B Representative of immunofluorescence detection of MAO - A / B αSMA in LAM lung lesions , nuclei stained with DAPI ( merged ) .

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

B Representative images of immunofluorescence detection of MAO-A/B and αSMA in LAM lung lesions, nuclei stained with DAPI (merged).


C Graph showing gene expression differences ( log10 - fold changes ) of defined genes ( X - axis ) in Tsc2 - deficient relative to wild - type MEFs grown in DMEM 10 % FBS . asterisks indicate significant differences with two - sided t - ( * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; Aldh1a3 P = 0 . 006 , Aldh2 P = 2x10 - 5 , Aldh3a1 P = 0 . 004 ; Aldh3b1 P = 0 . 006 ; Aldh3b2 , P = 0 . 17 , Maoa P = 7x10 - 5 , Maob P = 7x10 - 4 ; replicates / condition n = 3 , assays n = 4 ) . bars indicate mean ± SD . Dotted horizontal lines indicate 2 - fold ( top ) 0 . 5 - fold ( bottom ) .

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

C Graph showing gene expression differences (log10-fold changes) of defined genes (X-axis) in Tsc2-deficient relative to wild-type MEFs grown in DMEM 10% FBS. The asterisks indicate significant differences with two-sided t-test (**P < 0.01, ***P < 0.001, and ****P < 0.0001; Aldh1a3 P = 0.006, Aldh2 P = 2x10-5, Aldh3a1 P = 0.004; Aldh3b1 P = 0.006; Aldh3b2, P = 0.17, Maoa P = 7x10-5, and Maob P = 7x10-4; replicates/condition n = 3, assays n = 4). The bars indicate mean ± SD. Dotted horizontal lines indicate 2-fold (top) and 0.5-fold (bottom).


D Western blot results ( independent experiments n = 4 ) from Tsc2 - deficient wild - type MEFs exposed to DMSO or treated with 20 nM everolimus for 16 hours in DMEM 10 % FBS . Loading controls are shown . inferred expression level is indicated by between corresponding signal loading control , standardized to basal

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 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

D Western blot results (independent experiments n = 4) from Tsc2-deficient and wild-type MEFs exposed to DMSO or treated with 20 nM everolimus for 16 hours in DMEM 10% FBS. Loading controls are shown. The inferred protein expression level is indicated by the ratio between the corresponding signal and loading control, standardized to the basal setting


A No differences of Vdac1 / VDAC1 expression between MEF lines ( replicates / condition n = 3 independent experiments n = 2 ) . graph shows fold change expression ( mean ± ) in Tsc2 - deficient relative to wild - type MEFs .

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

A No differences of Vdac1/VDAC1 expression between MEF cell lines (replicates/condition n = 3 and independent experiments n = 2). The graph shows fold change expression (mean ± SEM) in Tsc2-deficient relative to wild-type MEFs.


B Flow cytometry results ( independent experiments n = 3 ) showing a higher of MitoSOX red - positive in Tsc2 - deficient MEFs . C Flow cytometry results ( independent experiments n = 3 ) showing higher ( X - axis , single channel FL3 670 nm ) of MitoTracker red - positive in Tsc2 - deficient MEFs .

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 B-CONTROLLED_VAR O O O O

B Flow cytometry results (independent experiments n = 3) showing a higher percentage of MitoSOX red-positive cells in Tsc2-deficient MEFs. C Flow cytometry results (independent experiments n = 3) showing higher intensity (X-axis, single channel intensity FL3 670 nm) of MitoTracker red-positive cells in Tsc2-deficient MEFs.


D Higher basal respiration ( as measured by oxygen consumption , Y - axis ) in Tsc2 - deficient MEFs . asterisks indicate significant difference with two - sided t - ( P = 5x10 - 4 ; replicates / condition n = 5 , independent experiments n = 3 ) .

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

D Higher basal cell respiration (as measured by oxygen consumption, Y-axis) in Tsc2-deficient MEFs. The asterisks indicate significant difference with two-sided t-test (P = 5x10-4; replicates/condition n = 5, and independent experiments n = 3).


