al, 2014), as well as ZIKV©, showing diverse characteristics on certain regions. Surfaces are colored by electrostatic potential at neutral pH from -2 kT (red) to +2 kT (blue) using PyMOL (http://pymol.org/).


A . The NS1 surface is colored using the ConSurf server ( Ashkenazy et al , 2016 ) according to sequence conservation from the most conserved ( dark magenta ) to the most divergent ( dark cyan ) based on an alignment of NS1 sequences from 73 flaviviruses .

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A. The NS1 surface is colored using the ConSurf server (Ashkenazy et al, 2016) according to sequence conservation from the most conserved (dark magenta) to the most divergent (dark cyan) based on an alignment of NS1 sequences from 73 flaviviruses.


B . Models of intertwined loop " spike " interacting with the cellular membrane . The disordered " spike " regions are indicated by dash lines in DENV2 and WNV NS1 structures , which consist of hydrophobic or positively charged amino acids .

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B. Models of intertwined loop "spike" interacting with the cellular membrane. The disordered "spike" regions are indicated by dash lines in DENV2 and WNV NS1 structures, which consist of hydrophobic or positively charged amino acids.


A . B - factor putty representation of the intertwined loop structure . Shades of blue and a narrower tube indicate regions with lower B - factors , whereas orange to red and a wider tube indicate regions with higher B - factors . The figure was prepared using PyMOL ( http : / / pymol . org / ) . B . The 2Fo - Fc electron density map for the intertwined loop ( yellow ) " spike " region residues contoured at 1 . 0 sigma is represented in blue . C . Detailed interactions of the highly conserved residues W115 and W118 with the neighboring residues to stabilize the conformation of the intertwined loop . W115 forms a hydrogen - bonding interaction with E74 from α2 helix . W118 inserts into the hydrophobic interspace between α1 and α2 helix , formed by P36 , L42 , V71 , L75 and I78 , and W118 also interact with P36 and D37 by hydrogen - bonding interactions .

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A. B-factor putty representation of the intertwined loop structure. Shades of blue and a narrower tube indicate regions with lower B-factors, whereas orange to red and a wider tube indicate regions with higher B-factors. The figure was prepared using PyMOL (http://pymol.org/).B. The 2Fo-Fc electron density map for the intertwined loop (yellow) "spike" region residues contoured at 1.0 sigma is represented in blue.C. Detailed interactions of the highly conserved residues W115 and W118 with the neighboring residues to stabilize the conformation of the intertwined loop. W115 forms a hydrogen-bonding interaction with E74 from α2 helix. W118 inserts into the hydrophobic interspace between α1 and α2 helix, formed by P36, L42, V71, L75 and I78, and W118 also interact with P36 and D37 by hydrogen-bonding interactions.


( a ) Ten - month - old adult mice and 20 - month - old aged mice were treated with an intraperitoneal injection of 5 mg kg - 1 CCCP and their hearts were collected 12 h later . Representative electron micrographs of hearts from CCCP - treated adult and aged mice ; original magnification × 5 , 000 ; scale bar , 2 μm . The magnified photograph represents an autophagic vacuole containing mitochondria ; original magnification × 12 , 000 ; scale bar , 500 nm . Mitochondria incorporated into an autophagic vacuole in baseline and CCCP - treated conditions were quantified blindly from 8 - 10 images from different fields ( original magnification × 5 , 000 ) ( n = 3 per group ) .

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(a) Ten-month-old adult mice and 20-month-old aged mice were treated with an intraperitoneal injection of 5 mg kg-1 CCCP and their hearts were collected 12 h later. Representative electron micrographs of hearts from CCCP-treated adult and aged mice; original magnification × 5,000; scale bar, 2 μm. The magnified photograph represents an autophagic vacuole containing mitochondria; original magnification × 12,000; scale bar, 500 nm. Mitochondria incorporated into an autophagic vacuole in baseline and CCCP-treated conditions were quantified blindly from 8-10 images from different fields (original magnification × 5,000) (n=3 per group).


( b , c ) CCCP - induced recruitment of Parkin and p62 to mitochondria was determined by immunoblotting of the heart mitochondria - rich fraction in adult and aged mice ( b ) , and DOX - treated mice ( c ) . Representative immunoblots are shown from four independent experiments .

