p62immunohistochemistry ( IHC ) staining in humanbrain tissue . G - I Hematoxylin and eosin ( H & amp ; amp ; E ) staining ( G ) and p62immunohistochemistry analysis ( H , I ) of a CCM surgical sample ( case n ° 6 in Appendix Table S1 ) containing normal vessels in the peri - lesional area , which served as an internal control . Notice marked p62 - positive staining in endothelial cells lining a CCM lesion ( H , arrows ) and p62 - negative staining in endothelial cells lining a normal peri - lesional vessel ( I , arrows ) . Scale bars : ( G ) 300 μm ; ( H , I ) 100 μm . Background staining in brainparenchyma surrounding CCM lesions may be attributed to either cell debris or p62 immunoreactivity in neuronal cells and glial cells .

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p62immunohistochemistry (IHC) staining in humanbrain tissue.G-I Hematoxylin and eosin (H&amp;E) staining (G) and p62immunohistochemistry analysis (H, I) of a CCM surgical sample (case n° 6 in Appendix Table S1) containing normal vessels in the peri-lesional area, which served as an internal control. Notice marked p62-positive staining in endothelial cells lining a CCM lesion (H, arrows) and p62-negative staining in endothelial cells lining a normal peri-lesional vessel (I, arrows). Scale bars: (G) 300 μm; (H, I) 100 μm. Background staining in brainparenchyma surrounding CCM lesions may be attributed to either cell debris or p62 immunoreactivity in neuronal cells and glial cells.


Depletion of RBM14 induces ectopic formation of centriolar protein complexesA - E Mitotic control U2OS cells or U2OS cells treated with RBM14 siRNA were stained with antibodies against centrin - 2 ( green ) and C - Nap1 ( magenta ) ( A ) , Centrobin ( green ) and C - Nap1 ( magenta ) ( B ) , centrin - 2 ( green ) and CPAP ( magenta ) ( C ) , acetylated - tubulin ( green ) and CP110 ( magenta ) ( D ) , and centrin - 2 ( green ) and γ - tubulin ( magenta ) ( E ) . DNA is shown in blue . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm . Histograms on the right represent frequency of mitotic cells with excess foci of the indicated proteins at spindle poles in each condition . Values are mean percentages ± standard error of mean ( SEM ) from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 , * * P < 0 . 01 , n . s . , not significant ( one - tailed t - test ) . Note that we counted the number of C - Nap1 foci after anaphase in mitosis when C - Nap1 signals recover from the reduction in prometaphase .

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Depletion of RBM14 induces ectopic formation of centriolar protein complexesA-E Mitotic control U2OS cells or U2OS cells treated with RBM14 siRNA were stained with antibodies against centrin-2 (green) and C-Nap1 (magenta) (A), Centrobin (green) and C-Nap1 (magenta) (B), centrin-2 (green) and CPAP (magenta) (C), acetylated-tubulin (green) and CP110 (magenta) (D), and centrin-2 (green) and γ-tubulin (magenta) (E). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms on the right represent frequency of mitotic cells with excess foci of the indicated proteins at spindle poles in each condition. Values are mean percentages ± standard error of mean (SEM) from three independent experiments (n = 30 for each condition). *P < 0.05, **P < 0.01, n.s., not significant (one-tailed t-test). Note that we counted the number of C-Nap1 foci after anaphase in mitosis when C-Nap1 signals recover from the reduction in prometaphase.


A U2OS cells or U2OS cells expressing FLAG - RBM14 full length ( FL , aa1 - 669 ) , N - terminal fragment ( RBM14 [ N ] , aa1 - 150 ) or C - terminal fragment ( RBM14 [ C ] , aa151 - 669 ) and treated with control siRNA or siRNA against 3 ′ UTR targeting endogenous RBM14 were stained with antibodies against FLAG as well as centrin - 2 . Histograms represent frequency of mitotic cells with excess centrin foci at spindle poles in each condition . Values are mean percentages ± SEM from three independent samples ( n = 30 for each condition ) . * * P < 0 . 01 , n . s . , not significant ( one - tailed t - test ) .

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A U2OS cells or U2OS cells expressing FLAG-RBM14 full length (FL, aa1-669), N-terminal fragment (RBM14[N], aa1-150) or C-terminal fragment (RBM14[C], aa151-669) and treated with control siRNA or siRNA against 3′ UTR targeting endogenous RBM14 were stained with antibodies against FLAG as well as centrin-2. Histograms represent frequency of mitotic cells with excess centrin foci at spindle poles in each condition. Values are mean percentages ± SEM from three independent samples (n = 30 for each condition). **P < 0.01, n.s., not significant (one-tailed t-test).


C , D Cytoplasmic RBM14 could suppress centriole amplification in HU - treated cells . ( C ) U2OS cells or U2OS cells expressing FLAG - RBM14 FL or [ C ] and treated with or without HU were stained with antibodies against centrin - 2 ( green ) and FLAG ( magenta ) . ( D ) U2OS cells or U2OS cells expressing FLAG - RBM14 FL , FLAG - RBM14 - NES or GFP - RBM14 - PACT and treated with or without HU were stained with antibodies against FLAG ( magenta , left ) or GFP ( magenta , right ) as well as centrin - 2 ( green ) . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 10 μm . Histograms represent frequency of cells in interphase with excess centrin foci in each condition . The percentages of U2OS cells with centrosomal localization of the RBM14 full - length protein or mutants are shown below the immunofluorescence images . We counted only cells that had adequate intensity of FLAG or GFP signals and did not find any significant difference in the total expression levels of the exogenous RBM14 proteins . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * * P < 0 . 01 ( one - tailed t - test ) . Please note that cytoplasmic expression levels of GFP - RBM14 - PACT are less than those of FLAG - RBM14 FL ( ˜ 0 . 5 - fold ) or FLAG - RBM14 - NES ( ˜ 0 . 7 - fold ) .

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C, D Cytoplasmic RBM14 could suppress centriole amplification in HU-treated cells. (C) U2OS cells or U2OS cells expressing FLAG-RBM14 FL or [C] and treated with or without HU were stained with antibodies against centrin-2 (green) and FLAG (magenta). (D) U2OS cells or U2OS cells expressing FLAG-RBM14 FL, FLAG-RBM14-NES or GFP-RBM14-PACT and treated with or without HU were stained with antibodies against FLAG (magenta, left) or GFP (magenta, right) as well as centrin-2 (green). Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 10 μm. Histograms represent frequency of cells in interphase with excess centrin foci in each condition. The percentages of U2OS cells with centrosomal localization of the RBM14 full-length protein or mutants are shown below the immunofluorescence images. We counted only cells that had adequate intensity of FLAG or GFP signals and did not find any significant difference in the total expression levels of the exogenous RBM14 proteins. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01 (one-tailed t-test). Please note that cytoplasmic expression levels of GFP-RBM14-PACT are less than those of FLAG-RBM14 FL (˜0.5-fold) or FLAG-RBM14-NES (˜0.7-fold).


A HeLa cells immunoprecipitated with control IgG or STIL antibodies . Soluble cytosolic fractions ( input ) and immunoprecipitates ( IPs ) were analyzed by Western blotting using RBM14 , STIL or CPAP antibodies .

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A HeLa cells immunoprecipitated with control IgG or STIL antibodies. Soluble cytosolic fractions (input) and immunoprecipitates (IPs) were analyzed by Western blotting using RBM14, STIL or CPAP antibodies.


B GST pull - down assay testing interactions between purified STIL [ N ] ( ˜ 5 μg , aa1 - 1018 ) and GST - RBM14 [ N ] or [ C ] . The asterisks indicate non - specific bands .

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B GST pull-down assay testing interactions between purified STIL[N] (˜5 μg, aa1-1018) and GST-RBM14 [N] or [C]. The asterisks indicate non-specific bands.


D Mitotic U2OS cells treated with control or RBM14 siRNA were stained with antibodies against centrin - 2 ( green ) and STIL ( magenta ) . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm . Histograms represent frequency of mitotic cells with excess STIL foci co - localized with centrin foci . Values are mean percentages ± SEM from three independent samples ( n = 30 for each condition ) . * * P < 0 . 01 ( one - tailed t - test ) .

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D Mitotic U2OS cells treated with control or RBM14 siRNA were stained with antibodies against centrin-2 (green) and STIL (magenta). Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent frequency of mitotic cells with excess STIL foci co-localized with centrin foci. Values are mean percentages ± SEM from three independent samples (n = 30 for each condition). **P < 0.01 (one-tailed t-test).


