α5 integrin ( ITGA5 ) labeling at P2 ( left image : section , right image : whole - mount ) . Scale bar : 100 μm . Blister edges ( epidermal tongue ) and HFs are indicated by arrowheads and arrows , respectively . Blisters are indicated by stars .

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α5 integrin (ITGA5) labeling at P2 (left image: section, right image: whole-mount). Scale bar: 100 μm. Blister edges (epidermal tongue) and HFs are indicated by arrowheads and arrows, respectively. Blisters are indicated by stars.


Sections of K14CreER : H2B - mCherry mice at P4 . Scale bar : 100 μm . ( Bottom ) Quantification of mCherry - positive cells ( n = 3 ) . . The data from individual mice are connected by lines . Student ' s t - test . NS , no significance . Whole - mount imaging of K14CreER : R26R - confetti samples at P4 . Scale bar : 200 μm . Sections of Lrig1CreER : H2B - mCherry mouse skin at P4 . Scale bar : 100 μm . ( Bottom ) Quantification of mCherry - positive cells ( n = 3 ) . The data from individual mice are connected by lines . * 0 . 01 test . Whole - mount imaging of Lrig1CreER : R26R - confetti mouse samples at P4 . Scale bar : 200 μm .

O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-ORGANISM B-TISSUE O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O

Sections of K14CreER:H2B-mCherry mice at P4. Scale bar: 100 μm. (Bottom) Quantification of mCherry-positive cells (n=3).. The data from individual mice are connected by lines. Student's t-test. NS, no significance. Whole-mount imaging of K14CreER:R26R-confetti samples at P4. Scale bar: 200 μm. Sections of Lrig1CreER:H2B-mCherry mouse skin at P4. Scale bar: 100 μm. (Bottom) Quantification of mCherry-positive cells (n=3). The data from individual mice are connected by lines. *0.01 test. Whole-mount imaging of Lrig1CreER:R26R-confetti mouse samples at P4. Scale bar: 200 μm.


H & E ( P2 , left ) , ITGA6 ( P2 , middle ) and LOR staining ( P4 , right ) of Col17a1 - / - mice ( top ) and littermate controls ( bottom ) . The regenerated epidermis is indicated by arrowheads . Scale bar : 200 μm .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O

H&E (P2, left), ITGA6 (P2, middle) and LOR staining (P4, right) of Col17a1-/- mice (top) and littermate controls (bottom) . The regenerated epidermis is indicated by arrowheads. Scale bar: 200 μm.


BrdU labeling of Col17a1 - / - skin at P2 . Scale bar : 100 μm . ( Bottom ) Quantification of BrdU - positive cells in the epidermis surrounding blisters ( n = 3 ( control ) and 4 ( Col17a1 - / - ) biological replicates ) . The data are shown as the mean ± SE . Student ' s t - test . NS , no significance .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-TISSUE O B-DISEASE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

BrdU labeling of Col17a1-/- skin at P2. Scale bar: 100 μm. (Bottom) Quantification of BrdU-positive cells in the epidermis surrounding blisters (n= 3 (control) and 4 (Col17a1-/-) biological replicates). The data are shown as the mean ± SE. Student's t-test. NS, no significance.


Lineage tracing of K14CreER : R26R - mCherry : Col17a1 - / - at P4 . Scale bar : 100 μm .

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Lineage tracing of K14CreER:R26R-mCherry:Col17a1-/- at P4. Scale bar: 100 μm.


K10 / K14 ( low magnification , left ) and K14 ( high magnification , middle and right ) labeling of Col7a1 - / - mouse ( bottom ) and littermate control ( top ) blistered skin at P2 . Scale bar : 30 μm . Quantification of BrdU - positive cells per μm HF length ( n = 55 HFs from three control and 143 HFs from four Col7a1 - / - mice ) . The data are shown as violin plots . Student ' s t - test . NS , no significance . Length of the major axis of keratinocytes in the regenerated epidermis ( n = 244 ( control , L ) , and 132 ( Col7a1 - / - , L ) cells from four mice , respectively ) and in the surrounding intact epidermis ( basal cells ; n = 200 ( control , NL ) , 299 ( Col7a1 - / - , NL ) , from four mice , respectively ) . NL : nonlesional area . L : lesional area . The data are shown as violin plots . * * * * p < 0 . 0001 , one - way ANOVA test , followed by Tukey ' s test . NS , no significance .

