Figure 7A Representative immunoblots for TMEM175 expression levels in the midbrains of mice of different ages ( W : weeks ; M : months ) . B Statistics of the relative TMEM175 protein levels normalized to GAPDH in ( A ) . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point . D TMEM175 mRNA levels in the midbrains of WT or TMEM175 KO mice were detected by RT - qPCR using SYBR Green PCR Master Mix . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point . E The knockout of TMEM175 in mouse was verified by Western blot of brain tissues . F Representative TMEM175 basal lysosomal currents recorded in macrophages of WT or TMEM175 - KO mice . Ψ is lysosomal membrane potential ( deﬁned as Vcytosol - Vlumen ) . The currents were elicited with ramp voltage protocols ( - 100 to + 100 mV in 1 s , Vh = 0 mV ) . G Statistics of the current amplitudes of ( F ) . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point . H , Quantification of rears in the cylinder ( H ) pre - and post - MPTP treatment . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point . I Quantification of the latency to fall from a wire hang apparatus ( I ) pre - and post - MPTP treatment . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point . J Swing Duration CV ( CV % ) ( Left panel ) and Ataxia Coefficient in Gait tests 7 days post - MPTP . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point . K Latency to fall in rotarod tests 7 days post - MPTP . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point . L Immunoblots of the TH levels post - MPTP treatment in WT and TMEM175 KO mouse midbrains . M Quantification of the relative TH levels normalized to GAPDH post - MPTP . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point . N TH immunofluorescent labeling on substantia nigra slices of WT or TMEM175 - KO mice post - MPTP treatment . Scale bars = 100 μm . O Statistics of the TH - positive neurons in ( N ) . Data information : The data are presented as the mean ± SEM . Statistical significance was analyzed with two - sided student ' s t - tests , and is indicated with NS for not significant ( p > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A Representative immunoblots for TMEM175 expression levels in the midbrains of mice of different ages (W: weeks; M: months). B Statistics of the relative TMEM175 protein levels normalized to GAPDH in (A). Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.D TMEM175 mRNA levels in the midbrains of WT or TMEM175 KO mice were detected by RT-qPCR using SYBR Green PCR Master Mix. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.E The knockout of TMEM175 in mouse was verified by Western blot of brain tissues.F Representative TMEM175 basal lysosomal currents recorded in macrophages of WT or TMEM175-KO mice. Ψ is lysosomal membrane potential (deﬁned as Vcytosol - Vlumen). The currents were elicited with ramp voltage protocols (-100 to +100 mV in 1 s, Vh = 0 mV). G Statistics of the current amplitudes of (F). Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.H, Quantification of rears in the cylinder (H) pre- and post-MPTP treatment. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.I Quantification of the latency to fall from a wire hang apparatus (I) pre- and post-MPTP treatment. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.J Swing Duration CV (CV%) (Left panel) and Ataxia Coefficient in Gait tests 7 days post-MPTP. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.K Latency to fall in rotarod tests 7 days post-MPTP. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.L Immunoblots of the TH levels post-MPTP treatment in WT and TMEM175 KO mouse midbrains. M Quantification of the relative TH levels normalized to GAPDH post-MPTP. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.N TH immunofluorescent labeling on substantia nigra slices of WT or TMEM175-KO mice post-MPTP treatment. Scale bars = 100 μm. O Statistics of the TH-positive neurons in (N). Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


Figure 2A , Heat map of all reported cancer blood metabolites ( each dark blue mark denotes that the metabolite was reported to be increased or decreased ) in various cancer types , illustrating that most individual studies report only a small subset of all previously measured metabolites . It is typically not described whether the metabolites that were not reported ( white " empty " space ) were not measured , or measured but not reported . Labelings : X - axis ( top ) : ribbon colour code , denoting the cancer type ( right ; indicated by red arrow ) ; X - axis ( bottom ) : cohorts , arranged from 1 to 71 ; Y - axis ( right ) : all 1 , 206 metabolites reported in at least one of the studies ; Y - axis ( left ) : ribbon colour code , denoting the metabolite class ( amino acids , carbohydrates , etc ; " other " refers to all other metabolites than the listed classes ) . B , Vote counting of cancer blood metabolites ( reported in at least six cohorts ) showed consistently deregulated metabolites . Blue bars : decreased metabolites ; red bars : increased metabolites . * ( asterisk ) in front of the name of the metabolite indicates a statistical trend ( p < 0 . 1 ) ; red arrowheads denote metabolites mentioned in the main text . C , D , Volcano plot of bloodmetabolites ( C ) and tissuemetabolites ( D ) , reported in at least six cancer studies , with the vote counting score on the X - axis and the - log10 adjusted p - value on the Y - axis . Cyan indicates deregulated metabolites that show a trend ( p < 0 . 1 ; for blood , corresponding to metabolites marked with * in panel 2B ; for tissue , corresponding to metabolites marked with * in Figure 3 ) or statistical significance ( p < 0 . 05 ; above black dashed horizontal line ) ; red indicates metabolites with a p - value > 0 . 1 . A subset of metabolites is annotated ( see Table EV4 ( blood ) and EV5 ( tissue ) for full annotation ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A, Heat map of all reported cancer blood metabolites (each dark blue mark denotes that the metabolite was reported to be increased or decreased) in various cancer types, illustrating that most individual studies report only a small subset of all previously measured metabolites. It is typically not described whether the metabolites that were not reported (white "empty" space) were not measured, or measured but not reported. Labelings: X-axis (top): ribbon colour code, denoting the cancer type (right; indicated by red arrow); X-axis (bottom): cohorts, arranged from 1 to 71; Y-axis (right): all 1,206 metabolites reported in at least one of the studies; Y-axis (left): ribbon colour code, denoting the metabolite class (amino acids, carbohydrates, etc; "other" refers to all other metabolites than the listed classes).B, Vote counting of cancer blood metabolites (reported in at least six cohorts) showed consistently deregulated metabolites. Blue bars: decreased metabolites; red bars: increased metabolites. * (asterisk) in front of the name of the metabolite indicates a statistical trend (p < 0.1); red arrowheads denote metabolites mentioned in the main text.C,D, Volcano plot of bloodmetabolites (C) and tissuemetabolites (D), reported in at least six cancer studies, with the vote counting score on the X-axis and the -log10 adjusted p-value on the Y-axis. Cyan indicates deregulated metabolites that show a trend (p < 0.1; for blood, corresponding to metabolites marked with * in panel 2B; for tissue, corresponding to metabolites marked with * in Figure 3) or statistical significance (p < 0.05; above black dashed horizontal line); red indicates metabolites with a p-value > 0.1. A subset of metabolites is annotated (see Table EV4 (blood) and EV5 (tissue) for full annotation).


