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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 .

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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 ) .

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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 ) .

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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 ) .

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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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 ) .

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(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 ) .

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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 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 .

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 .

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 .

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 .

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


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

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 ) .

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(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 .

(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 .

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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 .

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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 .

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 .

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(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 .

(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 .

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(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 ) .

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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 .

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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 .

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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 .

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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 .

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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.


( B ) Identification of wild - type ( WT ) , heterozygous ( HZ ) , and knockout ( KO ) mice by PCR and agarose gel electrophoresis .

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(B) Identification of wild-type (WT), heterozygous (HZ), and knockout (KO) mice by PCR and agarose gel electrophoresis.


( C ) Immunoblotting ( IB ) of ENKD1 and α - tubulin in wild - type and Enkd1 knockout mice .

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(C) Immunoblotting (IB) of ENKD1 and α-tubulin in wild-type and Enkd1 knockout mice.


( D - F ) Immunofluorescence images ( D ) and quantification of the density of cilia ( E , n = 20 fields from three mice ) and ciliary length ( F , n = 100 cilia from three mice ) in mouse retinas stained with antibodies against acetylated α - tubulin ( Ace - α - tubulin ) and γ - tubulin and DAPI . Scale bar , 2 µm .

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(D-F) Immunofluorescence images (D) and quantification of the density of cilia (E, n = 20 fields from three mice) and ciliary length (F, n = 100 cilia from three mice) in mouse retinas stained with antibodies against acetylated α-tubulin (Ace-α-tubulin) and γ-tubulin and DAPI. Scale bar, 2 µm.


( G - H ) ERG recording images ( G ) and a - b wave amplitudes ( H , n = 5 mice ) show the difference in the ERG a - b wave amplitude between wild - type and Enkd1 knockout mice .

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(G-H) ERG recording images (G) and a-b wave amplitudes (H, n = 5 mice) show the difference in the ERG a-b wave amplitude between wild-type and Enkd1 knockout mice.


( I - J ) VEP recording images ( I ) and N2 - P2 wave amplitudes ( J , n = 5 mice ) show the difference in the VEP N2 - P2 wave amplitude between wild - type and Enkd1 knockout mice .

(I-J) VEP recording images (I) and N2-P2 wave amplitudes (J, n = 5 mice) show the difference in the VEP N2-P2 wave amplitude between wild-type and Enkd1 knockout mice.


( K - M ) Immunofluorescence images ( K ) and quantification of the percentage of ciliated cells ( L , n = 3 mice ) and ciliary length ( M , n = 100 cilia from three mice ) in mouse kidneys stained with the antibody against acetylated α - tubulin and DAPI . To quantify the percentage of ciliated cells ( L ) , > 200 cells from 12 images were analyzed for each mouse . Scale bar , 3 µm .

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(K-M) Immunofluorescence images (K) and quantification of the percentage of ciliated cells (L, n = 3 mice) and ciliary length (M, n = 100 cilia from three mice) in mouse kidneys stained with the antibody against acetylated α-tubulin and DAPI. To quantify the percentage of ciliated cells (L), >200 cells from 12 images were analyzed for each mouse. Scale bar, 3 µm.


( N - Q ) Images ( N ) and quantification of the percentage of flagellated sperm ( O , n = 9 mice ) , flagellar length ( P , n = 100 sperm from nine mice ) , and the percentage of sperm with normal motility among flagellated sperm ( Q , n = 9 mice ) for wild - type and Enkd1 knockout mice . To quantify the percentage of flagellated sperm ( O ) , > 200 sperm were analyzed for each mouse . To quantify the percentage of sperm with normal motility among flagellated sperm ( Q ) , > 200 flagellated sperm were analyzed for each mouse . In panel N , the arrows indicate two spermatozoa without tails . Scale bar , 50 µm .