E In vitro competitive binding assay . GST pull - down experiment was performed as in ( B ) , with purified STIL [ N ] and GST - RBM14 [ C ] in the presence of the indicated amount of purified His - CPAP [ SBD ] , His - tagged STIL - Binding Domain of CPAP . The fraction of STIL [ N ] bound to GST - RBM14 [ C ] in such conditions was monitored by Western blotting using STIL antibodies which recognize the N - terminal region of STIL . Input materials were analyzed by Western blotting using the STIL or CPAP antibodies . The precipitated GST - RBM14 [ C ] was analyzed by SDS - PAGE , stained with SimplyBlue ™ Safe ( Invitrogen ) .

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E In vitro competitive binding assay. GST pull-down experiment was performed as in (B), with purified STIL[N] and GST-RBM14[C] in the presence of the indicated amount of purified His-CPAP[SBD], His-tagged STIL-Binding Domain of CPAP. The fraction of STIL[N] bound to GST-RBM14[C] in such conditions was monitored by Western blotting using STIL antibodies which recognize the N-terminal region of STIL. Input materials were analyzed by Western blotting using the STIL or CPAP antibodies. The precipitated GST-RBM14[C] was analyzed by SDS-PAGE, stained with SimplyBlue™ Safe (Invitrogen).


F Interaction between STIL and GFP - CPAP in the presence of FLAG - RBM14 FL or fragments . U2OS cells expressing GFP - CPAP were transfected with empty vector as a control , FLAG - RBM14 [ N ] , FLAG - RBM14 [ C ] or FLAG - RBM14 FL constructs , immunoprecipitated with STIL antibodies , and the resulting IPs were analyzed by Western blotting using STIL , CPAP or FLAG antibodies . Quantification of relative protein amounts of co - immunoprecipitated GFP - CPAP in each STIL - IP fraction , normalized with the amount of STIL precipitated , is shown below the panels . Means ± SEM were calculated from three independent experiments . * * P < 0 . 01 ( one - tailed t - test ) . Note that we did not find any significant effect of exogenous expression of RBM14 full - length protein or deletion mutants on the expression levels of GFP - CPAP or endogenous STIL proteins in this experiment .

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F Interaction between STIL and GFP-CPAP in the presence of FLAG-RBM14 FL or fragments. U2OS cells expressing GFP-CPAP were transfected with empty vector as a control, FLAG-RBM14[N], FLAG-RBM14[C] or FLAG-RBM14 FL constructs, immunoprecipitated with STIL antibodies, and the resulting IPs were analyzed by Western blotting using STIL, CPAP or FLAG antibodies. Quantification of relative protein amounts of co-immunoprecipitated GFP-CPAP in each STIL-IP fraction, normalized with the amount of STIL precipitated, is shown below the panels. Means ± SEM were calculated from three independent experiments. **P < 0.01 (one-tailed t-test). Note that we did not find any significant effect of exogenous expression of RBM14 full-length protein or deletion mutants on the expression levels of GFP-CPAP or endogenous STIL proteins in this experiment.


G U2OS cells treated with the indicated siRNAs were stained with antibodies against centrin - 2 ( green ) and C - Nap1 ( magenta ) . Histograms represent frequency of mitotic cells with excess centrin foci at spindle poles in each condition . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * * P < 0 . 01 ( one - tailed t - test ) . Representative mitotic cells treated with siRNAs against RBM14 alone , RBM14 + STIL or RBM14 + CPAP are shown below . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm .

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G U2OS cells treated with the indicated siRNAs were stained with antibodies against centrin-2 (green) and C-Nap1 (magenta). Histograms represent frequency of mitotic cells with excess centrin foci at spindle poles in each condition. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01 (one-tailed t-test). Representative mitotic cells treated with siRNAs against RBM14 alone, RBM14 + STIL or RBM14 + CPAP are shown below. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm.


A U2OS cells treated with control siRNA , RBM14 siRNA alone or RBM14 + HsSAS - 6 siRNAs were stained with antibodies against centrin - 2 ( green ) and HsSAS - 6 ( magenta ) . DNA is shown in blue . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm . Histograms represent frequency of mitotic cells with excess centrin foci in the indicated conditions . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . n . s . , not significant ( one - tailed t - test ) .

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A U2OS cells treated with control siRNA, RBM14 siRNA alone or RBM14 + HsSAS-6 siRNAs were stained with antibodies against centrin-2 (green) and HsSAS-6 (magenta). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent frequency of mitotic cells with excess centrin foci in the indicated conditions. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). n.s., not significant (one-tailed t-test).


B Live imaging of cycling RBM14 - depleted HeLa cells expressing GFP - centrin1 ( green ) and RFP - H2B ( magenta ) . Insets show approximately 1 . 5 - fold magnified images of fluorescent foci . Time is denoted in hh : min , and scale bar is 10 μm . Time zero corresponds to the onset of excess centrin foci formation . Quantification of the number and total signal intensity of centrin foci in HeLa cells treated with control or RBM14 siRNA over time . Means ± SEM are shown ( n = 5 ) .

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B Live imaging of cycling RBM14-depleted HeLa cells expressing GFP-centrin1 (green) and RFP-H2B (magenta). Insets show approximately 1.5-fold magnified images of fluorescent foci. Time is denoted in hh:min, and scale bar is 10 μm. Time zero corresponds to the onset of excess centrin foci formation. Quantification of the number and total signal intensity of centrin foci in HeLa cells treated with control or RBM14 siRNA over time. Means ± SEM are shown (n = 5).


C Ectopic formation of centriolar intermediates likely occurs from late G1 to S phase . Time - lapse recording across the cell cycle of HeLa cells expressing GFP - centrin ( green ) and RFP - H2B ( magenta ) and treated with RBM14 siRNA . Time is denoted in hh : min , and scale bar is 10 μm . Time zero corresponds to the metaphase onset .

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C Ectopic formation of centriolar intermediates likely occurs from late G1 to S phase. Time-lapse recording across the cell cycle of HeLa cells expressing GFP-centrin (green) and RFP-H2B (magenta) and treated with RBM14 siRNA. Time is denoted in hh:min, and scale bar is 10 μm. Time zero corresponds to the metaphase onset.


D Ectopic formation of centriolar protein complexes occurs during S phase arrest induced by HU treatment but not G1 phase arrest induced by lovastatin treatment . Insets show threefold magnified images of fluorescent foci . Scale bar , 5 μm . Histograms represent frequency of mitotic cells with excess centrin foci in the indicated conditions . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * * P < 0 . 01 , n . s . , not significant ( one - tailed t - test ) .

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D Ectopic formation of centriolar protein complexes occurs during S phase arrest induced by HU treatment but not G1 phase arrest induced by lovastatin treatment. Insets show threefold magnified images of fluorescent foci. Scale bar, 5 μm. Histograms represent frequency of mitotic cells with excess centrin foci in the indicated conditions. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01, n.s., not significant (one-tailed t-test).


E Amorphous electron - dense structures containing microtubules are formed in RBM14 - depleted cells ( see also Supplementary Fig S5I ) . Electron micrographs from U2OS cells depleted of RBM14 . Squares indicate the ectopic electron - dense structures , whereas circles indicate the pre - existing centrioles . Scale bar , 500 nm and 200 nm ( magnified views ) . Average diameter of pre - existing centrioles and the amorphous electron - dense structures are 225 ± 8 nm ( n = 10 ) and 130 + 11 nm ( n = 16 ) , respectively . Insets show magnified views of the squares . Note that microtubules ( black arrowheads ) are observed within the electron - dense structures ( white arrowheads ) .

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E Amorphous electron-dense structures containing microtubules are formed in RBM14-depleted cells (see also Supplementary Fig S5I). Electron micrographs from U2OS cells depleted of RBM14. Squares indicate the ectopic electron-dense structures, whereas circles indicate the pre-existing centrioles. Scale bar, 500 nm and 200 nm (magnified views). Average diameter of pre-existing centrioles and the amorphous electron-dense structures are 225 ± 8 nm (n = 10) and 130 + 11 nm (n = 16), respectively. Insets show magnified views of the squares. Note that microtubules (black arrowheads) are observed within the electron-dense structures (white arrowheads).


F Histograms represent frequency of the cells with ectopic foci of GFP - CPAP in the indicated conditions . U2OS cells expressing GFP - CPAP were fixed and stained 24 h after RNAi treatment and induction of GFP - CPAP . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * * P < 0 . 01 ( one - tailed t - test ) .

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F Histograms represent frequency of the cells with ectopic foci of GFP-CPAP in the indicated conditions. U2OS cells expressing GFP-CPAP were fixed and stained 24 h after RNAi treatment and induction of GFP-CPAP. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01 (one-tailed t-test).