Figure 3Vote counting of cancer tissue metabolites ( reported in at least six cohorts ) showed consistently deregulated metabolites . Blue bars : decreased metabolites ; red bars : increased metabolites . * ( asterisk ) in front of the name of the metabolite indicates a statistical trend ( p < 0 . 1 ) ; red arrows denote key metabolites mentioned in the main text ( see Table EV5 for full annotation ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Vote counting of cancer tissue metabolites (reported in at least six cohorts) showed consistently deregulated metabolites. Blue bars: decreased metabolites; red bars: increased metabolites. * (asterisk) in front of the name of the metabolite indicates a statistical trend (p < 0.1); red arrows denote key metabolites mentioned in the main text (see Table EV5 for full annotation).


Figure 1 ( B ) Genotyping of Brd4fl / fl mice with ERT2 - Cre ( Cre ) or without ERT2 - Cre ( + / + ) . Tamoxifen treatment deleted 2 . 2 kb Brd4fl / fl fragment from Cre / + and Cre / Cre mice ( top ) . Cre / + , Cre / Cre and + / + mice were confirmed by the presence of ERT2 - Cre band ( Cre ) , or its absence ( WT ) ( bottom ) . ( C ) WT and Brd4 KO cells were tested for mRNA ( left ) and the protein ( right ) by qRT - PCR and immune blot assays . Values represent the average of three experiments + / - SD . ( D ) Vav - cre mediated Brd4 depletion was verified for cells from Brd4fl / fl Vav - Cre mice ( fetal liver or spleen ) by qRT - PCR and Immunoblot ( left and right ) . Values represent the average of three experiments + / - SD . ( E ) Photographic images of E14 . 5 embryos from WT and KO mice . ( F ) Corresponding Flow cytometric profile of Lin - fetal liver cells stained with C - Kit and Sca - 1 antibodies examining early hematopoietic progenitor population ( LSK ) ( left ) . LSK populations were further divided by the Flt3 marker to detect HSCs and MPPs ( middle ) . The number of total cells , that of LSKs , HSCs , MPPs in a fetal liver were estimated on indicated days of embryonic development ( E13 . 5 , E14 . 5 and E17 . 5 ) . Values are the average of 3 - 5 embryos + / - S . D ( right ) . ( H ) Representative flow cytometric profile of CD71 - cells stained for the myeloid and lymphoid markers . Values are the average of 3 E17 . 5 embryos + / - S . D .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) Genotyping of Brd4fl/fl mice with ERT2-Cre (Cre) or without ERT2 -Cre (+/+). Tamoxifen treatment deleted 2.2 kb Brd4fl/fl fragment from Cre/+ and Cre/Cre mice (top). Cre/+, Cre/Cre and +/+ mice were confirmed by the presence of ERT2 -Cre band (Cre), or its absence (WT) (bottom).(C) WT and Brd4 KO cells were tested for mRNA (left) and the protein (right) by qRT-PCR and immune blot assays. Values represent the average of three experiments +/- SD.(D) Vav-cre mediated Brd4 depletion was verified for cells from Brd4fl/fl Vav-Cre mice (fetal liver or spleen) by qRT-PCR and Immunoblot (left and right). Values represent the average of three experiments +/- SD.(E) Photographic images of E14.5 embryos from WT and KO mice.(F) Corresponding Flow cytometric profile of Lin- fetal liver cells stained with C-Kit and Sca-1 antibodies examining early hematopoietic progenitor population (LSK) (left). LSK populations were further divided by the Flt3 marker to detect HSCs and MPPs (middle). The number of total cells, that of LSKs, HSCs, MPPs in a fetal liver were estimated on indicated days of embryonic development (E13.5, E14.5 and E17.5). Values are the average of 3-5 embryos +/- S.D (right).(H) Representative flow cytometric profile of CD71- cells stained for the myeloid and lymphoid markers. Values are the average of 3 E17.5 embryos +/- S.D.