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(b,c) CCCP-induced recruitment of Parkin and p62 to mitochondria was determined by immunoblotting of the heart mitochondria-rich fraction in adult and aged mice (b), and DOX-treated mice (c). Representative immunoblots are shown from four independent experiments.


( d ) Parkin was overexpressed in young and senescent MEFs using adenovirus - mediated transduction for the in vitro cell - based bioassay to assess the ability to clear damaged mitochondria . Mitochondrial content after 24 h of treatment with 20 μM CCCP was assessed by flow cytometry for MitoTracker Green FM . Results from four independent experiments performed in duplicate are shown . MEFs at passage 3 and passage 9 were used as young and senescent MEFs , respectively .

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(d) Parkin was overexpressed in young and senescent MEFs using adenovirus-mediated transduction for the in vitro cell-based bioassay to assess the ability to clear damaged mitochondria. Mitochondrial content after 24 h of treatment with 20 μM CCCP was assessed by flow cytometry for MitoTracker Green FM. Results from four independent experiments performed in duplicate are shown. MEFs at passage 3 and passage 9 were used as young and senescent MEFs, respectively.


( e ) Mitochondrial DNA content was assessed by real - time PCR . Results are shown from six - independent experiments .

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(e) Mitochondrial DNA content was assessed by real-time PCR. Results are shown from six-independent experiments.


( f ) Mitochondrial content was assessed by immunoblotting for the mitochondrial chaperone , GRP75 ( matrix protein ) and complex I subunit , NDUFA9 ( inner membrane protein ) . MEFs were treated with 100 nM bafilomycin - A1 , ( Baf - A1 , an inhibitor of the vacuolar - type proton ATPase ) to inhibit autophagy . Representative immunoblots are shown from three independent experiments .

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(f) Mitochondrial content was assessed by immunoblotting for the mitochondrial chaperone, GRP75 (matrix protein) and complex I subunit, NDUFA9 (inner membrane protein). MEFs were treated with 100 nM bafilomycin-A1, (Baf-A1, an inhibitor of the vacuolar-type proton ATPase) to inhibit autophagy. Representative immunoblots are shown from three independent experiments.


( g ) Representative images of GFP - LC3 expressing MEFs treated with 20 μM CCCP for 10 h before the immunostaining of mitochondria with anti - TOM20 ( red ) ; original magnification , × 400 and × 1 , 000 ; scale bar , 100 μm and scale bar , 15 μm . Line scans below the images indicate colocalization between LC3 ( green ) and mitochondria ( red ) and correlate to the arrows drawn in the images . WHL indicates whole - heart lysate . Data are shown as the means ± s . d . * P0 . 05 ; * * P0 . 01 ( two - tailed unpaired Student ' s t - test ) .

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(g) Representative images of GFP-LC3 expressing MEFs treated with 20 μM CCCP for 10 h before the immunostaining of mitochondria with anti-TOM20 (red); original magnification, × 400 and × 1,000; scale bar, 100 μm and scale bar, 15 μm. Line scans below the images indicate colocalization between LC3 (green) and mitochondria (red) and correlate to the arrows drawn in the images. WHL indicates whole-heart lysate. Data are shown as the means±s.d. *P0.05; **P0.01 (two-tailed unpaired Student's t-test).


( a ) The senescence - associated molecules p53 , p21 , p16 , H - Ras 12V and Rb were overexpressed in MEFs by retrovirus - mediated transduction . After puromycin selection , mitophagy induction was performed with treatment of 20 μM CCCP 24 h after adenovirus - mediated Parkin expression . Mitochondrial content 24 h after mitophagy induction was assessed by flow cytometry for MitoTracker Green FM . Results are shown from four independent experiments performed in duplicate .

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(a) The senescence-associated molecules p53, p21, p16, H-Ras 12V and Rb were overexpressed in MEFs by retrovirus-mediated transduction. After puromycin selection, mitophagy induction was performed with treatment of 20 μM CCCP 24 h after adenovirus-mediated Parkin expression. Mitochondrial content 24 h after mitophagy induction was assessed by flow cytometry for MitoTracker Green FM. Results are shown from four independent experiments performed in duplicate.


( b ) Representative images of MEFs infected with the GFP - LC3 adenovirus and stained with anti - TOM20 ( red ) to visualize mitochondria 10 h after mitophagy induction ; original magnification , × 400 and × 1 , 000 ; scale bar , 100 μm and scale bar , 15 μm .