G U2OS cells expressing GFP - CPAP ( green ) and treated with RBM14 siRNA were analyzed using CLEM . Separate z - plane images of the cell with GFP - CPAP foci and the threefold magnified images are aligned from bottom to top along z - axis ( left LM panels ) . White broken lines represent cell shapes , and blue broken lines represent nuclear shapes . Corresponding regions of EM images are shown on the right . White squares ( a - d ) represent the region around GFP - CPAP foci . Note that other than pre - existing centrioles ( a , 1 - 4 ) , ectopic small CPAP foci are recognizable as amorphous electron - dense structures ( b - d ) . One of pre - existing centrioles ( 1 ) is elongated because of exogenous GFP - CPAP expression . Scale bar , 10 μm ( LM ) , 2 μm ( EM , low magnified image ) , 500 nm [ magnified images of ( a ) and ( d ) ) , 200 nm ( magnified images of ( b ) and ( c ) ] .

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

G U2OS cells expressing GFP-CPAP (green) and treated with RBM14 siRNA were analyzed using CLEM. Separate z-plane images of the cell with GFP-CPAP foci and the threefold magnified images are aligned from bottom to top along z-axis (left LM panels). White broken lines represent cell shapes, and blue broken lines represent nuclear shapes. Corresponding regions of EM images are shown on the right. White squares (a-d) represent the region around GFP-CPAP foci. Note that other than pre-existing centrioles (a, 1-4), ectopic small CPAP foci are recognizable as amorphous electron-dense structures (b-d). One of pre-existing centrioles (1) is elongated because of exogenous GFP-CPAP expression. Scale bar, 10 μm (LM), 2 μm (EM, low magnified image), 500 nm [magnified images of (a) and (d)), 200 nm (magnified images of (b) and (c)].


B - E Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were cold - treated for 30 min , followed by 30 - 60 min incubation at 37 ° C and stained with antibodies against centrin ( magenta in ( B ) and ( D ) ) or γ - tubulin ( magenta in ( E ) ) as well as α - tubulin ( green ) . DNA is shown in blue . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm . Histograms represent the percentages of mitotic cells showing the indicated phenotype at each time point . Values are mean percentages ± standard error of mean ( SEM ) from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 , * * P < 0 . 01 ( one - tailed t - test ) .

O O O O B-CELL_LINE O O B-CELL_LINE 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 B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were cold-treated for 30 min, followed by 30-60 min incubation at 37°C and stained with antibodies against centrin (magenta in (B) and (D)) or γ-tubulin (magenta in (E)) as well as α-tubulin (green). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent the percentages of mitotic cells showing the indicated phenotype at each time point. Values are mean percentages ± standard error of mean (SEM) from three independent experiments (n = 30 for each condition). *P < 0.05, **P < 0.01 (one-tailed t-test).


A U2OS cells treated with RBM14 siRNA and stained with antibodies against centrin - 2 ( green ) as well as HsSAS - 6 ( magenta ) . DNA is shown in blue . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm . Histograms represent the frequency of mitotic cells with the indicated phenotype . Values are mean percentages ± SEM from three independent experiments ( n = 60 for each condition ) .

O B-CELL_LINE O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O 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 U2OS cells treated with RBM14 siRNA and stained with antibodies against centrin-2 (green) as well as HsSAS-6 (magenta). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent the frequency of mitotic cells with the indicated phenotype. Values are mean percentages ± SEM from three independent experiments (n = 60 for each condition).


B Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were stained with antibodies against HsSAS - 6 as well as centrin - 2 . Immunofluorescence micrographs are shown in Supplementary Fig S6D . When we counted HsSAS - 6 foci in this paper , we only counted cells before metaphase / anaphase transition considering the disappearance of HsSAS - 6 from anaphase in mitosis . Histograms represent frequency of mitotic cells with excess foci of the indicated proteins at spindle poles . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 ( one - tailed t - test ) .

O O B-CELL_LINE O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O 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 Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were stained with antibodies against HsSAS-6 as well as centrin-2. Immunofluorescence micrographs are shown in Supplementary Fig S6D. When we counted HsSAS-6 foci in this paper, we only counted cells before metaphase/anaphase transition considering the disappearance of HsSAS-6 from anaphase in mitosis. Histograms represent frequency of mitotic cells with excess foci of the indicated proteins at spindle poles. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). *P < 0.05 (one-tailed t-test).


C U2OS cells treated with the indicated siRNAs were stained with anti - α - tubulin antibodies and Hoechst 33258 . Histograms represent frequency of mitotic cells with the multipolar spindle in the indicated conditions . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 ( one - tailed t - test ) .

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

C U2OS cells treated with the indicated siRNAs were stained with anti-α-tubulin antibodies and Hoechst 33258. Histograms represent frequency of mitotic cells with the multipolar spindle in the indicated conditions. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). *P < 0.05 (one-tailed t-test).


D RBM14 - depleted HeLa cells expressing GFP - centrin ( green ) were stained with antibodies against CP110 ( magenta ) and HsSAS - 6 ( blue ) . Scale bar , 1 μm . A representative stained procentriole - like structure as well as endogenous centrioles is shown together with a schematic . Histograms represent length between the centers of the foci of the indicated proteins . Values are mean percentages ± SEM from seven individual cells .

O B-GENEPROD O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O 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 RBM14-depleted HeLa cells expressing GFP-centrin (green) were stained with antibodies against CP110 (magenta) and HsSAS-6 (blue). Scale bar, 1 μm. A representative stained procentriole-like structure as well as endogenous centrioles is shown together with a schematic. Histograms represent length between the centers of the foci of the indicated proteins. Values are mean percentages ± SEM from seven individual cells.


E , F Ectopic DsRed - centrin - 2 foci in RBM14 - depleted cells recruit HsSAS - 6 - GFP . ( E ) Live imaging of RBM14 - depleted U2OS cells expressing HsSAS - 6 - GFP ( green ) and DsRed - centrin - 2 ( magenta ) . Time is denoted in hh : min , and scale bar is 10 μm . Time zero corresponds to the onset of recording . Pre - existing HsSAS - 6 - GFP foci ( white arrowheads ) and ectopic HsSAS - 6 - GFP focus ( red arrowhead ) were traced throughout the time - lapse recording . White broken lines represent cell shapes , and blue broken lines represent nuclear shapes . ( F ) Histograms represent the percentages of ectopic DsRed - centrin - 2 foci with HsSAS - 6 - GFP recruitment ( n = 69 foci for control , and n = 67 foci for RBM14 siRNA ) .

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

E, F Ectopic DsRed-centrin-2 foci in RBM14-depleted cells recruit HsSAS-6-GFP. (E) Live imaging of RBM14-depleted U2OS cells expressing HsSAS-6-GFP (green) and DsRed-centrin-2 (magenta). Time is denoted in hh:min, and scale bar is 10 μm. Time zero corresponds to the onset of recording. Pre-existing HsSAS-6-GFP foci (white arrowheads) and ectopic HsSAS-6-GFP focus (red arrowhead) were traced throughout the time-lapse recording. White broken lines represent cell shapes, and blue broken lines represent nuclear shapes. (F) Histograms represent the percentages of ectopic DsRed-centrin-2 foci with HsSAS-6-GFP recruitment (n = 69 foci for control, and n = 67 foci for RBM14 siRNA).


G Histograms represent frequency of mitotic cells with excess foci of HsSAS - 6 in the indicated conditions . In this experiment , cells were treated with double amount of RBM14 siRNA ( 20 pmol ) compared to that in the other experiments . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 ( one - tailed t - test ) .

O O O O O O O O O O O O B-GENEPROD I-GENEPROD 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 O O O O O O O O O O O O O O O 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 Histograms represent frequency of mitotic cells with excess foci of HsSAS-6 in the indicated conditions. In this experiment, cells were treated with double amount of RBM14 siRNA (20 pmol) compared to that in the other experiments. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). *P < 0.05 (one-tailed t-test).


H HeLa cells expressing GFP - centrin ( green ) and RFP - H2B ( magenta ) and treated with RBM14 siRNA were analyzed using Live CLEM . Pre - existing GFP - centrin foci ( white arrowheads ) and ectopic GFP - centrin foci ( red arrowheads ) were traced throughout the time - lapse recording . Z - stacked confocal images spanning the entire height of the cells ( < 30 μm ) are shown ( upper panels ) . Time is denoted in hh : min . Time zero corresponds to the onset of ectopic formation of GFP - centrin foci . The cells fixed after live imaging were also analyzed for confirming the local correlation between LM and EM images ( lower panels ) . Red square ( ectopically formed GFP - centrin foci ) represents the region corresponding to that of the EM images acquired from serial sections . Note that ectopic GFP - centrin foci that form a spindle pole include not only electron - dense structures ( 1 , 3 ) but also a morphologically recognizable centriole - like structure ( 2 ) . Scale bars , 10 μm ( live imaging ) and 500 nm ( EM ) .