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(N-Q) Images (N) and quantification of the percentage of flagellated sperm (O, n = 9 mice), flagellar length (P, n = 100 sperm from nine mice), and the percentage of sperm with normal motility among flagellated sperm (Q, n = 9 mice) for wild-type and Enkd1 knockout mice. To quantify the percentage of flagellated sperm (O), >200 sperm were analyzed for each mouse. To quantify the percentage of sperm with normal motility among flagellated sperm (Q), >200 flagellated sperm were analyzed for each mouse. In panel N, the arrows indicate two spermatozoa without tails. Scale bar, 50 µm.


( R , S ) Scanning electron microscopy images of cilia ( R ) and quantification of the percentage of ciliated cells ( S , n = 9 mice ) in the mouse tracheal epithelium . To quantify the percentage of ciliated cells ( S ) , > 200 cells from six fields were analyzed for each mouse . Scale bar , 5 µm .

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(R, S) Scanning electron microscopy images of cilia (R) and quantification of the percentage of ciliated cells (S, n = 9 mice) in the mouse tracheal epithelium. To quantify the percentage of ciliated cells (S), >200 cells from six fields were analyzed for each mouse. Scale bar, 5 µm.


( A ) Immunoblot analysis of ENKD1 and GAPDH in MEFs cultured in serum - free medium for 48 h .

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(A) Immunoblot analysis of ENKD1 and GAPDH in MEFs cultured in serum-free medium for 48 h.


( B - D ) Immunofluorescence images ( B ) and quantification of the percentage of ciliated cells ( C , n = 3 independent experiments ) and ciliary length ( D , n = 70 cilia from three independent experiments ) for MEFs cultured in serum - free medium and stained with antibodies against acetylated α - tubulin and γ - tubulin and DAPI . To quantify the percentage of ciliated cells ( C ) , > 100 cells were analyzed for each experiment . Scale bar , 5 µm .

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(B-D) Immunofluorescence images (B) and quantification of the percentage of ciliated cells (C, n = 3 independent experiments) and ciliary length (D, n = 70 cilia from three independent experiments) for MEFs cultured in serum-free medium and stained with antibodies against acetylated α-tubulin and γ-tubulin and DAPI. To quantify the percentage of ciliated cells (C), >100 cells were analyzed for each experiment. Scale bar, 5 µm.


( E ) Immunoblot analysis of ENKD1 and β - actin in RPE1 cells transfected with control or ENKD1 siRNAs and cultured in serum - free medium for 48 h .

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(E) Immunoblot analysis of ENKD1 and β-actin in RPE1 cells transfected with control or ENKD1 siRNAs and cultured in serum-free medium for 48 h.


Immunofluorescence images ( F ) and quantification of the percentage of ciliated cells ( G , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium , and stained with antibodies against acetylated α - tubulin and γ - tubulin and DAPI . To quantify the percentage of ciliated cells ( G ) , > 150 cells were analyzed for each experiment . Scale bar , 5 µm .

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Immunofluorescence images (F) and quantification of the percentage of ciliated cells (G, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against acetylated α-tubulin and γ-tubulin and DAPI. To quantify the percentage of ciliated cells (G), >150 cells were analyzed for each experiment. Scale bar, 5 µm.


quantification of ciliary length ( H , n = 100 cilia from three independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free

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quantification of ciliary length (H, n = 100 cilia from three independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium


( I , J ) Immunofluorescence images ( I ) and quantification of the percentage of ciliated cells ( J , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium , and stained with antibodies against Arl13b and Centrin and DAPI . To quantify the percentage of ciliated cells ( J ) , > 80 cells were analyzed for each experiment . Scale bar , 2 µm .

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(I, J) Immunofluorescence images (I) and quantification of the percentage of ciliated cells (J, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against Arl13b and Centrin and DAPI. To quantify the percentage of ciliated cells (J), >80 cells were analyzed for each experiment. Scale bar, 2 µm.