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(b) Representative images of MEFs infected with the GFP-LC3 adenovirus and stained with anti-TOM20 (red) to visualize mitochondria 10 h after mitophagy induction; original magnification, × 400 and × 1,000; scale bar, 100 μm and scale bar, 15 μm.


( c ) Mitochondrial content of H - Ras 12 V expressing WT , p53 − / − and p21 − / − MEFs was assessed by flow cytometry 24 h after mitophagy induction . Results are shown from four independent experiments performed in duplicate .

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(c) Mitochondrial content of H-Ras 12 V expressing WT, p53−/− and p21−/− MEFs was assessed by flow cytometry 24 h after mitophagy induction. Results are shown from four independent experiments performed in duplicate.


( d ) Mitochondrial content of WT and p53 − / − MEFs was assessed by flow cytometry 24 h after mitophagy induction . WT and p53 − / − MEFs were transfected with control or p53 - specific siRNAs or cultured in complete medium for 24 h in the presence or absence of 20 μM pifithrin - α before mitophagy induction . Results are shown from four independent experiments performed in duplicate .

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(d) Mitochondrial content of WT and p53−/− MEFs was assessed by flow cytometry 24 h after mitophagy induction. WT and p53−/− MEFs were transfected with control or p53-specific siRNAs or cultured in complete medium for 24 h in the presence or absence of 20 μM pifithrin-α before mitophagy induction. Results are shown from four independent experiments performed in duplicate.


( e ) Murine HL - 1 cardiac myocytes were infected with retrovirus vectors encoding p53 , p21 or p16 and after puromycin selection , underwent mitophagy induction . Mitochondrial content is assessed by flow cytometry from four independent experiments performed in duplicate are shown .

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(e) Murine HL-1 cardiac myocytes were infected with retrovirus vectors encoding p53, p21 or p16 and after puromycin selection, underwent mitophagy induction. Mitochondrial content is assessed by flow cytometry from four independent experiments performed in duplicate are shown.


( f ) HL - 1 cells were transfected with control , p53 , p21 or p16 - specific siRNAs , followed by treatment with 0 . 02 μM DOX and mitophagy induction . Mitochondrial content is assessed by flow cytometry from four independent experiments performed in duplicate .

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(f) HL-1 cells were transfected with control, p53, p21 or p16-specific siRNAs, followed by treatment with 0.02 μM DOX and mitophagy induction. Mitochondrial content is assessed by flow cytometry from four independent experiments performed in duplicate.


( g ) CCCP - induced recruitment of Parkin and expression of Mfn1 and Drp1 in DOX - treated WT , p53 − / − and p21 − / − mice were determined by immunoblotting of the heartmitochondria - rich fraction . Representative immunoblots are shown from four independent experiments . Data are shown as the means ± s . d . * P0 . 05 ; * * P0 . 01 ( two - tailed unpaired Student ' s t - test ) .

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(g) CCCP-induced recruitment of Parkin and expression of Mfn1 and Drp1 in DOX-treated WT, p53−/− and p21−/− mice were determined by immunoblotting of the heartmitochondria-rich fraction. Representative immunoblots are shown from four independent experiments. Data are shown as the means±s.d. *P0.05; **P0.01 (two-tailed unpaired Student's t-test).


( a ) Whole - cell lysates of HL - 1 cells treated with 0 . 02 μM DOX and transfected with FLAG - tagged Parkin or the FLAG - empty vector were immunoprecipitated with the anti - Flag - M2 antibody and blotted with anti - p53 and Flag antibodies .

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(a) Whole-cell lysates of HL-1 cells treated with 0.02 μM DOX and transfected with FLAG-tagged Parkin or the FLAG-empty vector were immunoprecipitated with the anti-Flag-M2 antibody and blotted with anti-p53 and Flag antibodies.


( b ) Whole - cell lysates of rat neonatal cardiomyocytes treated with the indicated concentrations of H2O2 and DOX for 24 h were immunoprecipitated with the anti - Parkin antibody .

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(b) Whole-cell lysates of rat neonatal cardiomyocytes treated with the indicated concentrations of H2O2 and DOX for 24 h were immunoprecipitated with the anti-Parkin antibody.


( c ) Cytosolic heart lysates of adult and aged mice were immunoprecipitated with anti - Parkin , p53 and control IgG antibodies .