Figure 2 ( A ) Flow cytometry profiles of peritoneal cells from WT and KO mice stained for CD19 and F4 / 80 ( left ) . FACS profile of CD19 - subset showing CD11b + and F4 / 80 + subsets of cells from WT and KO macrophage populations ( middle ) . Cell numbers ( right ) represent the average of 4 WT and 4 Brd4 KO mice + / - S . D . Note a reduction in F4 / 80 + cells from Brd4 KO peritoneum . ( B ) Flow cytometry profiles of peritoneal cells from IL - 4C injected WT and KO mice ( left ) . Cell numbers ( right ) represent the average of 4 WT and 4 Brd4 KO mice + / - S . D . ( C ) Flow cytometry profiles of BrdU incorporation and Ki67 , ARG1 expression in peritoneal cells ( gated for F4 / 80 + , CD19 - macrophages ) from IL - 4C treated WT and KO mice ( left ) . The numbers of cells positive for BrdU , Ki67 and ARG1 ( right ) . The numbers represent the average of three experiments + / - S . D . ( D ) Microarray analysis of macrophage gene expression in IL - 4C treated WT or Brd4 KO mice . Macrophages were isolated from peritoneal cells by the MACS system . Differentially expressed genes were identified with a cut - off line of log 2fold change > 2 difference with p value of < 0 . 05 . Red dots indicate differentially expressed genes ( KO vs WT ) and gray dots are those below the cut - off line . ( F ) qRT - PCR confirmation of representative genes downregulated in KO macrophages from IL - 4C treated mice . Values represent the average of three independent experiments + / - S . D .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Flow cytometry profiles of peritoneal cells from WT and KO mice stained for CD19 and F4/80 (left). FACS profile of CD19- subset showing CD11b+ and F4/80+ subsets of cells from WT and KO macrophage populations (middle). Cell numbers (right) represent the average of 4 WT and 4 Brd4 KO mice +/- S.D. Note a reduction in F4/80+ cells from Brd4 KO peritoneum.(B) Flow cytometry profiles of peritoneal cells from IL-4C injected WT and KO mice (left). Cell numbers (right) represent the average of 4 WT and 4 Brd4 KO mice +/- S.D.(C) Flow cytometry profiles of BrdU incorporation and Ki67, ARG1 expression in peritoneal cells (gated for F4/80+, CD19- macrophages) from IL-4C treated WT and KO mice (left). The numbers of cells positive for BrdU, Ki67 and ARG1 (right). The numbers represent the average of three experiments +/- S.D.(D) Microarray analysis of macrophage gene expression in IL-4C treated WT or Brd4 KO mice. Macrophages were isolated from peritoneal cells by the MACS system. Differentially expressed genes were identified with a cut-off line of log 2fold change >2 difference with p value of < 0.05. Red dots indicate differentially expressed genes (KO vs WT) and gray dots are those below the cut-off line.(F) qRT-PCR confirmation of representative genes downregulated in KO macrophages from IL-4C treated mice. Values represent the average of three independent experiments +/- S.D.


Figure 3 ( B ) Flow cytometry profiles of CD11b + , F4 / 80 + macrophage population harvested on Day 7 from untreated ( WT ) or tamoxifen treated ( KO ) cultures ( left ) . Histogram depicting F4 / 80 expression levels in WT and KO macrophages ( middle ) . Total number of macrophage yields calculated from > 15 independent experiments . Values represent the average of 21 WT and 24 KO experiments + / - S . D . ( C ) MA plots of RNA - seq data from untreated ( UT ) or LPS treated ( 4h ) WT and KO macrophages . Differentially expressed genes were identified by a cut off line of log2FC > 1 ( FDR < 0 . 1 ) . ( D ) Venn diagram depicting the number of LPS induced genes in WT and KO macrophages . ( E ) qRT - PCR analysis of genes representative of the BRD4 independent group ( unaffected by KO ) , dependent group ( downregulated by KO ) or upregulated by KO . Data were obtained from three independent macrophage samples . Values are the average of 3 + / - S . D .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(B) Flow cytometry profiles of CD11b+, F4/80+ macrophage population harvested on Day 7 from untreated (WT) or tamoxifen treated (KO) cultures (left). Histogram depicting F4/80 expression levels in WT and KO macrophages (middle). Total number of macrophage yields calculated from > 15 independent experiments. Values represent the average of 21 WT and 24 KO experiments +/- S.D.(C) MA plots of RNA-seq data from untreated (UT) or LPS treated (4h) WT and KO macrophages. Differentially expressed genes were identified by a cut off line of log2FC>1 (FDR < 0.1). (D) Venn diagram depicting the number of LPS induced genes in WT and KO macrophages.(E) qRT-PCR analysis of genes representative of the BRD4 independent group (unaffected by KO), dependent group (downregulated by KO) or upregulated by KO. Data were obtained from three independent macrophage samples. Values are the average of 3+/- S.D.


Figure 4 ( B ) Heat maps of BRD4 occupancy on the promoter region ( TSS + / - 5kb ) , aligned by the degree of BRD4 signal intensity in UT and LPS treated macrophages . ( D ) Immunoblot detection of BRD4 protein levels in UT and LPS treated WT macrophages . Forty μg of whole cell extracts from UT or LPS treated ( 1h or 4h ) macrophages using antibody for BRD4 or β - actin ( loading control ) . ( E ) Binding of BRD4 and PolII and distribution of histone marks ( H3K27ac and H3K9ac ) on BRD4 dependent and independent genes ( from - 5kb of TSS to + 5kb of transcription end site ( TES ) ) in UT and LPS treated WT and KO macrophages . Normalized ChIP - seq signals ( rpm / bp ) are shown on the Y axis . Arrows show reduced signals in Brd4 KO macrophages . ( F ) Gene tracks of normalized island - filtered ChIP - seq peaks for BRD4 , Pol II , and indicated histone modification marks , along with RNA - seq peaks for a BRD4 independent gene ( Ccl9 ) and a BRD4 dependent gene ( Fcgr2b ) . Values on the Y axis are normalized ChIP - seq signals ( rpm / bp ) .