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

H HeLa cells expressing GFP-centrin (green) and RFP-H2B (magenta) and treated with RBM14 siRNA were analyzed using Live CLEM. Pre-existing GFP-centrin foci (white arrowheads) and ectopic GFP-centrin foci (red arrowheads) were traced throughout the time-lapse recording. Z-stacked confocal images spanning the entire height of the cells (< 30 μm) are shown (upper panels). Time is denoted in hh:min. Time zero corresponds to the onset of ectopic formation of GFP-centrin foci. The cells fixed after live imaging were also analyzed for confirming the local correlation between LM and EM images (lower panels). Red square (ectopically formed GFP-centrin foci) represents the region corresponding to that of the EM images acquired from serial sections. Note that ectopic GFP-centrin foci that form a spindle pole include not only electron-dense structures (1, 3) but also a morphologically recognizable centriole-like structure (2). Scale bars, 10 μm (live imaging) and 500 nm (EM).


( C - I ) BMDMs were primed with LPS for 4 h and then treated with LicoB for 1 h , prior to stimulation with nigericin for 45 min or ATP for 1 h . Western blot analyses of pro - caspase - 1 ( p45 ) , pro - IL - 1β , NLRP3 , and ASC ( the arrow indicates ASC in the whole cell lysate ( WCL ) ; activated caspase - 1 ( p20 ) and cleaved IL - 1β ( p17 ) in the culture supernatants ( SN ) of BMDMs ( C , G ) . Caspase - 1 activity ( D , H ) , IL - 1β secretion ( E , I ) , and LDH release ( F , J ) in the SN were measured . Coomassie Blue staining was used as the supernatant loading control , while lamin B was used as the lysate loading control .

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

(C-I) BMDMs were primed with LPS for 4 h and then treated with LicoB for 1 h, prior to stimulation with nigericin for 45 min or ATP for 1 h. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC (the arrow indicates ASC in the whole cell lysate (WCL); activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture supernatants (SN) of BMDMs (C, G). Caspase-1 activity (D, H), IL-1β secretion (E, I), and LDH release (F, J) in the SN were measured. Coomassie Blue staining was used as the supernatant loading control, while lamin B was used as the lysate loading control.


( A - C ) BMDMs were primed with LPS and then treated with LicoB ( 20 μΜ ) for 1 h , followed by stimulation with nigericin , ATP , poly ( I : C ) , or MSU . Pam3CSK4 - primed BMDMs were treated with LicoB ( 20 μM ) and then transfected with LPS . Western blot analyses of pro - caspase - 1 ( p45 ) , pro - IL - 1β , NLRP3 , and ASC in the whole cell lysate ( WCL ) ; and activated caspase - 1 ( p20 ) and cleaved IL - 1β ( p17 ) in the culture supernatants ( SN ) of BMDMs . ( A ) . Caspase - 1 activity ( B ) and IL - 1β secretion ( C ) in the SN were measured .

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

(A-C) BMDMs were primed with LPS and then treated with LicoB (20 μΜ) for 1 h, followed by stimulation with nigericin, ATP, poly (I:C), or MSU. Pam3CSK4-primed BMDMs were treated with LicoB (20 μM) and then transfected with LPS. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC in the whole cell lysate (WCL); and activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture supernatants (SN) of BMDMs. (A). Caspase-1 activity (B) and IL-1β secretion (C) in the SN were measured.


( D - F ) LPS - primed BMDMs were treated with LicoB ( 20 μM ) for 1 h and then stimulated with nigericin for 45 min , or poly ( dA : dT ) / Salmonella for 6 h . Western blot analyses of pro - caspase - 1 ( p45 ) , pro - IL - 1β , NLRP3 , and ASC in the whole cell lysate ( WCL ) ; and activated caspase - 1 ( p20 ) and cleaved IL - 1β ( p17 ) in the culture supernatants ( SN ) of BMDMs ( D ) . Caspase - 1 activity ( E ) and IL - 1β secretion ( F ) in the SN were measured .

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

(D-F) LPS-primed BMDMs were treated with LicoB (20 μM) for 1 h and then stimulated with nigericin for 45 min, or poly(dA:dT)/Salmonella for 6 h. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC in the whole cell lysate (WCL); and activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture supernatants (SN) of BMDMs (D). Caspase-1 activity (E) and IL-1β secretion (F) in the SN were measured.


( A - C ) LPS - primed BMDMs were pre - treated with the indicated dose of LicoB for 1 h and then stimulated with ATP for 1 h . Western blot analysis was used to detect cross - linked ASC in the Triton X - insoluble pellet , the arrow indicates ASC ( A ) . Caspase - 1 activity ( B ) and IL - 1β secretion ( C ) in the SN were measured again to verify NLRP3 activation .

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

(A-C) LPS-primed BMDMs were pre-treated with the indicated dose of LicoB for 1 h and then stimulated with ATP for 1 h. Western blot analysis was used to detect cross-linked ASC in the Triton X-insoluble pellet, the arrow indicates ASC (A). Caspase-1 activity (B) and IL-1β secretion (C) in the SN were measured again to verify NLRP3 activation.


( E ) Western blot analysis of cross - linked ASC in the Triton X - insoluble pellet from LPS - primed BMDMs pre - treated with LicoB ( 20 μM ) or vehicle and then stimulated with nigericin , poly ( dA : dT ) , or Salmonella .

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

(E) Western blot analysis of cross-linked ASC in the Triton X-insoluble pellet from LPS-primed BMDMs pre-treated with LicoB (20 μM) or vehicle and then stimulated with nigericin, poly (dA:dT), or Salmonella.


( A ) Western blot analysis of protein in whole cell lysates ( WCL ) from BMDMs stimulated with LPS for 3 h and then treated with LicoB for 1 h ( LicoB after LPS ) , or BMDMs first treated with LicoB for 1 h and then stimulated with LPS for 3 h ( LicoB before LPS ) .

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

(A) Western blot analysis of protein in whole cell lysates (WCL) from BMDMs stimulated with LPS for 3 h and then treated with LicoB for 1 h (LicoB after LPS), or BMDMs first treated with LicoB for 1 h and then stimulated with LPS for 3 h (LicoB before LPS).


( I ) Cell lysates of LPS - primed BMDMs treated with / without nigericin were incubated with sepharose or LicoB - sepharose . The pull - down samples and input were analysed using Western blot analysis . ( J ) Cell lysates of LPS - primed BMDMs were incubated with sepharose or LicoB - sepharose in the presence of different concentrations of free LicoB ( 0 . 5 mM and 1 mM ) . The pull - down samples and input were analysed using Western blot .

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(I) Cell lysates of LPS-primed BMDMs treated with/without nigericin were incubated with sepharose or LicoB-sepharose. The pull-down samples and input were analysed using Western blot analysis. (J) Cell lysates of LPS-primed BMDMs were incubated with sepharose or LicoB-sepharose in the presence of different concentrations of free LicoB (0.5 mM and 1 mM). The pull-down samples and input were analysed using Western blot.


( K ) HEK - 293T cells were transfected with Flag - NLRP3 or Flag - vector and then treated with LicoB ( 40 μM ) . Immunoprecipitation was performed with anti - DYKDDDDK ( Flag ) affinity gel agarose beads ; Western blot analysis has been shown .

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(K) HEK-293T cells were transfected with Flag-NLRP3 or Flag-vector and then treated with LicoB (40 μM). Immunoprecipitation was performed with anti-DYKDDDDK (Flag) affinity gel agarose beads; Western blot analysis has been shown.


( A ) Survival of WT mice pre - treated with vehicle , LicoB ( 20 mg / kg ) , LicoB ( 40 mg / kg ) , MCC950 ( 40 mg / kg ) , or LicoB ( 40 mg / kg ) + MCC950 ( 40 mg / kg ) , followed by i . p . injection with LPS ( 20 mg / kg ) ; mice survival was monitored for 72 h ( n = 10 mice per group ) .

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(A) Survival of WT mice pre-treated with vehicle, LicoB (20 mg/kg), LicoB (40 mg/kg), MCC950 (40 mg/kg), or LicoB (40 mg/kg) + MCC950 (40 mg/kg), followed by i.p. injection with LPS (20 mg/kg); mice survival was monitored for 72 h (n=10 mice per group).


C57BL / 6 mice received vehicle or LicoB ( 40 mg / kg ) for 17 consecutive doses , once every two days for 34 days . Following that treatment , the serum levels of ALT ( H ) ( n = 8 mice per group ) .

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C57BL/6 mice received vehicle or LicoB (40 mg/kg) for 17 consecutive doses, once every two days for 34 days. Following that treatment, the serum levels of ALT (H) (n=8 mice per group).


C57BL / 6 mice received vehicle or LicoB ( 40 mg / kg ) for 17 consecutive doses , once every two days for 34 days . Following that treatment , the serum levels of AST ( I ) , and CRE ( J ) were measured , ( n = 8 mice per group ) .