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(c) Cytosolic heart lysates of adult and aged mice were immunoprecipitated with anti-Parkin, p53 and control IgG antibodies.


( d ) Schematic representation of N - terminally FLAG - tagged WT Parkin and various mutants of Parkin . ( e ) The association of endogenous p53 with various Parkin mutants in DOX - treated HeLa cells . The immunoprecipitation assay revealed that the RING0 domain was essential for the interaction with p53 .

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(d) Schematic representation of N-terminally FLAG-tagged WT Parkin and various mutants of Parkin. (e) The association of endogenous p53 with various Parkin mutants in DOX-treated HeLa cells. The immunoprecipitation assay revealed that the RING0 domain was essential for the interaction with p53.


( f ) Schematic representation of the GST - p53 fusion proteins . ( g ) GST pull - down assays using GST - p53 fusion proteins and in vitro transcribed / translated FLAG - Parkin . Residues 81 - 160 of p53 were sufficient to bind Parkin .

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(f) Schematic representation of the GST-p53 fusion proteins. (g) GST pull-down assays using GST-p53 fusion proteins and in vitro transcribed/translated FLAG-Parkin. Residues 81-160 of p53 were sufficient to bind Parkin.


( a ) HL - 1 cells transfected with control or p53 - specific siRNAs no . 1 and cultured in complete medium for 24 h in the presence or absence of 0 . 02 μM DOX . After treatment with 20 μM CCCP for 6 h , the fractionation experiment was performed . Endogenous Parkin translocation to mitochondria and ubiquitination were assessed by immunoblotting . Representative immunoblots are shown from three independent experiments .

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(a) HL-1 cells transfected with control or p53-specific siRNAs no.1 and cultured in complete medium for 24 h in the presence or absence of 0.02 μM DOX. After treatment with 20 μM CCCP for 6 h, the fractionation experiment was performed. Endogenous Parkin translocation to mitochondria and ubiquitination were assessed by immunoblotting. Representative immunoblots are shown from three independent experiments.


( b ) These samples were subjected to immunoprecipitation with the anti - VDAC antibody to evaluate ubiquitination . Representative immunoblots are shown from three independent experiments .

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(b) These samples were subjected to immunoprecipitation with the anti-VDAC antibody to evaluate ubiquitination. Representative immunoblots are shown from three independent experiments.


( c , d ) Representative images of YFP - Parkin overexpressing p53 − / − HCT116 cells re - transfected with WT p53 and various mutants of p53 and treated with 60 μM CCCP for 8h ( c ) and 36 h ( d ) before the immunostaining of mitochondria with anti - TOM20 ( red ) and endogenous poly - ubiquitin with a specific antibody FK - 2 ( white ) ; original magnification , × 630 ; scale bar , 20 μm .

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(c,d) Representative images of YFP-Parkin overexpressing p53−/− HCT116 cells re-transfected with WT p53 and various mutants of p53 and treated with 60 μM CCCP for 8h (c) and 36 h (d) before the immunostaining of mitochondria with anti-TOM20 (red) and endogenous poly-ubiquitin with a specific antibody FK-2 (white); original magnification, × 630; scale bar, 20 μm.


( e - g ) Parkin mitochondrial translocation ( e ) and ubiquitination ( f ) 8 h after CCCP treatment and mitochondrial clearance ( g ) 36 h after CCCP treatment were quantified . A minimum of 300 - 400 YFP - positive cells were scored in three independent experiments . The single - letter amino - acid code is used . NES indicates nuclear export signal ; NLS , nuclear localization signal ; ER , endoplasmic reticulum ; Ub , ubiquitin . Data are shown as the means ± s . d . * P0 . 05 , compared with the corresponding control ( two - tailed unpaired Student ' s t - test ) .

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(e-g) Parkin mitochondrial translocation (e) and ubiquitination (f) 8 h after CCCP treatment and mitochondrial clearance (g) 36 h after CCCP treatment were quantified. A minimum of 300-400 YFP-positive cells were scored in three independent experiments. The single-letter amino-acid code is used. NES indicates nuclear export signal; NLS, nuclear localization signal; ER, endoplasmic reticulum; Ub, ubiquitin. Data are shown as the means±s.d. *P0.05, compared with the corresponding control (two-tailed unpaired Student's t-test).