O O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) Heat maps of BRD4 occupancy on the promoter region (TSS +/- 5kb), aligned by the degree of BRD4 signal intensity in UT and LPS treated macrophages.(D) Immunoblot detection of BRD4 protein levels in UT and LPS treated WT macrophages. Forty μg of whole cell extracts from UT or LPS treated (1h or 4h) macrophages using antibody for BRD4 or β-actin (loading control).(E) Binding of BRD4 and PolII and distribution of histone marks (H3K27ac and H3K9ac) on BRD4 dependent and independent genes (from -5kb of TSS to +5kb of transcription end site (TES)) in UT and LPS treated WT and KO macrophages. Normalized ChIP-seq signals (rpm/bp) are shown on the Y axis. Arrows show reduced signals in Brd4 KO macrophages.(F) Gene tracks of normalized island-filtered ChIP-seq peaks for BRD4, Pol II, and indicated histone modification marks, along with RNA-seq peaks for a BRD4 independent gene (Ccl9) and a BRD4 dependent gene (Fcgr2b). Values on the Y axis are normalized ChIP-seq signals (rpm/bp).


Figure 5 ( A ) BRD4 bound enhancers within 12 . 5kb were stitched together and ranked by BRD4 ChIP - seq signals ( rpm ) in untreated ( UT , left ) and LPS treated ( middle ) WT macrophages . The dotted line distinguished SEs from TEs . Gene names with the ranking in parenthesis are among genes closest to SEs . Venn diagram ( right ) showing nearest genes common in UT and LPS treated macrophages . ( B ) Genome - wide gain and loss of BRD4 SEs upon LPS stimulation . Based on SE coordinates ( rpm from LPS treated samples / rpm in untreated samples ) , changes greater or less than 2 folds ( Log2 ) were plotted as gain or loss . ( C ) Examples of nearest genes that gained or lost SEs upon LPS treatment . Gene tracks for gained and lost loci ( near Tnf and Csf1r ) showing clusters of BRD4 , Pol II and H3K27ac signals . Red bar represents SE length . ( D ) Meta - gene alignment of BRD4 signals on BRD4 ranked SEs . Pol II and H3K27ac ChIP - seq reads were aligned centering on BRD4 SEs of LPS treated WT macrophages . ( E ) Enhancer plots obtained by the H3K27ac - ranking in LPS stimulated WT and KO macrophages .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) BRD4 bound enhancers within 12.5kb were stitched together and ranked by BRD4 ChIP-seq signals (rpm) in untreated (UT, left) and LPS treated (middle) WT macrophages. The dotted line distinguished SEs from TEs. Gene names with the ranking in parenthesis are among genes closest to SEs. Venn diagram (right) showing nearest genes common in UT and LPS treated macrophages.(B) Genome-wide gain and loss of BRD4 SEs upon LPS stimulation. Based on SE coordinates (rpm from LPS treated samples/rpm in untreated samples), changes greater or less than 2 folds (Log2) were plotted as gain or loss.(C) Examples of nearest genes that gained or lost SEs upon LPS treatment. Gene tracks for gained and lost loci (near Tnf and Csf1r) showing clusters of BRD4, Pol II and H3K27ac signals. Red bar represents SE length.(D) Meta-gene alignment of BRD4 signals on BRD4 ranked SEs. Pol II and H3K27ac ChIP-seq reads were aligned centering on BRD4 SEs of LPS treated WT macrophages. (E) Enhancer plots obtained by the H3K27ac-ranking in LPS stimulated WT and KO macrophages.


Figure 6 ( A ) Heat maps of p65 binding centered at the TSS on BRD4 enriched promoters . ( B ) Meta - gene profile of p65 ChIP - seq signals ( rpm / bp ) from the TSS to TES and + / - 5kb on BRD4 independent genes ( top ) and BRD4 dependent genes ( bottom ) . ( C ) Gene tracks of BRD4 and p65 ChIP - seq peaks and RNA - seq profiles on a BRD4 independent gene ( Il1b ) , a gene upregulated in Brd4KO macrophages ( Ccl7 ) , and a BRD4 dependent gene ( Ptgs2 ) . ( D ) Enhancers ranked by increasing p65 signals in WT ( left ) and Brd4 KO macrophages ( right ) . ( E ) Meta - gene analysis of p65 occupancy on enhancers in WT ( red ) and KO macrophages ( Blue ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Heat maps of p65 binding centered at the TSS on BRD4 enriched promoters.(B) Meta-gene profile of p65 ChIP-seq signals (rpm/bp) from the TSS to TES and +/- 5kb on BRD4 independent genes (top) and BRD4 dependent genes (bottom).(C) Gene tracks of BRD4 and p65 ChIP-seq peaks and RNA-seq profiles on a BRD4 independent gene (Il1b), a gene upregulated in Brd4KO macrophages (Ccl7), and a BRD4 dependent gene (Ptgs2).(D) Enhancers ranked by increasing p65 signals in WT (left) and Brd4 KO macrophages (right).(E) Meta-gene analysis of p65 occupancy on enhancers in WT (red) and KO macrophages (Blue).