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C57BL/6 mice received vehicle or LicoB (40 mg/kg) for 17 consecutive doses, once every two days for 34 days. Following that treatment, the serum levels of AST (I), and CRE (J) were measured, (n=8 mice per group).


( A - D ) Mice were pre - treated with LicoB or MCC950 for 1 h , then i . p . injected with MSU ( 50 mg / kg ) and treated for 6 h ( n = 6 mice per group ) . The levels of IL - 1β in the peritoneal lavage fluid ( A ) and serum ( B ) were measured using ELISA . Quantification of peritoneal exudate cells ( PECs ) ( C ) and neutrophils ( Ly6G and CD11b ) ( D ) using flow cytometry .

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(A-D) Mice were pre-treated with LicoB or MCC950 for 1 h, then i.p. injected with MSU (50 mg/kg) and treated for 6 h (n=6 mice per group). The levels of IL-1β in the peritoneal lavage fluid (A) and serum (B) were measured using ELISA. Quantification of peritoneal exudate cells (PECs) (C) and neutrophils (Ly6G and CD11b) (D) using flow cytometry.


Under the same growth conditions ( permitted water , free food , and a 12 h / 12 h dark / light cycle at 20 ± 2 ° C ) , eight - week - old male C57BL / 6 mice were continuously fed with methionine - and choline - supplemented ( MCS ) or methionine - and choline - deficient ( MCD ) diets for 6 weeks , and at the same time , gavaged with LicoB , MCC950 , or a combination of LicoB and MCC950 ( n = 8 mice per group ( B , C ( A ) Representative liver section images stained with H & E , Sirius Red , and Masson ' s trichrome stains . Scale bar : 0 . 5 cm ( top row ) and 200 μm ( 3 bottom ows ) . ( B , C ) The serum levels of ALT and AST ( n = 8 mice per group ) were measured using ELISA .

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Under the same growth conditions (permitted water, free food, and a 12 h/12 h dark/light cycle at 20±2°C), eight-week-old male C57BL/6 mice were continuously fed with methionine- and choline-supplemented (MCS) or methionine- and choline-deficient (MCD) diets for 6 weeks, and at the same time, gavaged with LicoB, MCC950, or a combination of LicoB and MCC950 (n=8 mice per group (B, C (A) Representative liver section images stained with H&E, Sirius Red, and Masson's trichrome stains. Scale bar: 0.5 cm (top row) and 200 μm (3 bottom ows). (B, C) The serum levels of ALT and AST (n=8 mice per group) were measured using ELISA.


Under the same growth conditions ( permitted water , free food , and a 12 h / 12 h dark / light cycle at 20 ± 2 ° C ) , eight - week - old male C57BL / 6 mice were continuously fed with methionine - and choline - supplemented ( MCS ) or methionine - and choline - deficient ( MCD ) diets for 6 weeks , and at the same time , gavaged with LicoB , MCC950 n = 6 mice per group Real - time quantitative PCR was used to detect the mRNA levels of Col1a1 ( E ) , TNF - α ( F ) in the mice livers , as described in ( A ) ( n = 6 mice per group ) .

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

Under the same growth conditions (permitted water, free food, and a 12 h/12 h dark/light cycle at 20±2°C), eight-week-old male C57BL/6 mice were continuously fed with methionine- and choline-supplemented (MCS) or methionine- and choline-deficient (MCD) diets for 6 weeks, and at the same time, gavaged with LicoB, MCC950 n=6 mice per group Real-time quantitative PCR was used to detect the mRNA levels of Col1a1 (E), TNF-α (F) in the mice livers, as described in (A) (n=6 mice per group).


Under the same growth conditions ( permitted water , free food , and a 12 h / 12 h dark / light cycle at 20 ± 2 ° C ) , eight - week - old male C57BL / 6 mice were continuously fed with methionine - and choline - supplemented ( MCS ) or methionine - and choline - deficient ( MCD ) diets for 6 weeks , and at the same time , gavaged with LicoB , MCC950 n = 6 mice per group Real - time quantitative PCR was used to detect the mRNA levels of IL - 1β ( G ) , and IL - 18 ( H ) in the mice livers , as described in ( A ) ( n = 6 mice per group ) .

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Under the same growth conditions (permitted water, free food, and a 12 h/12 h dark/light cycle at 20±2°C), eight-week-old male C57BL/6 mice were continuously fed with methionine- and choline-supplemented (MCS) or methionine- and choline-deficient (MCD) diets for 6 weeks, and at the same time, gavaged with LicoB, MCC950 n=6 mice per group Real-time quantitative PCR was used to detect the mRNA levels of IL-1β (G), and IL-18 (H) in the mice livers, as described in (A) (n=6 mice per group).


A . Flow cytometry of cells isolated from heart tissue when scaled against TMRM staining and also FSC and SSC parameters of flow cytometry .

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A. Flow cytometry of cells isolated from heart tissue when scaled against TMRM staining and also FSC and SSC parameters of flow cytometry.


B . Cardiac troponin I ( cTnI ) and α - Sarcomeric actinin ( α - SA ) staining of the TMRM + + population ( CMs ) and Vimentin staining of the TMRM + population ( NCs ) . The scale bar represents 100 µm .

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B. Cardiac troponin I (cTnI) and α-Sarcomeric actinin (α-SA) staining of the TMRM++ population (CMs) and Vimentin staining of the TMRM+ population (NCs). The scale bar represents 100 µm.


D . EGFP expression in iPSC - like colonies derived from CMs or NCs isolated from triple transgenic mice .

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D. EGFP expression in iPSC-like colonies derived from CMs or NCs isolated from triple transgenic mice.


E . EGFP expression combined with immunofluorescence staining of SSEA - 1 and DAPI of iPSC - like colonies derived from CMs isolated from triple transgenic mice .

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E. EGFP expression combined with immunofluorescence staining of SSEA-1 and DAPI of iPSC-like colonies derived from CMs isolated from triple transgenic mice.


F . Morphological changes of isolated CMs and NCs at different time points before AP positive colony formation at day 6 . The scale bar represents 100 µm .

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F. Morphological changes of isolated CMs and NCs at different time points before AP positive colony formation at day 6. The scale bar represents 100 µm.


A . Gene profiling of CM and NC at D0 - D6 as shown clustered into 8 categories . Red indicates up - regulated genes while green indicates down - regulated genes ( n = 3 , n = 2 for NC - D6 ) .

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A. Gene profiling of CM and NC at D0-D6 as shown clustered into 8 categories. Red indicates up-regulated genes while green indicates down-regulated genes (n=3, n=2 for NC-D6).


B . Expression patterns of all genes per cluster by grey lines . Black line shows the mean value .

B. Expression patterns of all genes per cluster by grey lines. Black line shows the mean value.


D . Correlative analysis representing the relationships between CM and NC reprogramming specifically at days 2 , 4 and 6 . Red indicates closely related groups , while green indicates significantly different groups .

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D. Correlative analysis representing the relationships between CM and NC reprogramming specifically at days 2, 4 and 6. Red indicates closely related groups, while green indicates significantly different groups.


A . RNA expression ratio ( D2 - to - D0 ) of candidates selected from day 2 microarray data .

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A. RNA expression ratio (D2-to-D0) of candidates selected from day 2 microarray data.


C . Ki67 , α - MHC , and DAPI staining and quantification of CMs treated with control ( Ctrl ) , or combinations of a FoxM1 up - regulation adenovirus ( F ) , Id1 up - regulation adenovirus ( I ) , Hmgb2 up - regulation adenovirus ( H ) or a small molecule Jnk3 inhibitor ( Ji ) .

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C. Ki67, α-MHC, and DAPI staining and quantification of CMs treated with control (Ctrl), or combinations of a FoxM1 up-regulation adenovirus (F), Id1 up-regulation adenovirus (I), Hmgb2 up-regulation adenovirus (H) or a small molecule Jnk3 inhibitor (Ji).


D . Ki67 , α - MHC , and DAPI staining and quantification of CMs treated with Ctrl or combination of FI with Jnk3 - shRNAs in place of the small molecule ( Ji ) . The arrow head indicates Ki67 + / α - MHC + proliferated CMs .

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D. Ki67, α-MHC, and DAPI staining and quantification of CMs treated with Ctrl or combination of FI with Jnk3-shRNAs in place of the small molecule (Ji). The arrow head indicates Ki67+/α-MHC+ proliferated CMs.


E . Morphology and quantification of FIJs - treated CMs undergoing cytokinesis by H3P and Aurora kinase B ( AURKB ) staining .

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E. Morphology and quantification of FIJs-treated CMs undergoing cytokinesis by H3P and Aurora kinase B (AURKB) staining.


G . RNA expression ratio of Oct4 , FoxM1 , Id1 , or Jnk3 in doxycycline - injected versus control adult CMs isolated from reprogrammable mice .