( K , L ) Immunofluorescence images ( K ) and quantification of the percentage of ciliated cells ( L , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs and plasmids expressing GFP , GFP - ENKD1 , GFP - ENKD1 - N , GFP - ENKD1 - M , or GFP - ENKD1 - C , cultured in serum - free medium , and stained with the antibody against acetylated α - tubulin and DAPI . The siRNA - resistant forms of ENKD1 were used for these rescue experiments . To quantify the percentage of ciliated cells ( L ) , > 120 cells were analyzed for each experiment . Scale bar , 3 µm .

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(K, L) Immunofluorescence images (K) and quantification of the percentage of ciliated cells (L, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs and plasmids expressing GFP, GFP-ENKD1, GFP-ENKD1-N, GFP-ENKD1-M, or GFP-ENKD1-C, cultured in serum-free medium, and stained with the antibody against acetylated α-tubulin and DAPI. The siRNA-resistant forms of ENKD1 were used for these rescue experiments. To quantify the percentage of ciliated cells (L), >120 cells were analyzed for each experiment. Scale bar, 3 µm.


( A ) Immunofluorescence images of RPE1 cells cultured in normal serum medium , and stained with antibodies against ENKD1 and Centrin and DAPI . Scale bar , 1 µm .

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(A) Immunofluorescence images of RPE1 cells cultured in normal serum medium, and stained with antibodies against ENKD1 and Centrin and DAPI. Scale bar, 1 µm.


( B ) Immunofluorescence images and fluorescence intensity profiles of ENKD1 and CEP164 in serum - starved RPE1 cells . The arrow marks the centriole subjected to the line scan . Scale bar , 1 µm .

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(B) Immunofluorescence images and fluorescence intensity profiles of ENKD1 and CEP164 in serum-starved RPE1 cells. The arrow marks the centriole subjected to the line scan. Scale bar, 1 µm.


( C ) Immunofluorescence images and fluorescence intensity profiles of ENKD1 and Ninein in serum - starved RPE1 cells . The arrow marks the centriole subjected to the line scan . Scale bar , 2 µm .

(C) Immunofluorescence images and fluorescence intensity profiles of ENKD1 and Ninein in serum-starved RPE1 cells. The arrow marks the centriole subjected to the line scan. Scale bar, 2 µm.


( D - F ) 3D - SIM super - resolution images ( D ) , quantification of the outer toroid diameters ( E , n = 20 cells from three independent experiments ) , and a model illustrating the relative positions of γ - tubulin , Ninein , ENKD1 , CEP164 , CEP912 , and Centrin ( F ) within a centriole . RPE1 cells were serum - starved and stained with antibodies against the indicated proteins . Scale bar , 1 µm .

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(D-F) 3D-SIM super-resolution images (D), quantification of the outer toroid diameters (E, n = 20 cells from three independent experiments), and a model illustrating the relative positions of γ-tubulin, Ninein, ENKD1, CEP164, CEP912, and Centrin (F) within a centriole. RPE1 cells were serum-starved and stained with antibodies against the indicated proteins. Scale bar, 1 µm.


( G ) Immunofluorescence images of RPE1 cells cultured in normal serum or serum - free medium , and stained with antibodies against ENKD1 and γ - tubulin and DAPI . Scale bar , 1 µm .

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(G) Immunofluorescence images of RPE1 cells cultured in normal serum or serum-free medium, and stained with antibodies against ENKD1 and γ-tubulin and DAPI. Scale bar, 1 µm.


( H ) Immunofluorescence images of RPE1 cells cultured in serum - free medium , and stained with antibodies against ENKD1 and acetylated α - tubulin and DAPI . Scale bar , 1 µm .

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(H) Immunofluorescence images of RPE1 cells cultured in serum-free medium, and stained with antibodies against ENKD1 and acetylated α-tubulin and DAPI. Scale bar, 1 µm.


( I ) Immunofluorescence images of RPE1 cells transfected with GFP - ENKD1 or GFP vector , cultured in a serum - starved condition , and stained with the antibody against acetylated α - tubulin and DAPI . Scale bar , 1 µm .