F Left panel , western blot results showing overexpression of catalase ( CAT ) in Tsc2 - deficient MEFs , unaffected by exposure to everolimus ( independent experiments n = 2 ) . CAT expression level is indicated by of corresponding signal relative to loading control basal setting ( noted as 1 ( ref ) ) . Right panel , Cat overexpression ( log10 - fold change ) in Tsc2 - deficient MEFs . asterisks indicate significant difference with two - sided t - ( P = 1x10 - 2 ; replicates / condition n = 3 , independent experiments n = 2 ) . bars indicate mean ± SD . Dotted horizontal lines indicate 2 - fold ( top ) 0 . 5 - fold ( bottom ) .

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

F Left panel, western blot results showing overexpression of catalase (CAT) in Tsc2-deficient MEFs, and unaffected by exposure to everolimus (independent experiments n = 2). The CAT expression level is indicated by the ratio of the corresponding signal relative to loading control and basal setting (noted as 1(ref)). Right panel, Cat overexpression (log10-fold change) in Tsc2-deficient MEFs. The asterisks indicate significant difference with two-sided t-test (P = 1x10-2; replicates/condition n = 3, and independent experiments n = 2). The bars indicate mean ± SD. Dotted horizontal lines indicate 2-fold (top) and 0.5-fold (bottom).


G Representative of immunohistochemical detection of acrolein in LAM lung lesions .

G Representative images of immunohistochemical detection of acrolein in LAM lung lesions.


A Overabundance of histamine in Tsc2 wild - type relative to Tsc2 - deficient MEFs , both growth in DMEM 10 % FBS . asterisks indicate significant difference with two - sided t - ( P = 0 . 017 ) ; replicates / condition n = 5 , independent experiments n = 2 ) . Average values are indicated with lilac - colored lines .

O O O B-MEASURED_VAR O B-CONTROLLED_VAR 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

A Overabundance of histamine in Tsc2 wild-type relative to Tsc2-deficient MEFs, both growth in DMEM 10% FBS. The asterisks indicate significant difference with two-sided t-test (P = 0.017); replicates/condition n = 5, and independent experiments n = 2). Average values are indicated with lilac-colored lines.


D Western blot results of HRH1 , SLC22A3 , loading control from Tsc2 - deficient wild - type MEFs exposed to DMSO or treated with 20 nM everolimus for 16 hours in DMEM 10 % or 0 . 5 % FBS ( independent experiments n = 2 ) . expression are indicated by of corresponding signal relative to loading control basal

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

D Western blot results of HRH1, SLC22A3, and loading control from Tsc2-deficient and wild-type MEFs exposed to DMSO or treated with 20 nM everolimus for 16 hours in DMEM 10% or 0.5% FBS (independent experiments n = 2). The expression levels are indicated by the ratio of the corresponding signal relative to loading control and basal setting


E Representative of immunohistochemical detection of HRH1 in LAM lung lesions .

E Representative images of immunohistochemical detection of HRH1 in LAM lung lesions.


F Evaluation of viability inhibition by loratadine alone ( concentrations shown on X - axis , from 0 to 100 μM ) or combined with everolimus ( fixed to 20 nM ) in Tsc2 - deficient wild - type MEF cultures grown in DMEM 10 % FBS for 72 hours . synergistic combination index ( CIx < 1 ) is shown ( replicates / condition n = 3 , independent experiments n = 3 ) . Each data point represents mean SD .

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 B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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 Evaluation of cell viability inhibition by loratadine alone (concentrations shown on X-axis, from 0 to 100 μM) or combined with everolimus (fixed to 20 nM) in Tsc2-deficient and wild-type MEF cultures grown in DMEM 10% FBS for 72 hours. The synergistic combination index (CIx < 1) is shown (replicates/condition n = 3, independent experiments n = 3). Each data point represents the mean and SD.


H Percentages of viability of Tsc2 - dificient wild - type MEFs exposed to DMEM 10 % or 0 . 5 % FBS with or without L - histidine for 72 hours . asterisks indicate significant differences with one - sided t - ( 10 % FBS , P = 0 . 017 ; 0 . 5 % FBS , P = 5x10 - 4 ; replicates / condition n = 4 - 6 , independent experiments n = 2 ) . bars indicate mean ± SD .