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G. RNA expression ratio of Oct4, FoxM1, Id1, or Jnk3 in doxycycline-injected versus control adult CMs isolated from reprogrammable mice.


H . Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from control or doxycycline injected mice .

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H. Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from control or doxycycline injected mice.


B . The size of hearts isolated from Ctrl or FIJs - treated mice ; heart / body weight ratio of Ctrl or FIJs - treated mice .

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B. The size of hearts isolated from Ctrl or FIJs-treated mice; heart/body weight ratio of Ctrl or FIJs-treated mice.


C . H3P , cTnI , and DAPI staining and quantification of heart tissues in Ctrl or FIJs - treated mice . The arrowhead indicates H3P + / cTnI + proliferated CMs and the scale bar represents 50 µm .

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C. H3P, cTnI, and DAPI staining and quantification of heart tissues in Ctrl or FIJs-treated mice. The arrowhead indicates H3P+/cTnI+ proliferated CMs and the scale bar represents 50 µm.


E . Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from adeno - Ctrl or adeno - FIJs injected mice .

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E. Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from adeno-Ctrl or adeno-FIJs injected mice.


F . Quantification of BrdU + / EGFP + population % in isolated CMs from adeno - Ctrl or adeno - FIJs injected M / Z mice by flow cytometry .

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F. Quantification of BrdU+/EGFP+ population % in isolated CMs from adeno-Ctrl or adeno-FIJs injected M/Z mice by flow cytometry.


B . RNA expressional changes of FoxM1 , Id1 , and Jnk3 after adeno - Ctrl or adeno - FIJs treatment after MI .

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B. RNA expressional changes of FoxM1, Id1, and Jnk3 after adeno-Ctrl or adeno-FIJs treatment after MI.


C . Immunofluorescence staining of BrdU or H3P and cTnI or EGFP and quantification in the heart tissues after MI . The arrow head indicates cTnI + / BrdU + , cTnI + / H3P + or EGFP + / BrdU + , EGFP + / H3P + proliferated CMs . The scale bar represents 50 µm .

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C. Immunofluorescence staining of BrdU or H3P and cTnI or EGFP and quantification in the heart tissues after MI. The arrow head indicates cTnI+/BrdU+, cTnI+/H3P+ or EGFP+/BrdU+, EGFP+/H3P+proliferated CMs. The scale bar represents 50 µm.


D . Ejection fraction and fraction shortening percentage in Ctrl or FIJs - treated mice 21 days post - MI .

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D. Ejection fraction and fraction shortening percentage in Ctrl or FIJs-treated mice 21 days post-MI.


E . Catheterization in Ctrl or FIJs - treated mice 21 days post - MI under baseline or inferior vena cava occlusion condition .

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E. Catheterization in Ctrl or FIJs-treated mice 21 days post-MI under baseline or inferior vena cava occlusion condition.


F . Trichrome staining and quantification showing percentage of fibrotic tissue ( blue ) in Ctrl or FIJs - treated mice 21 days after MI .

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F. Trichrome staining and quantification showing percentage of fibrotic tissue (blue) in Ctrl or FIJs-treated mice 21 days after MI.


A . RNA expression of mitosis checkpoint genes in CMs with FoxM1 or Id1 overexpression , Jnk3 - shRNA , or combined treatment .

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A. RNA expression of mitosis checkpoint genes in CMs with FoxM1 or Id1 overexpression, Jnk3-shRNA, or combined treatment.


B . Transcriptional expression of cyclin - dependent kinases in FoxM1 or Id1 up - regulated , Jnk3 down - regulated , or combined treated CMs .

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B. Transcriptional expression of cyclin-dependent kinases in FoxM1 or Id1 up-regulated, Jnk3 down-regulated, or combined treated CMs.


C . Real - time RT - PCR analysis of CDK inhibitor expression in CMs with FoxM1 or Id1 overexpression , Jnk3 - shRNA or combined treatment .

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C. Real-time RT-PCR analysis of CDK inhibitor expression in CMs with FoxM1 or Id1 overexpression, Jnk3-shRNA or combined treatment.


a , Starvation ( 3 h ) - induced autophagy results in a downregulation of H4K16ac in histone extracts of MEF cells .

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a, Starvation (3 h)-induced autophagy results in a downregulation of H4K16ac in histone extracts of MEF cells.


b , Upon rapamycin treatment ( 300 nM ) LC3 conversion and downregulation of H4K16ac are observed in WT MEF cells but not in the autophagy - deficient atg5 − / − and atg7 − / − MEF cells .

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b, Upon rapamycin treatment (300 nM) LC3 conversion and downregulation of H4K16ac are observed in WT MEF cells but not in the autophagy-deficient atg5−/− and atg7−/− MEF cells.


c , Rapamycin treatment increased the LC3 - II / LC3 - I ratio and promoted H4K16ac decrease in sirt1 − / − and WT MEF cells .

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c, Rapamycin treatment increased the LC3-II/LC3-I ratio and promoted H4K16ac decrease in sirt1−/− and WT MEF cells.


d , Rapamycin - induced autophagy led to downregulation of H4K16ac at 48 h in histone extracts of HeLa and U20S cells , and after 6 h in U1810 cells .

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d, Rapamycin-induced autophagy led to downregulation of H4K16ac at 48 h in histone extracts of HeLa and U20S cells, and after 6 h in U1810 cells.


e , Quantification of H4K16 acetylation by immunoblotting is depicted for rapamycin - treated cells . Data are expressed as mean ± s . e . m . ( n = 3 - 5 ) ; * P 0 . 05 ; * * P 0 . 01 .

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e, Quantification of H4K16 acetylation by immunoblotting is depicted for rapamycin-treated cells. Data are expressed as mean ± s.e.m. (n = 3-5); *P 0.05; **P 0.01.


Rapamycin treatment ( 48 h ) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in MEF ( a )

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Rapamycin treatment (48 h) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in MEF (a)


Rapamycin treatment ( 48 h ) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in transfected HeLa cells ( b ) .

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Rapamycin treatment (48 h) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in transfected HeLa cells (b).


VPA ( 1 mM ) treatment counteracted rapamycin - induced H4K16ac downregulation ( c ) and decreased the LC3 ratio ( d ) .

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VPA (1 mM) treatment counteracted rapamycin-induced H4K16ac downregulation (c) and decreased the LC3 ratio (d).


e , Co - treatment with chloroquine ( CQ , 10 µM ) showed that the decrease in LC3 ratio was a result of an increase in autophagic flux .

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e, Co-treatment with chloroquine (CQ, 10 µM) showed that the decrease in LC3 ratio was a result of an increase in autophagic flux.


f , Inhibition of autophagy by CQ after hMOF overexpression shows that hMOF does not inhibit autophagic flux .

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f, Inhibition of autophagy by CQ after hMOF overexpression shows that hMOF does not inhibit autophagic flux.


g , The yeast homologue of hMOF , Sas2 , tagged with 3 × HA showed a complete disappearance of HA signal upon autophagy induction after 3 h .

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g, The yeast homologue of hMOF, Sas2, tagged with 3× HA showed a complete disappearance of HA signal upon autophagy induction after 3 h.


h , Overexpression of Sas2 repressed the downregulation of H4K16ac upon rapamycin treatment .

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h, Overexpression of Sas2 repressed the downregulation of H4K16ac upon rapamycin treatment.


a , VPA increases the autophagic flux in rapamycin - treated HeLa cells transfected with the mRFP - GFP - LC3 tandem reporter construct which allows distinction between autophagosomes ( GFP + / RFP + yellow puncta ) and autolysosomes ( GFP − / RFP + red puncta ) .

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a, VPA increases the autophagic flux in rapamycin-treated HeLa cells transfected with the mRFP-GFP-LC3 tandem reporter construct which allows distinction between autophagosomes (GFP+/RFP+ yellow puncta) and autolysosomes (GFP−/RFP+ red puncta).


b , Confocal microscopy image of a cell treated with rapamycin and VPA depicting a high ratio of red to green LC3 puncta indicating an increase in autophagic flux .

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b, Confocal microscopy image of a cell treated with rapamycin and VPA depicting a high ratio of red to green LC3 puncta indicating an increase in autophagic flux.


c , Co - treatment with VPA and rapamycin led to increased cell death .

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c, Co-treatment with VPA and rapamycin led to increased cell death.


d , Co - treatment with CQ abrogated VPA + rapamycin - induced cell death .

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d, Co-treatment with CQ abrogated VPA + rapamycin-induced cell death.


e , f , Increasing H4K16ac levels by either overexpression of hMOF , inhibition of SIRT1 by siRNA or the chemical inhibitor Ex527 promoted cell death upon rapamycin treatment .