Figure 1A . Expression of endothelial Notch target genes in microvascular endothelial cells isolated from skeletal muscle of mice kept on control diet ( CD , 10 % fat , 70 % carbohydrates ) or high - fat diet ( HFD , 60 % fat , 20 % carbohydrates ) or high - fat and sugar diet ( HFS , 60 % fat , 20 % carbohydrates and 42 g / l sucrose in drinking water ) . n = 4 , data represent mean ± SEM , unpaired t - test . B . Expression of endothelial Notch target genes in microvascular endothelial cells isolated from skeletal muscle of mice fasted for 24 hours ( Fasted group ) and then refed for 4 hours ( Fasted + Refed group ) normalized to mice fed for 24 hours ( Fed group ) . n = 5 , data represent mean ± SEM , unpaired t - test . C . Blood glucose levels of control ( n = 5 ) and NICDiOE - EC ( n = 5 ) mice five weeks after recombination . Data represent mean ± SEM , unpaired t - test . D . Plasma insulin levels of control ( n = 5 ) and NICDiOE - EC ( n = 5 ) mice five weeks after recombination . Data represent mean ± SEM , Welch ' s t - test . E . Blood glucose levels for insulin tolerance test of control ( n = 7 ) and NICDiOE - EC control ( n = 7 ) mice . Data represent mean ± SEM , unpaired t - test . F . Quantification of area under curve for insulin tolerance test in E . Data represent mean ± SEM , Welch ' s t - test . G . Blood glucose levels for glucose tolerance test of control ( n = 6 ) or NICDiOE - EC ( n = 7 ) mice . Data represent mean ± SEM , unpaired t - test . H . Quantification of area under the curve for glucose tolerance test in G . Data represent mean ± SEM , unpaired t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Expression of endothelial Notch target genes in microvascular endothelial cells isolated from skeletal muscle of mice kept on control diet (CD, 10% fat, 70% carbohydrates) or high-fat diet (HFD, 60% fat, 20% carbohydrates) or high-fat and sugar diet (HFS, 60% fat, 20% carbohydrates and 42 g/l sucrose in drinking water). n=4, data represent mean ± SEM, unpaired t-test.B. Expression of endothelial Notch target genes in microvascular endothelial cells isolated from skeletal muscle of mice fasted for 24 hours (Fasted group) and then refed for 4 hours (Fasted+Refed group) normalized to mice fed for 24 hours (Fed group). n=5, data represent mean ± SEM, unpaired t-test.C. Blood glucose levels of control (n=5) and NICDiOE-EC (n=5) mice five weeks after recombination. Data represent mean ± SEM, unpaired t-test.D. Plasma insulin levels of control (n=5) and NICDiOE-EC (n=5) mice five weeks after recombination. Data represent mean ± SEM, Welch's t-test.E. Blood glucose levels for insulin tolerance test of control (n=7) and NICDiOE-EC control (n=7) mice. Data represent mean ± SEM, unpaired t-test.F. Quantification of area under curve for insulin tolerance test in E. Data represent mean ± SEM, Welch's t-test.G. Blood glucose levels for glucose tolerance test of control (n=6) or NICDiOE-EC (n=7) mice. Data represent mean ± SEM, unpaired t-test. H. Quantification of area under the curve for glucose tolerance test in G. Data represent mean ± SEM, unpaired t-test.


Figure 2A . Blood glucose levels of control ( n = 8 ) and Rbpji ∆ EC ( n = 8 ) mice five weeks after recombination . Data represent mean ± SEM , unpaired t - test . B . Plasma insulin levels of control ( n = 8 ) and Rbpji ∆ EC ( n = 8 ) mice five weeks after recombination . Data represent mean ± SEM , unpaired t - test . C . Blood glucose levels for insulin tolerance test of control ( n = 10 ) and Rbpji ∆ EC ( n = 7 ) mice . Data represent mean ± SEM , unpaired t - test . D . Quantification of area under curve for insulin tolerance test in C . Data represent mean ± SEM , Welch ' s t - test . E . Blood glucose levels for glucose tolerance test of control ( n = 6 ) or RbpjiΔEC ( n = 5 ) mice . Data represent mean ± SEM , unpaired t - test . F . Quantification of area under the curve for glucose tolerance test in E . Data represent mean ± SEM , Welch ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Blood glucose levels of control (n=8) and Rbpji∆EC (n=8) mice five weeks after recombination. Data represent mean ± SEM, unpaired t-test.B. Plasma insulin levels of control (n=8) and Rbpji∆EC (n=8) mice five weeks after recombination. Data represent mean ± SEM, unpaired t-test.C. Blood glucose levels for insulin tolerance test of control (n=10) and Rbpji∆EC (n=7) mice. Data represent mean ± SEM, unpaired t-test.D. Quantification of area under curve for insulin tolerance test in C. Data represent mean ± SEM, Welch's t-test.E. Blood glucose levels for glucose tolerance test of control (n=6) or RbpjiΔEC (n=5) mice. Data represent mean ± SEM, unpaired t-test. F. Quantification of area under the curve for glucose tolerance test in E. Data represent mean ± SEM, Welch's t-test.


Figure 3A . Uptake of FITC - insulin and CAV1 expression in primary human umbilical venous ECs ( HUVECs ) upon Notch induction . Scale bar 20 µm . B . Uptake of FITC - insulin and CAV1 expression in ( HUVECs ) upon Notch blockage . Scale bar 20 µm . C . Representative western blot of AKT phosphorylation in gastrocnemius muscle 7 minutes after intravenous injection of PBS or 1 . 5 U / kg insulin in control ( n = 3 ) and Rbpji ∆ EC ( n = 3 ) mice five weeks after recombination . D . Densitometric analysis of western blot in C . n = 3 , data represent mean ± SEM , unpaired t - test . E . Representative western blot of AKT phosphorylation in liver 7 minutes after intravenous injection of PBS or 1 . 5 U / kg insulin in control ( n = 3 ) and Rbpji ∆ EC ( n = 3 ) mice five weeks after recombination . F . Densitometric analysis of western blot in E . n = 3 , data represent mean ± SEM , unpaired t - test .