B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O B-DISEASE B-TISSUE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-TISSUE B-EXP_ASSAY O O O O B-TISSUE O O O O O B-TISSUE O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-CELL_TYPE O O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O O O B-TISSUE O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K10/K14 (low magnification, left) and K14 (high magnification, middle and right) labeling of Col7a1-/- mouse (bottom) and littermate control (top) blistered skin at P2. Scale bar: 30 μm. Quantification of BrdU-positive cells per μm HF length (n=55 HFs from three control and 143 HFs from four Col7a1-/- mice). The data are shown as violin plots. Student's t-test. NS, no significance. Length of the major axis of keratinocytes in the regenerated epidermis (n=244 (control, L), and 132 (Col7a1-/-, L) cells from four mice, respectively) and in the surrounding intact epidermis (basal cells; n=200 (control, NL), 299 (Col7a1-/-, NL), from four mice, respectively). NL: nonlesional area. L: lesional area. The data are shown as violin plots. ****p<0.0001, one-way ANOVA test, followed by Tukey's test. NS, no significance.


K10 / K14 ( left ) and K14 ( high magnification , right ) labeling of WT blistered skin treated with CaCl2 ( middle and bottom ) or PBS ( top ) at P2 . Scale bar : 30 μm . Quantification of BrdU - positive cells per μm HF length ( n = 83 ( PBS ) , 95 ( 1 . 8 mM CaCl2 ) , and 97 ( 9 . 0 mM CaCl2 ) HFs from four mice ) . One - way ANOVA test , followed by Tukey ' s test . NS , no significance . Length of the major axis of keratinocytes in the regenerated epidermis ( n = 433 ( PBS , L ) , 451 ( 1 . 8 mM CaCl2 , L ) , and 425 ( 9 . 0 mM CaCl2 , L ) cells from four mice ) and in the surrounding intact epidermis ( basal cells ; n = 311 ( PBS , NL ) , 279 ( 1 . 8 mM CaCl2 , NL ) , 302 ( 9 . 0 mM CaCl2 , NL ) cells from four mice ) . NL : nonlesional area . L : lesional area . The data are shown as violin plots . * * * * p < 0 . 0001 , one - way ANOVA test , followed by Tukey ' s test . NS , no significance .

B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-DISEASE B-TISSUE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-CELL_TYPE O O O B-TISSUE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O B-TISSUE O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K10/K14 (left) and K14 (high magnification, right) labeling of WT blistered skin treated with CaCl2 (middle and bottom) or PBS (top) at P2. Scale bar: 30 μm. Quantification of BrdU-positive cells per μm HF length (n=83 (PBS), 95 (1.8 mM CaCl2), and 97 (9.0 mM CaCl2) HFs from four mice). One-way ANOVA test, followed by Tukey's test. NS, no significance. Length of the major axis of keratinocytes in the regenerated epidermis (n=433 (PBS, L), 451 (1.8 mM CaCl2, L), and 425 (9.0 mM CaCl2, L) cells from four mice) and in the surrounding intact epidermis (basal cells; n=311 (PBS, NL), 279 (1.8 mM CaCl2, NL), 302 (9.0 mM CaCl2, NL) cells from four mice). NL: nonlesional area. L: lesional area. The data are shown as violin plots. ****p<0.0001, one-way ANOVA test, followed by Tukey's test. NS, no significance.


C Overabundance of MIAA in LAM plasma . Top panel shows each control and patient group ( number ( n ) of samples are indicated ) ; bottom panel shows aggregation of related pulmonary diseases . The asterisks indicate significant differences based on two - sided Mann - Whitney tests ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ; top panel , control - LAM P = 0 . 048 , LAM - Langerhans P = 1x10 - 4 , LAM - Sjögren P = 1x10 - 4 , LAM - lupus P = 1x10 - 3 , LAM - emphysema P = 0 . 041 ; bottom panel , LAM - Langerhans P = 7x10 - 6 and control - other P = 0 . 031 ) . Average values are indicated with lilac - colored lines . Comparison of the three groups : Kruskal - Wallis test P = 6x10 - 4 ( top panel ) and P = 3x10 - 5 ( bottom panel ) .