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e, f, Increasing H4K16ac levels by either overexpression of hMOF, inhibition of SIRT1 by siRNA or the chemical inhibitor Ex527 promoted cell death upon rapamycin treatment.


Co - treatment with the autophagy inhibitor bafilomycin A1 ( BafA , 40 nM ) ( g ) or 3 - methyladenine ( 3MA , 5 mM ) ( h ) abrogated VPA + rapamycin - induced cell death .

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Co-treatment with the autophagy inhibitor bafilomycin A1 (BafA, 40 nM) (g) or 3-methyladenine (3MA, 5 mM) (h) abrogated VPA + rapamycin-induced cell death.


Treatment of HeLa cells upon amino - acid starvation with VPA induced cell death ( i ) , which was rescued when cells were co - treated with CQ ( j ) . Data are expressed as mean ± s . e . m . ( n = 3 ) .

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Treatment of HeLa cells upon amino-acid starvation with VPA induced cell death (i), which was rescued when cells were co-treated with CQ (j). Data are expressed as mean ± s.e.m. (n = 3).


Male mice , housed at 30 ° C or 6 ° C for 10 days , were intraperitoneally ( IP ) administered with [ U - 13C ] glucose ( 2 g / kg ) . 15 minutes after injection , BAT was harvested for metabolic enrichment assay . ( A ) m + 6 glucose enrichment in serum and BAT . ( B ) relative glucose abundance in serum and BAT .

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Male mice, housed at 30°C or 6°C for 10 days, were intraperitoneally (IP) administered with [U-13C]glucose (2 g/kg). 15 minutes after injection, BAT was harvested for metabolic enrichment assay. (A) m+6 glucose enrichment in serum and BAT. (B) relative glucose abundance in serum and BAT.


( D , E ) Metabolic 13C enrichments in BAT of male mice are shown as m + 3 glycolytic intermediates ( D ) , m + 2 TCA cycle intermediates ( E ) .

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(D,E) Metabolic 13C enrichments in BAT of male mice are shown as m+3 glycolytic intermediates (D), m+2 TCA cycle intermediates (E).


( A ) Representative immunofluorescence staining shows UCP1 ( green ) , perilipin ( red ) and DAPI ( blue ) in sWAT of mice housed at 6 ° C .

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(A) Representative immunofluorescence staining shows UCP1 (green), perilipin (red) and DAPI (blue) in sWAT of mice housed at 6°C.


( B - G ) Male mice , housed at 30 ° C or 6 ° C for 10 days , were administered with [ U - 13C ] glucose ( 2 g / kg , IP ) . 15 minutes after injection , sWAT and gWAT were harvested for metabolic enrichment assay . Metabolic 13C enrichments in sWAT of male mice are shown as m + 6 glucose ( B ) , m + 3 glycolytic intermediates ( C ) , and m + 2 TCA cycle intermediates ( D ) . Metabolic 13C enrichments in gWAT of male mice are shown as m + 6 glucose ( E ) , m + 3 glycolysis intermediates ( F ) , and m + 2 TCA cycle intermediates ( G ) .

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

(B-G) Male mice, housed at 30°C or 6°C for 10 days, were administered with [U-13C]glucose (2 g/kg, IP). 15 minutes after injection, sWAT and gWAT were harvested for metabolic enrichment assay. Metabolic 13C enrichments in sWAT of male mice are shown as m+6 glucose (B), m+3 glycolytic intermediates (C), and m+2 TCA cycle intermediates (D). Metabolic 13C enrichments in gWAT of male mice are shown as m+6 glucose (E), m+3 glycolysis intermediates (F), and m+2 TCA cycle intermediates (G).


( H ) Plasma 13CO2 enrichment , after normalizing to the [ U - 13C ] glucose tracer enrichment .

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(H) Plasma 13CO2 enrichment, after normalizing to the [U-13C]glucose tracer enrichment.


Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions ( SVF ) of the interscapular BAT . ( A ) Upper : Oil red staining of the cells during differentiation . Lower : Relative mRNA levels of brown adipocyte markers were measured by qPCR .

B-ORGANISM B-CELL_TYPE I-CELL_TYPE 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O B-EXP_ASSAY O

Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions (SVF) of the interscapular BAT. (A) Upper: Oil red staining of the cells during differentiation. Lower: Relative mRNA levels of brown adipocyte markers were measured by qPCR.


Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions ( SVF ) of the interscapular BAT . ( B ) Glycolytic intermediates were analyzed by GC / MS , after cultured with medium containing 10 mM [ U - 13C ] glucose for 2 hours .

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

Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions (SVF) of the interscapular BAT. (B) Glycolytic intermediates were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose for 2 hours.


Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions ( SVF ) of the interscapular BAT . ( C ) TCA cycle intermediates were analyzed by GC / MS , after cultured with medium containing 10 mM [ U - 13C ] glucose for 2 hours .

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

Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions (SVF) of the interscapular BAT. (C) TCA cycle intermediates were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose for 2 hours.


( A ) Fully differentiated brown adipocytes were treated with β3 - AR agonist CL316 , 243 ( 10 µM ) . Relative mRNA levels of thermogenic markers were measured by qPCR .

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

(A) Fully differentiated brown adipocytes were treated with β3-AR agonist CL316,243 (10 µM). Relative mRNA levels of thermogenic markers were measured by qPCR.


( B ) Pyruvate and lactate enrichment were analyzed by GC / MS , after cultured with medium containing 10 mM [ U - 13C ] glucose and 10 µM CL316 , 243 for 2 hours .

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(B) Pyruvate and lactate enrichment were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose and 10 µM CL316,243 for 2 hours.


( C ) Citrate and malate enrichment were analyzed by GC / MS , after cultured with medium containing 10 mM [ U - 13C ] glucose and 10 µM CL316 , 243 for 2 hours .

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(C) Citrate and malate enrichment were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose and 10 µM CL316,243 for 2 hours.


( A ) oxygen consumption rate ( OCR ) of mouse brown adipocytes treated with MPC inhibitor CHC ( 2 mM ) or UK5099 ( 2 µM ) , n = 6 - 7 biological repeats .

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(A) oxygen consumption rate (OCR) of mouse brown adipocytes treated with MPC inhibitor CHC (2 mM) or UK5099 (2 µM), n=6-7 biological repeats.


( B ) Differentiated mouse brown adipocytes were pre - treated with β3 - AR agonist CL316 , 243 for 4 hours , before cultured with medium containing 10 mM [ U - 13C ] glucose for 2 hours with CHC or UK5099 . Metabolic 13C enrichments in brown adipocytes were shown as m + 3 glycolytic intermediates , m + 2 and m + 3 TCA cycle intermediates , n = 3 biological replicates .

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

(B) Differentiated mouse brown adipocytes were pre-treated with β3-AR agonist CL316,243 for 4 hours, before cultured with medium containing 10 mM [U-13C]glucose for 2 hours with CHC or UK5099. Metabolic 13C enrichments in brown adipocytes were shown as m+3 glycolytic intermediates, m+2 and m+3 TCA cycle intermediates, n=3 biological replicates.


( C ) After housed at 6 ° C for 10 days , mice were IP injected with PBS or CHC ( 500 mg / kg ) . 30 minutes after CHC treatment , mice were IP administered with [ U - 13C ] glucose ( 2 g / kg ) . Metabolic 13C enrichments in BAT of male mice are shown as m + 3 glycolytic intermediates , m + 2 and m + 3 TCA cycle intermediates , n = 7 .

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(C) After housed at 6°C for 10 days, mice were IP injected with PBS or CHC (500 mg/kg). 30 minutes after CHC treatment, mice were IP administered with [U-13C]glucose (2 g/kg). Metabolic 13C enrichments in BAT of male mice are shown as m+3 glycolytic intermediates, m+2 and m+3 TCA cycle intermediates, n=7.


( D ) Body temperature of the CHC ( left : 400 mg / kg , n = 5 ) and ( right : 500 mg / kg , n = 7 ) treated mice .

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(D) Body temperature of the CHC (left: 400 mg/kg, n=5) and (right: 500 mg/kg, n=7) treated mice.


A , Unsupervised cluster analysis of patients with cutaneous malignant melanoma based on global gene expression ( RNA - seq ) in metastases ( n = 367 ) . Partitioning around medoids clustering algorithm was used ( k = 2 ) .

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A, Unsupervised cluster analysis of patients with cutaneous malignant melanoma based on global gene expression (RNA-seq) in metastases (n=367). Partitioning around medoids clustering algorithm was used (k=2).


B , Heat - map displaying the expression of bromodomain genes in metastases from patients with cutaneous malignant melanoma ( n = 367 ) . BRD4 , SMARCA4 , TRIM28 and BRPF1 are highlighted by a black bar . Gene expression is represented by z - score .