O B-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Uptake of FITC-insulin and CAV1 expression in primary human umbilical venous ECs (HUVECs) upon Notch induction. Scale bar 20 µm.B. Uptake of FITC-insulin and CAV1 expression in (HUVECs) upon Notch blockage. Scale bar 20 µm.C. Representative western blot of AKT phosphorylation in gastrocnemius muscle 7 minutes after intravenous injection of PBS or 1.5 U/kg insulin in control (n=3) and Rbpji∆EC (n=3) mice five weeks after recombination. D. Densitometric analysis of western blot in C. n=3, data represent mean ± SEM, unpaired t-test. E. Representative western blot of AKT phosphorylation in liver 7 minutes after intravenous injection of PBS or 1.5 U/kg insulin in control (n=3) and Rbpji∆EC (n=3) mice five weeks after recombination. F. Densitometric analysis of western blot in E. n=3, data represent mean ± SEM, unpaired t-test.


Figure 4B . AKT phosphorylation in myogenic C2C12 cells treated with different doses of insulin . n = 3 , data represent mean ± SEM , unpaired t - test . C . Representative western blot of AKT phosphorylation in C2C12 cells upon Cav1 knockdown in mouse cardiac endothelial cells ( MCECs ) . D . Densitometric analysis of western blot in C . Data represented as fold change over control . n = 3 , data represent mean ± SEM , unpaired t - test . E . Proximity ligation assay showing the association of CAV1 and INSR ( insulin receptor ) in MCECs upon stimulation with insulin . n = 86 - 120 cells from 8 microscopic high power fields per time point , data represent mean ± SEM , unpaired t - test , * * * means p < 0 . 001 .

O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B. AKT phosphorylation in myogenic C2C12 cells treated with different doses of insulin. n=3, data represent mean ± SEM, unpaired t-test.C. Representative western blot of AKT phosphorylation in C2C12 cells upon Cav1 knockdown in mouse cardiac endothelial cells (MCECs). D. Densitometric analysis of western blot in C. Data represented as fold change over control. n=3, data represent mean ± SEM, unpaired t-test. E. Proximity ligation assay showing the association of CAV1 and INSR (insulin receptor) in MCECs upon stimulation with insulin. n=86-120 cells from 8 microscopic high power fields per time point, data represent mean ± SEM, unpaired t-test, *** means p<0.001.


Figure 5A . Quantitative RT - PCR detection of CAV1 , CAV2 and CAVIN1 in primary human umbilical venous endothelial cells ( HUVECs ) upon Notch blockade ( dnMAML ) and induction ( NICD ) . n = 3 , data represent mean ± SEM , unpaired t - test . B . Representative western blot of CAV1 expression in HUVECs upon Notch manipulation . C . Densitometric analysis of western blot in B . Data represented as fold change over GFP control . n = 3 , data represent mean ± SEM , unpaired t - test . D . Representative western blot of CAVIN1 expression in HUVECs upon Notch induction . E . Densitometric analysis of western blot in D . Data represented as fold change over LacZ control . n = 5 , data represent mean ± SEM , unpaired t - test . F . Expression of CAV1 in HUVECs upon Notch manipulation and HEY1 induction . n = 3 , data represent mean ± SEM , unpaired t - test . G . Expression of Cav1 , Cav2 , Cavin1 in primary microvascular ECs freshly isolated from skeletal muscle of RbpjiΔEC mice compared to control mice . n = 5 , data represent mean ± SEM , unpaired t - test . H . Expression of Cav1 , Cav2 , Cavin1 in primary microvascular ECs freshly isolated from skeletal muscle of NICDiOE - EC mice compared to control mice . n = 4 , data represent mean ± SEM , unpaired t - test . I . Expression of Cav1 , Cav2 , Cavin1 in microvascular endothelial cells isolated from skeletal muscle of mice kept on control diet ( CD , 10 % fat , 70 % carbohydrates ) or high - fat diet ( HFD , 60 % fat , 20 % carbohydrates ) or high - fat and sugar diet ( HFS , 60 % fat , 20 % carbohydrates and 42 g / l sucrose in drinking water ) . n = 4 , data represent mean ± SEM , unpaired t - test . J , K . Quantification of number of caveolae attached to apical membrane in EC of cardiac capillaries from NICDiOE - EC mice ( 6 segments of 1000 nm length from 3 hearts per genotype ) or RbpjiΔEC ( 6 segments of 1000 nm length from 8 hearts per genotype ) based on electron microscopy . Box plot depicts median and percentiles ( 10th , 25th , 75th , 90th ) , unpaired t - test , * means p < 0 . 05 . Representative immune - histochemistry of Cav1 staining in heart sections from control and RbpjiΔEC mice . n = 3 , scale bar 50 µm . Representative confocal images of Cav1 staining in heart sections from control and RbpjiΔEC mice . n = 3 , scale bar 50 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. Quantitative RT-PCR detection of CAV1, CAV2 and CAVIN1 in primary human umbilical venous endothelial cells (HUVECs) upon Notch blockade (dnMAML) and induction (NICD). n=3, data represent mean ± SEM, unpaired t-test.B. Representative western blot of CAV1 expression in HUVECs upon Notch manipulation. C. Densitometric analysis of western blot in B. Data represented as fold change over GFP control. n=3, data represent mean ± SEM, unpaired t-test. D. Representative western blot of CAVIN1 expression in HUVECs upon Notch induction. E. Densitometric analysis of western blot in D. Data represented as fold change over LacZ control. n=5, data represent mean ± SEM, unpaired t-test. F. Expression of CAV1 in HUVECs upon Notch manipulation and HEY1 induction. n=3, data represent mean ± SEM, unpaired t-test.G. Expression of Cav1, Cav2, Cavin1 in primary microvascular ECs freshly isolated from skeletal muscle of RbpjiΔEC mice compared to control mice. n=5, data represent mean ± SEM, unpaired t-test.H. Expression of Cav1, Cav2, Cavin1 in primary microvascular ECs freshly isolated from skeletal muscle of NICDiOE-EC mice compared to control mice. n=4, data represent mean ± SEM, unpaired t-test.I. Expression of Cav1, Cav2, Cavin1 in microvascular endothelial cells isolated from skeletal muscle of mice kept on control diet (CD, 10% fat, 70% carbohydrates) or high-fat diet (HFD, 60% fat, 20% carbohydrates) or high-fat and sugar diet (HFS, 60% fat, 20% carbohydrates and 42 g/l sucrose in drinking water). n=4, data represent mean ± SEM, unpaired t-test.J, K. Quantification of number of caveolae attached to apical membrane in EC of cardiac capillaries from NICDiOE-EC mice (6 segments of 1000 nm length from 3 hearts per genotype) or RbpjiΔEC (6 segments of 1000 nm length from 8 hearts per genotype) based on electron microscopy. Box plot depicts median and percentiles (10th, 25th, 75th, 90th), unpaired t-test, * means p<0.05.Representative immune-histochemistry of Cav1 staining in heart sections from control and RbpjiΔEC mice. n=3, scale bar 50 µm.Representative confocal images of Cav1 staining in heart sections from control and RbpjiΔEC mice. n=3, scale bar 50 µm.