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

H Percentages of viability of Tsc2-dificient and wild-type MEFs exposed to DMEM 10% or 0.5% FBS with or without L-histidine for 72 hours. The asterisks indicate significant differences with one-sided t-test (10% FBS, P = 0.017; 0.5% FBS, P = 5x10-4; replicates/condition n = 4-6, independent experiments n = 2). The bars indicate mean ± SD.


B Inhibition of Tsc2 - deficient 105K growth with different monotherapies , as indicated in inset . Asterisks indicate significant reductions relative to vehicle ( two - way ANOVA ; clorgyline P = 0 . 015 , loratadine P = 0 . 018 , rasagiline P = 0 . 049 ) . Each data point represents mean .

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

B Inhibition of Tsc2-deficient 105K tumor growth with different monotherapies, as indicated in the inset. Asterisks indicate significant reductions relative to vehicle (two-way ANOVA; clorgyline P = 0.015, loratadine P = 0.018, and rasagiline P = 0.049). Each data point represents the mean and SEM.


C Further reduction of Tsc2 - deficient 105K growth by rapamycin combined with clorgyline or loratadine , relative to rapamycin alone . Asterisks indicate significant reductions relative to rapamycin alone ( two - way ANOVA ; clorgyline P = 0 . 035 , loratadine P = 0 . 045 ) . Each data point represents mean .

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

C Further reduction of Tsc2-deficient 105K tumor growth by rapamycin combined with clorgyline or loratadine, relative to rapamycin alone. Asterisks indicate significant reductions relative to rapamycin alone (two-way ANOVA; clorgyline P = 0.035, loratadine P = 0.045). Each data point represents the mean and SEM.


E Inhibition of Tsc2 - deficient 105K growth with Maoa expression depletion using shRNAs . Asterisks indicate significant reductions relative to control pLKO ( two - way ANOVA ; P = 1x10 - 3 ) . Each data point represents mean .

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

E Inhibition of Tsc2-deficient 105K tumor growth with Maoa expression depletion using shRNAs. Asterisks indicate significant reductions relative to control pLKO (two-way ANOVA; P = 1x10-3). Each data point represents the mean and SEM.


F Inhibition of Tsc2 - deficient 105K growth with Hrh1 expression depletion . Asterisk indicates significant reduction relative to control pLKO ( two - way ANOVA ; P = 0 . 035 ) . Each data point represents mean . G Inhibition of Tsc2 - deficient 105K growth with administration of α - methyl - DL - histidine . Asterisk indicates significant reduction relative to vehicle ( two - way ANOVA ; P = 0 . 028 ) . Each data point represents mean .

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

F Inhibition of Tsc2-deficient 105K tumor growth with Hrh1 expression depletion. Asterisk indicates significant reduction relative to control pLKO (two-way ANOVA; P = 0.035) . Each data point represents the mean and SEM. G Inhibition of Tsc2-deficient 105K tumor growth with administration of α-methyl-DL-histidine. Asterisk indicates significant reduction relative to vehicle (two-way ANOVA; P = 0.028). Each data point represents the mean and SEM.


A Top panels , western blot results of HRH1 phospho - Ser616 DRP1 expression in vehicle control single - drug treated tumors . Bottom panels show quantifications ; significant differences correspond to one - sided t - ( * P = 0 . 011 * * P = 2x10 - 3 ; tumors / group n = 4 ) . Average values are indicated with lilac - colored lines . B Western blot results showing increased HRH1 expression in combinations relative to rapamycin alone , reduced expression of S6 total in rapamycin - loratadine combination , raised phospho - Ser616 DRP1 expression in this combination . Bottom panels show quantifications significant differences with one - sided t - ( HRH1 , clorgyline P = 0 . 011 , loratadine P = 0 . 049 , rasagiline P = 0 . 021 ; total S6 , loratadine P = 4x10 - 4 ; phospho - Ser616 DRP1 , loratadine P = 0 . 037 ; tumors / group n = 4 ) . Average values are indicated with lilac - colored lines .