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B, Heat-map displaying the expression of bromodomain genes in metastases from patients with cutaneous malignant melanoma (n=367). BRD4, SMARCA4, TRIM28 and BRPF1 are highlighted by a black bar. Gene expression is represented by z-score.


C , Kaplan - Meier analysis of overall survival of patients with stage III melanoma in cluster C1 ( median survival 79 . 5 months ) and C2 ( median survival 25 . 9 months ) , and of patients with stage III melanoma with high ( median survival 61 . 5 months ) or low ( median survival 107 months ) TRIM28 expression . The log - rank test was used for statistical testing of survival data .

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C, Kaplan-Meier analysis of overall survival of patients with stage III melanoma in cluster C1 (median survival 79.5 months) and C2 (median survival 25.9 months), and of patients with stage III melanoma with high (median survival 61.5 months) or low (median survival 107 months) TRIM28 expression. The log-rank test was used for statistical testing of survival data.


C , GSEA plot of RAS signature genes in in TRIM28high and TRIM28low metastatic tumors ( n = 367 ) , and in A375 cells transduced with shT28 - 1 , shT28 - 2 or shNTC ( shLUC and shSCR ) ( n = 3 per construct ) .

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C, GSEA plot of RAS signature genes in in TRIM28high and TRIM28low metastatic tumors (n=367), and in A375 cells transduced with shT28-1, shT28-2 or shNTC (shLUC and shSCR) (n=3 per construct).


C , Metagene profiles after CUT & RUN sequencing of TRIM28 in untransduced A375 cells and of RNAPII in A375 cells transduced with shT28 - 1 or shSCR lentivirus . The lower panel is focused on the TRIM28 metagene profile to clearer depict its binding profile around TSS .

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C, Metagene profiles after CUT&RUN sequencing of TRIM28 in untransduced A375 cells and of RNAPII in A375 cells transduced with shT28-1 or shSCR lentivirus. The lower panel is focused on the TRIM28 metagene profile to clearer depict its binding profile around TSS.


E , TRIM28 and RNAPII occupancy at EGR1 and JUNB in A375 as determined by CUT & RUN sequencing ( PI = length - normalized pause index ) .

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E, TRIM28 and RNAPII occupancy at EGR1 and JUNB in A375 as determined by CUT&RUN sequencing (PI = length-normalized pause index).


F , Immunoblots against TRIM28 and JUNB in A375 , A2058 and SK - MEL - 28 cells transduced with shSCR , shT28 - 1 or shT28 - 2 lentivirus . Results are expressed as mean ± SEM from three biological replicates ( n = 3 ) . G , Densitometry of JUNB and TRIM28 protein levels ( relative to β - actin ) in A375 , A2058 and SK - MEL - 28 melanoma cells based on the immunoblots in F . One - way ANOVA with Dunnett ' s post hoc test was used for statistical testing .

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F, Immunoblots against TRIM28 and JUNB in A375, A2058 and SK-MEL-28 cells transduced with shSCR, shT28-1 or shT28-2 lentivirus. Results are expressed as mean ±SEM from three biological replicates (n=3). G, Densitometry of JUNB and TRIM28 protein levels (relative to β-actin) in A375, A2058 and SK-MEL-28 melanoma cells based on the immunoblots in F. One-way ANOVA with Dunnett's post hoc test was used for statistical testing.


A , Immunoblotting to determine the overexpression of JUNB in A375 cells transduced with pBABE - JUNB .

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A, Immunoblotting to determine the overexpression of JUNB in A375 cells transduced with pBABE-JUNB.


B , Volcano plot displaying differentially expressed genes after RNA - seq analysis of JUNB overexpressing A375 cells ( pBABE - JUNB ) and control A375 cells transduced with empty pBABE vector ( EV ) ( n = 4 ) . Genes induced after JUNB overexpression are highlighted in yellow , and gene suppressed after JUNB overexpression are highlighted in blue .

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B, Volcano plot displaying differentially expressed genes after RNA-seq analysis of JUNB overexpressing A375 cells (pBABE-JUNB) and control A375 cells transduced with empty pBABE vector (EV) (n=4). Genes induced after JUNB overexpression are highlighted in yellow, and gene suppressed after JUNB overexpression are highlighted in blue.


D , Heatmap displaying the induction of CXCL1 and CXCL8 , and suppression of YAP1 target genes , in A375 cells overexpressing JUNB .

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D, Heatmap displaying the induction of CXCL1 and CXCL8, and suppression of YAP1 target genes, in A375 cells overexpressing JUNB.


E , Expression of YAP1 signature genes in A375 cells after transduction with dCas9 - KRAB lentiviruses expressing gRNA targeting JUNB ( gJUNB - 1 or gJUNB - 2 ) or control gRNA ( gEGFP ) . qRT - PCR was used to determine expression levels , and results are expressed as mean ± SEM from three biological replicates ( n = 3 ) .

O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O B-ORGANISM O O O B-GENEPROD 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 O 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, Expression of YAP1 signature genes in A375 cells after transduction with dCas9-KRAB lentiviruses expressing gRNA targeting JUNB (gJUNB-1 or gJUNB-2) or control gRNA (gEGFP). qRT-PCR was used to determine expression levels, and results are expressed as mean ±SEM from three biological replicates (n=3).


F , Quantification of Matrigel invasion experiments with A375 cells transduced with pBABE - JUNB ( JUNB ) or pBABE empty vector ( EV ) retrovirus . Results are expressed as mean ± SEM from three biological replicates ( n = 3 ) and the two - sided unpaired t - test was used . G , Representative images from Matrigel invasion assays in ( F ) . Scale bar is 60 μm .

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

F, Quantification of Matrigel invasion experiments with A375 cells transduced with pBABE-JUNB (JUNB) or pBABE empty vector (EV) retrovirus. Results are expressed as mean ±SEM from three biological replicates (n=3) and the two-sided unpaired t-test was used. G, Representative images from Matrigel invasion assays in (F). Scale bar is 60 μm.


H , Quantification of Matrigel invasion experiments with A375 cells transduced with dCas9 - KRAB lentiviruses expressing gRNA targeting JUNB ( gJUNB - 1 ) or control gRNA ( gEGFP ) . Results are expressed as mean ± SEM from three biological replicates ( n = 3 ) and the two - sided unpaired t - test was used . I , Representative images from Matrigel invasion assays in ( H ) . Scale bar is 60 μm .

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

H, Quantification of Matrigel invasion experiments with A375 cells transduced with dCas9-KRAB lentiviruses expressing gRNA targeting JUNB (gJUNB-1) or control gRNA (gEGFP). Results are expressed as mean ±SEM from three biological replicates (n=3) and the two-sided unpaired t-test was used. I, Representative images from Matrigel invasion assays in (H). Scale bar is 60 μm.


J , Quantification of Matrigel invasion with A375 cells transduced with dCas9 - KRAB lentiviruses expressing gRNA targeting JUNB ( gJUNB - 1 ) or a control gRNA ( gEGFP ) , and then transfected with non - targeting siRNA ( siNTC ) or siRNA targeting YAP1 ( siYAP1 ) and TAZ ( siTAZ ) . Results are expressed as mean ± SEM from three biological replicates ( n = 3 ) . One - way ANOVA and the Tukey post hoc test were used for statistical testing .

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

J, Quantification of Matrigel invasion with A375 cells transduced with dCas9-KRAB lentiviruses expressing gRNA targeting JUNB (gJUNB-1) or a control gRNA (gEGFP), and then transfected with non-targeting siRNA (siNTC) or siRNA targeting YAP1 (siYAP1) and TAZ (siTAZ). Results are expressed as mean ±SEM from three biological replicates (n=3). One-way ANOVA and the Tukey post hoc test were used for statistical testing.


K , Tumor growth after subcutaneous injection of 2 . 0x106 A375 cells transduced with pBABE - JUNB ( JUNB ) or pBABE empty vector ( EV ) retrovirus ( n = 10 mice per group ) . Results are expressed as mean ± SEM . Repeated - measures ANOVA was used for the statistical test of tumor growth . L , Tumor weight after the subcutaneous injection of A375 cells as shown in ( K ) . Tumor weight was analyzed 20 days after subcutaneous injection . Results are expressed as mean ± SEM . The two - sided Mann - Whitney U - test was used for the statistical test of tumor weight .

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

K, Tumor growth after subcutaneous injection of 2.0x106 A375 cells transduced with pBABE-JUNB (JUNB) or pBABE empty vector (EV) retrovirus (n=10 mice per group). Results are expressed as mean ±SEM. Repeated-measures ANOVA was used for the statistical test of tumor growth. L, Tumor weight after the subcutaneous injection of A375 cells as shown in (K). Tumor weight was analyzed 20 days after subcutaneous injection. Results are expressed as mean ±SEM. The two-sided Mann-Whitney U-test was used for the statistical test of tumor weight.