Figure 6B . Blood glucose levels of control and NICDiOE - EC mice during the 2 - DG uptake assay . n = 5 , data represent mean ± SEM , unpaired t - test . C . Plasma insulin levels of control and NICDiOE - EC mice during the 2 - DG uptake assay . n = 5 , data represent mean ± SEM , unpaired t - test . D . 2 - DG uptake levels in skeletal muscle of control and NICDiOE - EC mice . n = 5 , data represent mean ± SEM , unpaired t - test . E . 2 - DG uptake levels in visceral white adipose tissue ( vWAT ) of control ( n = 4 ) and NICDiOE - EC ( n = 5 ) mice . Data represent mean ± SEM , unpaired t - test . G . Blood glucose levels of control and RbpjiΔEC mice during the 2 - DG uptake assay . n = 5 , data represent mean ± SEM , unpaired t - test . H . Plasma insulin levels of control and RbpjiΔEC mice during the 2 - DG uptake assay . n = 5 , data represent mean ± SEM , unpaired t - test . I . 2 - DG uptake levels in skeletal muscle of control and RbpjiΔEC mice . n = 4 , data represent mean ± SEM , unpaired t - test . J . 2 - DG uptake levels in vWAT of control and RbpjiΔEC mice . n = 5 , data represent mean ± SEM , unpaired t - test .

O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6B. Blood glucose levels of control and NICDiOE-EC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.C. Plasma insulin levels of control and NICDiOE-EC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.D. 2-DG uptake levels in skeletal muscle of control and NICDiOE-EC mice. n=5, data represent mean ± SEM, unpaired t-test. E. 2-DG uptake levels in visceral white adipose tissue (vWAT) of control (n=4) and NICDiOE-EC (n=5) mice. Data represent mean ± SEM, unpaired t-test. G. Blood glucose levels of control and RbpjiΔEC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.H. Plasma insulin levels of control and RbpjiΔEC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.I. 2-DG uptake levels in skeletal muscle of control and RbpjiΔEC mice. n=4, data represent mean ± SEM, unpaired t-test. J. 2-DG uptake levels in vWAT of control and RbpjiΔEC mice. n=5, data represent mean ± SEM, unpaired t-test.


Figure 7B . Blood glucose levels for glucose tolerance test ( GTT ) of control ( n = 4 ) or RbpjiΔEC ( n = 5 ) mice kept on high - fat diet ( HFD ) . Data represent unpaired t - test , mean ± SEM . C . Quantification of area under curve ( AUC ) for GTT in B . Data represent mean ± SEM , unpaired t - test . D . Blood glucose levels for insulin tolerance test ( ITT ) of control ( n = 4 ) or RbpjiΔEC ( n = 3 ) mice kept on HFD . Data represent unpaired t - test , mean ± SEM . E . Quantification of AUC for ITT in D . Data represent mean ± SEM , unpaired t - test . F . HbA1c levels of control ( n = 4 ) or RbpjiΔEC ( n = 6 ) mice kept on HFD seven weeks after recombination . Data represent mean ± SEM , unpaired t - test . H . Blood glucose levels of control and RbpjiΔEC mice on HFD during 2 - DG uptake assay . n = 5 , data represent mean ± SEM , unpaired t - test . I . Plasma insulin levels of control and RbpjiΔEC mice on HFD during 2 - DG uptake assay . n = 5 , data represent mean ± SEM , unpaired t - test . J . 2 - DG uptake levels in skeletal muscle of control ( n = 5 ) and RbpjiΔEC ( n = 4 ) mice on HFD . Data represent mean ± SEM , unpaired t - test . K . 2 - DG uptake levels in vWAT of control and RbpjiΔEC mice . n = 4 , data represent mean ± SEM , unpaired t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7B. Blood glucose levels for glucose tolerance test (GTT) of control (n=4) or RbpjiΔEC (n=5) mice kept on high-fat diet (HFD). Data represent unpaired t-test, mean ± SEM. C. Quantification of area under curve (AUC) for GTT in B. Data represent mean ± SEM, unpaired t-test. D. Blood glucose levels for insulin tolerance test (ITT) of control (n=4) or RbpjiΔEC (n=3) mice kept on HFD. Data represent unpaired t-test, mean ± SEM. E. Quantification of AUC for ITT in D. Data represent mean ± SEM, unpaired t-test. F. HbA1c levels of control (n=4) or RbpjiΔEC (n=6) mice kept on HFD seven weeks after recombination. Data represent mean ± SEM, unpaired t-test.H. Blood glucose levels of control and RbpjiΔEC mice on HFD during 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.I. Plasma insulin levels of control and RbpjiΔEC mice on HFD during 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.J. 2-DG uptake levels in skeletal muscle of control (n=5) and RbpjiΔEC (n=4) mice on HFD. Data represent mean ± SEM, unpaired t-test. K. 2-DG uptake levels in vWAT of control and RbpjiΔEC mice. n=4, data represent mean ± SEM, unpaired t-test.