O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_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

A Top panels, western blot results of HRH1 and phospho-Ser616 DRP1 expression in vehicle control and single-drug treated tumors. Bottom panels show quantifications; significant differences correspond to one-sided t-test (*P = 0.011 and **P = 2x10-3; tumors/group n = 4). Average values are indicated with lilac-colored lines. B Western blot results showing increased HRH1 expression in combinations relative to rapamycin alone, reduced expression of S6 total in the rapamycin-loratadine combination, and raised phospho-Ser616 DRP1 expression in this combination. Bottom panels show quantifications and significant differences with one-sided t-test (HRH1, clorgyline P = 0.011, loratadine P = 0.049, rasagiline P = 0.021; total S6, loratadine P = 4x10-4; phospho-Ser616 DRP1, loratadine P = 0.037; tumors/group n = 4). Average values are indicated with lilac-colored lines.


E Representative of hematoxylin - eosin - stained Tsc2 - deficient 105K tumors treated with vehicle or monotherapies . Tumors treated with rapamycin tend to have a fascicular growth pattern with bundles of spindle foci of fibrosis , whereas most of tumors of other treatment groups showed extensive epithelioid . Scale bars are shown .

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

E Representative images of hematoxylin-eosin-stained Tsc2-deficient 105K tumors treated with vehicle or monotherapies. Tumors treated with rapamycin tend to have a fascicular growth pattern with bundles of spindle cells and foci of fibrosis, whereas most of the tumors of the other treatment groups showed extensive epithelioid morphology. Scale bars are shown.


F Representative of hematoxylin - eosin - stained Tsc2 - deficient 105K tumors treated with rapamycin alone or rapamycin combinations . With addition of a second drug to rapamycin , tumors more frequently tended to show glandular differentiation less atypia . Scale bars are shown .

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

F Representative images of hematoxylin-eosin-stained Tsc2-deficient 105K tumors treated with rapamycin alone or rapamycin combinations. With the addition of a second drug to rapamycin, tumors more frequently tended to show glandular differentiation and less atypia. Scale bars are shown.


( B ) Violin plots showing expression of prostaglandin ( PG ) receptors in pre - adipocytes mature adipocytes from human deep - neck BAT ( E - MTAB - 8564 ) .

O O O O O O O O O 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) Violin plots showing the expression of prostaglandin (PG) receptors in pre-adipocytes and mature adipocytes from human deep-neck BAT(E-MTAB-8564).


( A ) Oil Red O of mouse BAs differentiated from normal or EP3 - deficient brown pre - adipocytes . Scale bar : 50 μm . ( B ) Mean lipid droplet of differentiated BAs from normal or EP3 - deficient brown pre - adipocytes . * P < 0 . 05 vs WT ( n = 6 ) . Data information : Data represent mean ± standard error of mean ( ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by Mann - Whitney U ( B , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ns , not significant ( P > 0 .

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 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

(A) Oil Red O staining of mouse BAs differentiated from normal or EP3-deficient brown pre-adipocytes. Scale bar: 50 μm. (B) Mean lipid droplet area of differentiated BAs from normal or EP3-deficient brown pre-adipocytes. *P < 0.05 vs WT (n = 6). 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 Mann-Whitney U tests (B, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05


( D ) Representative image of hematoxylin eosin ( H & E ) for iBAT from EP3F / F EP3F / FMyf5Cre mice . Scale bar : 50 μm . ( E ) Mean adipocyte of BAs in iBAT from EP3F / F EP3F / FMyf5Cre mice ( n = 8 ) . Data information : Data represent mean ± standard error of mean ( ) . Data are pooled from two independent experiments with biological replicates Statistical significance was evaluated by Mann - Whitney U E , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ns , not significant ( P > 0 . 05 ) .

O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative image of hematoxylin and eosin (H&E) staining for iBAT from EP3F/F and EP3F/FMyf5Cre mice. Scale bar: 50 μm. (E) Mean adipocyte area of BAs in iBAT from EP3F/F and EP3F/FMyf5Cre mice (n = 8). Data information: Data represent the mean ± standard error of mean (SEM). Data are pooled from two independent experiments with biological replicates Statistical significance was evaluated by Mann-Whitney U tests E, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( K ) Representative image of H & E of iBAT from EP3F / F EP3F / FMyf5Cre mice fed HFD . Scale bar : 50 μm . ( L ) Mean adipocyte of BAs in iBAT from EP3F / F EP3F / FMyf5Cre mice fed HFD ( n = 8 ) . Data information : Data represent mean ± standard error of mean ( ) . Data are pooled from two independent experiments with biological replicates L , Statistical significance was evaluated by Mann - Whitney U L , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ns , not significant ( P > 0 . 05 ) .