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(I) Immunofluorescence images of RPE1 cells transfected with GFP-ENKD1 or GFP vector, cultured in a serum-starved condition, and stained with the antibody against acetylated α-tubulin and DAPI. Scale bar, 1 µm.


( K ) 3D - SIM images of ENKD1 localization at the centrosome in RPE1 cells cultured in serum - free medium ( 0 , 12 , 24 , or 48 h of serum starvation ) , and stained with antibodies against ENKD1 and acetylated α - tubulin . Scale bar , 1 µm .

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(K) 3D-SIM images of ENKD1 localization at the centrosome in RPE1 cells cultured in serum-free medium (0, 12, 24, or 48 h of serum starvation), and stained with antibodies against ENKD1 and acetylated α-tubulin. Scale bar, 1 µm.


Immunofluorescence images ( B ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium , and stained with antibodies against CP110 and acetylated α - tubulin and DAPI .

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Immunofluorescence images (B) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI.


quantification of the percentage of cells with one CP110 dot ( C , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs To quantify the percentage of cells with one CP110 dot ( C ) , > 120 cells were analyzed for each experiment .

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quantification of the percentage of cells with one CP110 dot (C, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs To quantify the percentage of cells with one CP110 dot (C), >120 cells were analyzed for each experiment.


( D , E ) Immunofluorescence images ( D ) and quantification of the percentage of cells with one CEP97 dot ( E , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium , and stained with antibodies against CEP97 and acetylated α - tubulin and DAPI . To quantify the percentage of cells with one CEP97 dot ( E ) , > 150 cells were analyzed for each experiment . Scale bar , 1 µm .

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(D, E) Immunofluorescence images (D) and quantification of the percentage of cells with one CEP97 dot (E, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against CEP97 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CEP97 dot (E), >150 cells were analyzed for each experiment. Scale bar, 1 µm.


( F - H ) Immunoblot analysis ( F ) , immunofluorescence images ( G ) , and quantification of the percentage of cells with one CP110 dot ( H , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium for 120 h , and stained with antibodies against CP110 and acetylated α - tubulin and DAPI . To quantify the percentage of cells with one CP110 dot ( C ) , > 80 cells were analyzed for each experiment . Scale bar , 1 µm .

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(F-H) Immunoblot analysis (F), immunofluorescence images (G), and quantification of the percentage of cells with one CP110 dot (H, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium for 120 h, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CP110 dot (C), >80 cells were analyzed for each experiment. Scale bar, 1 µm.


( A - C ) Immunoblot analysis ( A ) , immunofluorescence images ( B ) and quantification of the percentage of cells with one CP110 dot ( C , n = 3 independent experiments ) for RPE1 cells transfected with the indicated siRNAs , cultured in serum - free medium for 48 h , and stained with antibodies against CP110 and acetylated α - tubulin and DAPI . To quantify the percentage of cells with one CP110 dot ( C ) , > 180 cells were analyzed for each experiment . Scale bar , 1 µm .

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(A-C) Immunoblot analysis (A), immunofluorescence images (B) and quantification of the percentage of cells with one CP110 dot (C, n = 3 independent experiments) for RPE1 cells transfected with the indicated siRNAs, cultured in serum-free medium for 48 h, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CP110 dot (C), >180 cells were analyzed for each experiment. Scale bar, 1 µm.


( D ) Immunoblot analysis of CP110 and β - actin in RPE1 cells transfected with control or CP110 siRNAs and cultured in serum - free medium for 48 h .

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(D) Immunoblot analysis of CP110 and β-actin in RPE1 cells transfected with control or CP110 siRNAs and cultured in serum-free medium for 48 h.


( E , F ) Immunofluorescence images ( E ) and quantification of the percentage of ciliated cells ( F , n = 3 independent experiments ) for RPE1 cells transfected with the indicated siRNAs , cultured in a serum - starved condition , and stained with antibodies against γ - tubulin and acetylated α - tubulin and DAPI . To quantify the percentage of ciliated cells ( F ) , > 120 cells were analyzed for each experiment . Scale bar , 10 µm .