O O O B-SMALL_MOLECULE O B-DISEASE B-TISSUE O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-DISEASE O O O O O O B-DISEASE O B-TISSUE O O O O O O B-DISEASE O B-DISEASE O O O O O O B-DISEASE O B-DISEASE O O O O O O B-DISEASE O B-DISEASE O O O O O O O O O B-DISEASE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Overabundance of MIAA in LAM plasma. Top panel shows each control and patient group (number (n) of samples are indicated); bottom panel shows aggregation of related pulmonary diseases. The asterisks indicate significant differences based on two-sided Mann-Whitney tests (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; top panel, control-LAM P = 0.048, LAM-Langerhans P = 1x10-4, LAM-Sjögren P = 1x10-4, LAM-lupus P = 1x10-3, LAM-emphysema P = 0.041; bottom panel, LAM-Langerhans P = 7x10-6 and control-other P = 0.031). Average values are indicated with lilac-colored lines. Comparison of the three groups: Kruskal-Wallis test P = 6x10-4 (top panel) and P = 3x10-5 (bottom panel).


E Overabundance of MIAA in LAM patients not treated with rapamycin relative to rapamycin - treated ( number ( n ) of samples are indicated ) . The asterisk indicates significant difference based on two - sided Mann - Whitney test ( P = 0 . 043 ) . Average values are indicated with lilac - colored lines .

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E Overabundance of MIAA in LAM patients not treated with rapamycin relative to rapamycin-treated (number (n) of samples are indicated). The asterisk indicates significant difference based on two-sided Mann-Whitney test (P = 0.043). Average values are indicated with lilac-colored lines.


G Plasma levels of three other metabolic products derived from monoamine metabolism and showing significant differences from controls and / or between LAM and related pulmonary diseases ( individual samples indicated in panel C ) . The asterisks indicate significant differences based on two - sided Mann - Whitney tests ( * P < 0 . 05 , * * P < 0 . 01 , and * * * * P < 0 . 0001 ; 4 - HPA , control - LAM P = 0 . 024 and LAM - other P = 0 . 089 ; HVA , control - LAM P = 0 . 008 and LAM - other P = 0 . 013 ; and VMA , control - LAM P = 1x10 - 4 and LAM - other P = 0 . 10 ) . Average values are indicated with lilac - colored lines . Comparison of the three groups : Kruskal - Wallis test ; 4 - HPA P = 0 . 095 ; HVA P = 0 . 013 ; and VMA P = 0 . 005 .

O B-TISSUE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-DISEASE O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-DISEASE O O O O O O B-DISEASE O O O O O O O O B-SMALL_MOLECULE O O O B-DISEASE O O O O O O B-DISEASE O O O O O O O O O B-SMALL_MOLECULE O O O B-DISEASE O O O O O O B-DISEASE O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O

G Plasma levels of three other metabolic products derived from monoamine metabolism and showing significant differences from controls and/or between LAM and related pulmonary diseases (individual samples indicated in panel C). The asterisks indicate significant differences based on two-sided Mann-Whitney tests (*P < 0.05, **P < 0.01, and ****P < 0.0001; 4-HPA, control-LAM P = 0.024 and LAM-other P = 0.089; HVA, control-LAM P = 0.008 and LAM-other P = 0.013; and VMA, control-LAM P = 1x10-4 and LAM-other P = 0.10). Average values are indicated with lilac-colored lines. Comparison of the three groups: Kruskal-Wallis test; 4-HPA P = 0.095; HVA P = 0.013; and VMA P = 0.005.