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

(K) Representative image of H&E staining of iBAT from EP3F/F and EP3F/FMyf5Cre mice fed HFD. Scale bar: 50 μm. (L) Mean adipocyte area of BAs in iBAT from EP3F/F and EP3F/FMyf5Cre mice fed HFD (n = 8). Data information: Data represent the mean ± standard error of mean (SEM). Data are pooled from two independent experiments with biological replicates L, Statistical significance was evaluated by Mann-Whitney U tests L, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( A ) N6 - methyladenosine ( m6A ) modification of mRNAs in normal EP3 - deficient brown pre - adipocytes with or without brown adipogenesis induction , detected by m6A Methylation Kit ( Colorimetric ) ( n = 4 - 6 ) . Data information : Data represent mean ± standard error of mean ( ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two - student ' s t ( A * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ns , not significant ( P > 0 . 05 ) .

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

(A) N6-methyladenosine (m6A) modification levels of mRNAs in normal and EP3-deficient brown pre-adipocytes with or without brown adipogenesis induction, detected by m6A RNA Methylation Quantification Kit (Colorimetric) (n = 4-6). 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 unpaired two-tailed student's t test (A *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( B ) qRT - PCR of relative mRNA of m6A writers ( METTL3 , METTL4 , METTL14 WTAP ) erasers ( FTO ALKBH5 ) in normal EP3 - deficient brown pre - adipocytes with or without brown adipogenesis induction ( n = 6 - 9 ) . Data information : Data represent mean ± standard error of mean ( ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two - student ' s t B , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ns , not significant ( P > 0 . 05 ) .

O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_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 O O O O O O 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) qRT-PCR analysis of the relative mRNA levels of m6A writers (METTL3, METTL4, METTL14 and WTAP) and erasers (FTO and ALKBH5) in normal and EP3-deficient brown pre-adipocytes with or without brown adipogenesis induction (n = 6-9). 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 unpaired two-tailed student's t test B, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( C ) Western blotting of m6A writers erasers in normal EP3 - deficient brown pre - adipocytes with or without brown adipogenesis induction .

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

(C) Western blotting analysis of m6A writers and erasers in normal and EP3-deficient brown pre-adipocytes with or without brown adipogenesis induction.


( D ) of in C ( n = 4 - 6 ) . Data information : Data represent mean ± standard error of mean ( ) . Data are representative of two independent experiments with biological replicates , D , Statistical significance was evaluated by unpaired two - student ' s t D , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ns , not significant ( 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

(D) Quantification of protein levels in C (n = 4-6). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates , D, Statistical significance was evaluated by unpaired two-tailed student's t test D, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( E ) Western blotting of WTAP in iBAT from EP3F / F EP3F / FMyf5Cre mice fed HFD for 1 week .

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

(E) Western blotting analysis of WTAP in iBAT from EP3F/F and EP3F/FMyf5Cre mice fed HFD for 1 week.


( F ) Western blotting of WTAP in BAs differentiated from EP3γ - expressed brown pre - adipocytes .

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

(F) Western blotting analysis of WTAP in BAs differentiated from EP3γ-expressed brown pre-adipocytes.


( G ) Relative mRNA of WTAP gene in BAs differentiated from EP3γ - expressed brown pre - adipocytes detected by RT - qPCR ( n = 6 - 7 ) . Data information : Data represent mean ± standard error of mean ( ) . Data are representative of two independent experiments with biological replicates G , Statistical significance was evaluated by two - way ANOVA followed by Tukey ' s for post - hoc comparisons ( G , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ns , not significant ( P > 0 . 05 ) .

O O O O O O B-MEASURED_VAR 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 O O

(G) Relative mRNA levels of WTAP gene in BAs differentiated from EP3γ-expressed brown pre-adipocytes detected by RT-qPCR (n = 6-7). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates G, Statistical significance was evaluated by two-way ANOVA followed by Tukey's test for post-hoc comparisons (G, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


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

O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR 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 O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 the relative mRNA levels of EP3γ gene in BAs differentiated from EP3γ-expressed EP3-/- brown pre-adipocytes after transfection with WTAP siRNA (n = 8). (I) qRT-PCR analysis of the relative mRNA levels of WTAP gene in BAs differentiated from EP3γ-expressed EP3-/- brown pre-adipocytes after transfection with WTAP siRNA (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 unpaired two-tailed student's t test H), two-way ANOVA followed by Tukey's test for post-hoc comparisons I, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( 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 .