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(E, F) Immunofluorescence images (E) and quantification of the percentage of ciliated cells (F, n = 3 independent experiments) for RPE1 cells transfected with the indicated siRNAs, cultured in a serum-starved condition, and stained with antibodies against γ-tubulin and acetylated α-tubulin and DAPI. To quantify the percentage of ciliated cells (F), >120 cells were analyzed for each experiment. Scale bar, 10 µm.


( G , H ) Immunoprecipitation and immunoblotting ( G ) and quantification ( H , n = 3 mice ) showing the CP110 - CEP97 interaction in the retinal tissues of wild - type and Enkd1 knockout mice . The intensity of each CEP97 band was normalized to that of the β - actin band .

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(G, H) Immunoprecipitation and immunoblotting (G) and quantification (H, n = 3 mice) showing the CP110-CEP97 interaction in the retinal tissues of wild-type and Enkd1 knockout mice. The intensity of each CEP97 band was normalized to that of the β-actin band.


( I , J ) Immunoprecipitation and immunoblotting ( I ) and quantification ( J , n = 3 independent experiments ) showing the CP110 - CEP97 interaction in serum - starved RPE1 cells transfected with control or ENKD1 siRNAs . The intensity of each CEP97 band was normalized to that of the β - actin band .

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(I, J) Immunoprecipitation and immunoblotting (I) and quantification (J, n = 3 independent experiments) showing the CP110-CEP97 interaction in serum-starved RPE1 cells transfected with control or ENKD1 siRNAs. The intensity of each CEP97 band was normalized to that of the β-actin band.


( A ) Immunoprecipitation and immunoblotting showing the interaction between endogenous ENKD1 and CP110 in mouse retinal tissues . Immunoprecipitation was performed with the anti - ENKD1 ( upper panel ) or anti - CP110 ( lower panel ) antibody or the corresponding control IgG .

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(A) Immunoprecipitation and immunoblotting showing the interaction between endogenous ENKD1 and CP110 in mouse retinal tissues. Immunoprecipitation was performed with the anti-ENKD1 (upper panel) or anti-CP110 (lower panel) antibody or the corresponding control IgG.


( B - D ) Immunoprecipitation and immunoblotting showing the interaction of GFP - ENKD1 with Flag - CP110 ( B and upper panel of D ) or HA - CP110 ( C and bottom panel of D ) in HEK293T cells . Immunoprecipitation was performed with antibodies against GFP ( B and C ) , Flag ( upper panel of D ) , or HA ( bottom panel of D ) .

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(B-D) Immunoprecipitation and immunoblotting showing the interaction of GFP-ENKD1 with Flag-CP110 (B and upper panel of D) or HA-CP110 (C and bottom panel of D) in HEK293T cells. Immunoprecipitation was performed with antibodies against GFP (B and C), Flag (upper panel of D), or HA (bottom panel of D).


( E ) Identification of the domains of ENKD1 mediating its interaction with CP110 .

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(E) Identification of the domains of ENKD1 mediating its interaction with CP110.


( F ) Identification of the domains of CP110 mediating its interaction with ENKD1 .

(F) Identification of the domains of CP110 mediating its interaction with ENKD1.


( H ) GST pull - down showing the interaction of purified GST - CP110 - N with purified His - ENKD1 - M .

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(H) GST pull-down showing the interaction of purified GST-CP110-N with purified His-ENKD1-M.


( I , J ) Immunoprecipitation and immunoblotting ( I ) and quantification ( J , n = 3 independent experiments ) showing the interaction of HA - CP110 with Flag - CEP97 in HEK293T cells transfected with the indicated plasmids . The intensity of each Flag - CEP97 band was normalized to that of the β - actin band .

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(I, J) Immunoprecipitation and immunoblotting (I) and quantification (J, n = 3 independent experiments) showing the interaction of HA-CP110 with Flag-CEP97 in HEK293T cells transfected with the indicated plasmids. The intensity of each Flag-CEP97 band was normalized to that of the β-actin band.