A Overabundance of MIAA in LAM plasma ( UK cohort not treated with rapamycin ) relative to healthy women . The number ( n ) of individuals in each group is indicated ; one of 20 healthy controls did not pass the quality controls for LC - MS / MS . Asterisk indicates significance using two - sided Mann - Whitney test ( P = 0 . 018 ) . Average values are indicated with lilac - colored lines . B Overabundance of MIAA in plasma of LAM patients with AMLs . Asterisk indicates significance using two - sided Mann - Whitney test ( P = 0 . 042 ) . The number ( n ) of individuals in each group is indicated . Average values are indicated with lilac - colored lines .

O O O B-SMALL_MOLECULE O B-DISEASE B-TISSUE O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O 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-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O 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-SMALL_MOLECULE O B-TISSUE O B-DISEASE B-ORGANISM O B-DISEASE O O O O O O O O O O O O O O O O O O O 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 MIAA in LAM plasma (UK cohort not treated with rapamycin) relative to healthy women. The number (n) of individuals in each group is indicated; one of 20 healthy controls did not pass the quality controls for LC-MS/MS. Asterisk indicates significance using two-sided Mann-Whitney test (P = 0.018). Average values are indicated with lilac-colored lines. B Overabundance of MIAA in plasma of LAM patients with AMLs. Asterisk indicates significance using two-sided Mann-Whitney test (P = 0.042). The number (n) of individuals in each group is indicated. Average values are indicated with lilac-colored lines.


A Representative images of immunohistochemical positivity of MAO - A / B in LAM lung lesions ( top panels , two patients ) and lung tissue from healthy ( non - LAM ) individuals ( bottom panels ) . In total , seven LAM patients and three healthy controls were analyzed . Brown - stained cells , counter - stained with hematoxylin , are considered positive . Scale bars are shown .

O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-DISEASE B-TISSUE O O O O O O O O O B-TISSUE O O O O O O B-DISEASE O O O O O O O O O O O B-DISEASE B-ORGANISM O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O

A Representative images of immunohistochemical positivity of MAO-A/B in LAM lung lesions (top panels, two patients) and lung tissue from healthy (non-LAM) individuals (bottom panels). In total, seven LAM patients and three healthy controls were analyzed. Brown-stained cells, counter-stained with hematoxylin, are considered positive. Scale bars are shown.


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

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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 . 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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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 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

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O B-TISSUE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 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 .

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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 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 .

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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 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 ) .

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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 , 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 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O 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-GENEPROD O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of immunohistochemical detection of acrolein in LAM lung lesions .

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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 . 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 .

O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O 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 , 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

O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-TISSUE O O 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 images of immunohistochemical detection of HRH1 in LAM lung lesions .

O O O O B-EXP_ASSAY O O B-GENEPROD O B-DISEASE B-TISSUE O O

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


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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O B-TISSUE O O O O O O O O O O O O O O O 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 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 .

O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O O B-TISSUE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O B-TISSUE O O O O O O 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 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 .

O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O 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 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 .

O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O 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 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 .

O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O 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 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 .

O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O 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-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O 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 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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-SMALL_MOLECULE B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE 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 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 .

O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O B-CELL_TYPE B-EXP_ASSAY 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 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 .

O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O B-DISEASE 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 the expression of prostaglandin ( PG ) receptors in pre - adipocytes and mature adipocytes from human deep - neck BAT ( E - MTAB - 8564 ) .

O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-ORGANISM O O B-TISSUE I-TISSUE 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 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 .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-ORGANISM B-CELL_TYPE B-EXP_ASSAY O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY B-CELL_TYPE O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-TISSUE O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O B-CELL_TYPE O O B-CELL_TYPE O B-TISSUE O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O B-EXP_ASSAY O B-TISSUE O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O B-CELL_TYPE O O B-CELL_TYPE O B-TISSUE O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 analysis of m6A writers and erasers in normal and EP3 - deficient brown pre - adipocytes with or without brown adipogenesis induction .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 ) 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 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 analysis of WTAP in iBAT from EP3F / F and EP3F / FMyf5Cre mice fed HFD for 1 week .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-TISSUE O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM 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 analysis of WTAP in BAs differentiated from EP3γ - expressed brown pre - adipocytes .

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


( 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 ) .