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 O O O O O O O

(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 of relative mRNA 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 mean ± standard error of mean ( ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two - way ANOVA followed by Tukey ' s for post - hoc comparisons K - Q ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ns , not significant ( 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 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

(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 of ubiquitination of WTAP in control EP3 - deficient brown pre - adipocytes with or without sulprostone treatment . Data information : Data are representative of two independent experiments with biological

O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR 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) 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 of ERK1 / 2 phosphorylation in control EP3 - deficient brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological

O O O O O O B-MEASURED_VAR 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

(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

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

(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 , melittin on ubiquitin - dependent degradation of WTAP in sulprostone - treated brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological

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

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

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

(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

O O O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O B-MEASURED_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

(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 B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR 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

(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

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

(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 of ERK1 / 2 , WTAP , ZFP410 , PRDM16 , 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 B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR 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 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 .

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


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

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

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

(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 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 mean ± standard error of mean ( ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two - way ANOVA followed by Tukey ' s for multiple comparisons ( E , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ns , not significant ( P > 0 . 05 ) .

O O O O O B-CONTROLLED_VAR 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

(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 mean ± standard error of mean ( ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by , two - way ANOVA followed by Tukey ' s for multiple comparisons F , G ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ns , not significant ( P > 0 . 05 ) .

O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 of ERK1 / 2 , WTAP , ZNF410 , PRDM16 , UCP1 in hESC - derived BAs treated with L798106 alone or combined with H89 .

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

(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 invasion abilities between T - B - lines . PC3 T - B - were incubated for migration ( 16h ) invasion ( 24h ) . Data shown represent means ± SD ( n = 3 biological replicates ) . Student ' s t - was used for statistical ( * P < 0 . 05 ; * * 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

(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 proliferation rate between T - B - lines . proliferation was performed by MTS reagents . Data are 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

(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 of endogenous CTH in PC3 orthotopically implanted - derived ( T2 T3 - lines ) bone - metastases - derived cancer ( B2 B3 - lines ) .

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

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

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

(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 , B3 were orthotopically injected into mouse prostate for 15 - 20 days . Incidences of paraaortic lymph nodes metastasis ( E ) bone metastasis ( F ) are shown ( n = 6 - 8 mice per group ) . Student ' s t - was used for statistical ( * P < 0 . 05 ; * * 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

(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 of commercial tissue arrays . Samples were sub - grouped by TNM stages . representative from different stages are shown . S : Stroma ; Scale bars : 250μm . Bottom : statistical significance was determined using χ2

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

(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


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

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

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 of PC with high or low CTH expression . statistical significance was determined using χ2 .

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

(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 of CTH level in PC3 - T2 with CTH overexpression by pCMV - CTH - HA or PC3 - B2 with CTH knockdown by siCTH - 1 .

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

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

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

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

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

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

(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 of CTH in PC3 transfected with HA - CTH or pCMV - HA vector control .

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

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


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

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

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

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 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

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

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

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

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

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

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 . representative are shown . Scale bars : 25μm .

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

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

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

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

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

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

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

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 were incubated for migration ( 16h ) invasion ( 24h ) . DMEM / 10 % FBS , together with various of NaHS ( 10nM - 1mM ) acting as a chemoattractant Data information Data shown represent means ± SD ( n = 3 biological replicates ) ANOVA followed by Tukey ' s post - hoc tes was used for statistical ( * 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 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

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

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 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

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

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

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

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

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

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

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

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

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

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

O O O O O B-CONTROLLED_VAR 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

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

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

O O O B-CONTROLLED_VAR 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

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


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

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

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


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

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

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

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

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

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

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

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

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

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

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).


for apical marker CD13 on liver tissue sections from untreated mice or 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 CV ( orange ) PV ( blue ) areas are shown as magnifications on left right , respectively .