A - D Mesenteric blood ( PECAM1 , red ) and lymphatic vessels ( LYVE1 , green ; PROX1 , gray ) . Asterisks indicate lymphatic valves , arrow indicates a lymphatic vessel stub , and arrowhead indicates an isolated lymphatic vessel fragmentE - H Blood vessels ( PECAM1 , green ) and lymphatic vessels ( VEGFR - 3 , red ) in the small intestinal wall .

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A-D Mesenteric blood (PECAM1, red) and lymphatic vessels (LYVE1, green; PROX1, gray). Asterisks indicate lymphatic valves, arrow indicates a lymphatic vessel stub, and arrowhead indicates an isolated lymphatic vessel fragmentE-H Blood vessels (PECAM1, green) and lymphatic vessels (VEGFR-3, red) in the small intestinal wall.


I , J Detection of chylous ascites ( arrows ) in the VCiΔR26 mice at P6 .

I, J Detection of chylous ascites (arrows) in the VCiΔR26 mice at P6.


K , L LYVE1 staining of intestinal lymphatic vessels at P6 .

K, L LYVE1 staining of intestinal lymphatic vessels at P6.


M - T LYVE1 staining of lymphatic vessels in the intestinal wall in adult mice . Genotypes and deletion lengths are indicated in ( U ) . U Quantification of LYVE1 areas in ( M - T ) . Length of the i ∆ Vegfc gene deletion is indicated in months ( mo ) , and Vegfd indicates the VEGF - D genotype . Data are represented as mean ± SEM . Significant differences were determined using one - way ANOVA and Bonferroni post hoc analysis compared to WT intestine represented in ( M ) . * P = 0 . 003 , * * P = 0 . 001 , * * * P = 0 . 0008 , ♯ P = 0 . 0002 , § P = 0 . 0001 .

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M-T LYVE1 staining of lymphatic vessels in the intestinal wall in adult mice. Genotypes and deletion lengths are indicated in (U).U Quantification of LYVE1 areas in (M-T). Length of the i∆Vegfc gene deletion is indicated in months (mo), and Vegfd indicates the VEGF-D genotype. Data are represented as mean ± SEM. Significant differences were determined using one-way ANOVA and Bonferroni post hoc analysis compared to WT intestine represented in (M). *P = 0.003, **P = 0.001, ***P = 0.0008, ♯P = 0.0002, §P = 0.0001.


Overview of the small intestine cross section stained with nuclear red and highlighting the location of higher magnification images in ( B - F ) ; ( I ) for the entire villus and ( II ) for the villus base . β - Gal staining pattern of the villus in wild - type ( Ctrl ) , Vegfc / LacZ ( VC ) , Vegfr3 / LacZ ( VR - 3 ) , and Vegfr2 / LacZ ( VR - 2 ) mice . Higher magnification images representing β - Gal staining of the villus base in Vegfc / LacZmice . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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Overview of the small intestine cross section stained with nuclear red and highlighting the location of higher magnification images in (B-F); (I) for the entire villus and (II) for the villus base. β-Gal staining pattern of the villus in wild-type (Ctrl), Vegfc/LacZ (VC), Vegfr3/LacZ (VR-3), and Vegfr2/LacZ (VR-2) mice.Higher magnification images representing β-Gal staining of the villus base in Vegfc/LacZmice.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Vegfc / LacZβ - Gal staining reaction with smooth muscle actin ( SMA ) peroxidase staining . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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Vegfc/LacZβ-Gal staining reaction with smooth muscle actin (SMA) peroxidase staining.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Vegfc / LacZβ - Gal staining and LYVE1 peroxidase staining . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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Vegfc/LacZβ-Gal staining and LYVE1 peroxidase staining.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Surface image of Vegfc / LacZβ - Gal - stained intestine ( left ) and cross section counterstaining with PECAM1 ( right ) . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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Surface image of Vegfc/LacZβ-Gal-stained intestine (left) and cross section counterstaining with PECAM1 (right).Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.