O O O O O O O B-GENEPROD O O B-CELL_TYPE B-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY B-CELL_TYPE B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD 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 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 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-CELL_TYPE O O B-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-SMALL_MOLECULE 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 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

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-EXP_ASSAY O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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-GENEPROD O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 , 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

O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 ) 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

O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O 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 B-SUBCELLULAR O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY B-CELL_TYPE B-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O B-SMALL_MOLECULE O O B-SMALL_MOLECULE 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 assay of ERK1 / 2 , WTAP , ZFP410 , PRDM16 , and UCP1 in BAs differentiated from sulprostone - treated brown pre - adipocyte with treatment of PTX , dbcAMP , or ravoxertinib . Data information : Data are representative of two independent experiments with biological

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O B-SMALL_MOLECULE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O B-SMALL_MOLECULE O O B-SMALL_MOLECULE 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 B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY B-ORGANISM B-CELL_TYPE B-EXP_ASSAY O B-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE 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 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 ) .

O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O B-CELL_TYPE O B-CELL_TYPE B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CELL_TYPE O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE 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 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 ) .

O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-CELL_TYPE O O B-CELL_TYPE O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-EXP_ASSAY O B-CELL_TYPE O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-EXP_ASSAY O B-CELL_TYPE O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 assay of ERK1 / 2 , WTAP , ZNF410 , PRDM16 , and UCP1 in hESC - derived BAs treated with L798106 alone or combined with H89 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE 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 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 ) .

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE 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 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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-CELL_LINE O O O O O 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 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 ) .

O O O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O 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 , 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 ) .

O O O O O O B-CELL_LINE O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O B-TISSUE I-TISSUE I-TISSUE B-EXP_ASSAY O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O O O O O 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 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

O O O O O B-EXP_ASSAY O B-GENEPROD O B-DISEASE I-DISEASE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-TISSUE 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


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 ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-DISEASE O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O B-DISEASE B-TISSUE 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 analysis of PC patients with high or low CTH expression . The statistical significance was determined using the χ2 test .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-DISEASE B-ORGANISM O O O O B-GENEPROD O O O 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O B-GENEPROD 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 : 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 ) .

O O O O O O B-SMALL_MOLECULE O O O O O O B-CELL_LINE O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O 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 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 ) .

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 analysis of the CTH levels in PC3 cells transfected with HA - CTH or pCMV - HA vector control .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD 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 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 ) .

O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O B-CELL_LINE O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O B-CELL_LINE O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-EXP_ASSAY 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 ) 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 ) .

O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O O O O B-EXP_ASSAY O B-SUBCELLULAR O B-EXP_ASSAY O O B-GENEPROD O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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

O O O B-CELL_LINE O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-CELL_LINE O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-CELL_LINE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

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


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

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

O B-CELL_LINE O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O O O O B-TISSUE B-EXP_ASSAY O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O B-ORGANISM O O B-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY B-TISSUE O O O O

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


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

O B-CELL_LINE O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O O O O B-TISSUE B-EXP_ASSAY O O B-EXP_ASSAY B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O B-CELL_LINE O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O O O O B-TISSUE B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O O O B-GENEPROD O O

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


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

O B-CELL_LINE O O O O B-GENEPROD O O O O O O B-ORGANISM B-TISSUE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-ORGANISM O O O O 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 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 ) .

O B-CELL_LINE O O O O B-GENEPROD O O O O O O B-ORGANISM B-TISSUE O O O O O O B-TISSUE I-TISSUE I-TISSUE B-EXP_ASSAY O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-ORGANISM O O O O 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 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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY B-DISEASE I-DISEASE O B-GENEPROD O O O O O O O B-TISSUE B-EXP_ASSAY O O 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-ORGANISM O O O O 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 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 ) .

B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY B-DISEASE I-DISEASE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-TISSUE B-EXP_ASSAY O O 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-ORGANISM O O O O 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).


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 .

B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-GENEPROD O B-TISSUE O O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O B-TISSUE O O O B-TISSUE O O O O O O O O B-TISSUE O O O O B-TISSUE 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.