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

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.


of BC diameter within 18 zones along CV - PV axis ( zone 1 , peri - central ; zone 18 , peri - portal ) in livers from untreated or mice at indicated timepoints post PH . diameter was measured from 3D BC network reconstructions of IF image stacks of CD13 as shown in ( a ) . zones directly adjacent to CV PV were excluded from ( ~ 1 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 .

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

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

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


of BC perimeter ( d ) total BC ( e ) from EM as representatively shown in ( c ) . Box - whisker plot with median , 25 - 75 quartiles 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 of untreated vs . sham condition , p = 0 . 86 ( n . s . ) ; Untreated vs . PH condition , p = 0 . 03 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 CV ( left , orange ) PV ( right , blue ) region . in ( a ) ( f ) are background - subtracted . Scale bars , 50 µm ( a ) , 1 µm ( c ) , 5 µm ( f ) .

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

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).


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

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

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.


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

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

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.


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

O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_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 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.


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

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

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.


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

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

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 of mean YAP from as representatively shown in ( a ) Appendix Fig . S4A , at indicated time points post PH ( solid ) or sham OP ( dashed ) in 10 zones within CV - PV axis ( zone 1 , CV ; zone 10 , PV ) . Data is normalized to untreated ( not shown ) . Mean ± s . e . m , n = 3 - 5 mice per timepoint . YAP 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


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

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

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

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

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 apical ( a ' ) basolateral ( a ' ' ) of hepatocyte are shown as magnification . Arrows indicate gold particles .

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

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

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

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.


stainings for YAP ( green ) , F - actin ( magenta ) with marker DAPI ( blue ) of 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 .

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

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.


of mean YAP from of hepatocytes treated with DMSO ( control ) , Y27 , DCA or DCA + Y27 for ~ 18 h as representatively shown in ( A ) . Data is normalized to untreated ( 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 B-MEASURED_VAR 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 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 GAPDH ( loading control ) of hepatocyte culture lysates . were untreated or incubated for 18 h with indicated

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

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


stainings of hepatocytes for YAP ( green ) , F - actin ( magenta ) with marker DAPI ( blue ) . 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 dilation of canaliculi ( asterisks ) fragmentation of F - actin ( arrows ) upon CytoD treatment .

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


of mean YAP from of 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 other conditions . Inhibitors affecting similar actin processes are displayed in same grey level . Saline , Fasudil , CK666 LatA conditions are not shown in ( d ) . Data is normalized to untreated ( 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 B-MEASURED_VAR 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 B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 GAPDH ( loading control ) in untreated or actin inhibitor - treated hepatocyte culture lysates . Inhibitor treatments are same as in ( E ) . Scale bars , 20 µm ( A , D ) .

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

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).


stainings for apical marker CD13 on liver tissue sections from mice at 2 d post sham OP or PH , treated with saline ( control ) or Rho kinase inhibitor Fasudil for 1 h . Shown are maximum projections of 50 µm stacks in PV . of BC diameter within 18 zones along 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 ) or Fasudil ( dashed ) . Diameter was measured from 3D BC network reconstructions of image stacks as representatively shown in ( a ) . zones directly adjacent to CV PV were excluded from ( ~ 1 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 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 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 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 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.


stainings for YAP , PCNA with marker DAPI on liver tissue sections from untreated mice or 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 . of mean YAP within 10 zones along CV - PV axis , ( zone 1 , peri - central ; zone 10 , peri - portal ) from IF as representatively shown in ( c ) . Data was normalized to untreated . Mean ± s . e . m , n = 7 - 8 mice per condition from 3 independent experiments . YAP of sham operated mice treated with saline vs . Fasudil , p = 0 . 13 ( n . s . ) ; YAP of PH mice treated with saline vs . Fasudil , p = 0 . 0004 .

O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR 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 were infected with SARS - CoV - 2 as per Figure 1A . were washed , fixed stained with DAPI ( blue ) to visualise nuclei incubated with anti - N ( red ) to visualise SARS - CoV - 2 infected . Scale bar denotes 10 µM .

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

b) 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 methods ) calculated from six fields of view per time point per biological replicate . Data point indicates sample means error bars standard error of mean ( ) ( n = 3 ) . d ) SARS - CoV - 2 detected plotted as a function of time where each data point denotes a different replicate ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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).