Figure 1c and d . Representation and quantification of cell length in epidermal ( e ) and cortical ( c ) cell files . Optical , longitudinal sections of 5 DAG old Col - 0 roots 12 HAT to ammonium ( C ) or nitrate ( D ) supplemented media . The first 20 epidermal ( e ) and cortex ( c ) cells ( from quiescent center ( QC ) ) are highlighted in grey and in red on ammonium ( C ) , and in blue and green on nitrate ( D ) , respectively . Scale bar = 30 µm . Column bars denote the geometric mean of the cell lengths at the respective positions . Lines represent a polynomial regression fit , with calculated slopes between cells 10 and 20 of 3 . 32639 + 0 . 17172 ( ammonium , epidermis ) , 1 . 22033 + 0 . 08754 ( ammonium , cortex ) and 1 . 70502 + 0 . 09532 ( nitrate , epidermis ) , 0 . 82342 + 0 . 06973 ( nitrate , cortex ) . Data are derived from 3 biological replicates , n = 18 roots in each case .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1c and d. Representation and quantification of cell length in epidermal (e) and cortical (c) cell files. Optical, longitudinal sections of 5 DAG old Col-0 roots 12 HAT to ammonium (C) or nitrate (D) supplemented media. The first 20 epidermal (e) and cortex (c) cells (from quiescent center (QC)) are highlighted in grey and in red on ammonium (C), and in blue and green on nitrate (D), respectively. Scale bar=30 µm. Column bars denote the geometric mean of the cell lengths at the respective positions. Lines represent a polynomial regression fit, with calculated slopes between cells 10 and 20 of 3.32639+0.17172 (ammonium, epidermis), 1.22033+0.08754 (ammonium, cortex) and 1.70502+0.09532 (nitrate, epidermis), 0.82342+0.06973 (nitrate, cortex). Data are derived from 3 biological replicates, n=18 roots in each case.


Figure 2a and b . Maximum intensity Z - stack projection images of 5 DAG old roots expressing the R2D2 auxin signaling reporter 12 HAT to ammonium ( a ) or nitrate ( b ) supplemented media . White arrows mark the position of the 10th and 20th cells from QC ; " e " and " c " mark epidermis and cortex , respectively . Scale bar = 50 µm . Graphs denote normalized relative auxin levels at the respective positions . Lines represent polynomial regression fit with 95 % confidence band . Data are derived from 5 roots per condition from 3 biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2a and b. Maximum intensity Z-stack projection images of 5 DAG old roots expressing the R2D2 auxin signaling reporter 12 HAT to ammonium (a) or nitrate (b) supplemented media. White arrows mark the position of the 10th and 20th cells from QC; "e" and "c" mark epidermis and cortex, respectively. Scale bar=50 µm. Graphs denote normalized relative auxin levels at the respective positions. Lines represent polynomial regression fit with 95% confidence band. Data are derived from 5 roots per condition from 3 biological replicates.


Figure 3b and c . Representation and quantification of cell length in epidermal ( e ) and cortical ( c ) cell files . Optical , longitudinal sections of 5 DAG eir1 - 4 roots 12 HAT to ammonium ( B ) or nitrate ( C ) supplemented media . The first 20 - 20 epidermal and cortex cells ( from quiescent center ( QC ) ) are highlighted in grey and in red on ammonium ( B ) and in blue and green on nitrate ( C ) , respectively . Scale bar = 30 µm . Column bars denote the geometric mean of cell length at the respective positions . Lines represent a polynomial regression fit , with calculated slopes between cells 10 and 20 of 0 . 75884 + 0 . 02624 ( ammonium , epidermis ) , 1 . 13088 + 0 . 08446 ( ammonium , cortex ) and 2 . 06912 + 0 . 10341 ( nitrate , epidermis ) , 0 . 99878 + 0 . 07278 ( nitrate , cortex ) . Data are derived from 3 biological replicates , n = 9 ( ammonium ) and 8 ( nitrate ) roots .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3b and c. Representation and quantification of cell length in epidermal (e) and cortical (c) cell files. Optical, longitudinal sections of 5 DAG eir1-4 roots 12 HAT to ammonium (B) or nitrate (C) supplemented media. The first 20-20 epidermal and cortex cells (from quiescent center (QC)) are highlighted in grey and in red on ammonium (B) and in blue and green on nitrate (C), respectively. Scale bar=30 µm. Column bars denote the geometric mean of cell length at the respective positions. Lines represent a polynomial regression fit, with calculated slopes between cells 10 and 20 of 0.75884+0.02624 (ammonium, epidermis), 1.13088+0.08446 (ammonium, cortex) and 2.06912+0.10341 (nitrate, epidermis), 0.99878+0.07278 (nitrate, cortex). Data are derived from 3 biological replicates, n=9 (ammonium) and 8 (nitrate) roots.


Figure 4b . Higher magnification of pseudo - colored confocal images of 5 DAG old roots expressing PIN2 - GFP 12 HAT to ammonium or nitrate supplemented media . " e " denotes epidermis and " c " cortex , respectively . Color code represents GFP intensity from low ( blue ) to high ( white ) values . Scale bar = 12 µm . White stars mark PIN2 - GFP protein localization on the lateral membranes . Box plots display lateralization index ( fluorescent signal detected on apical / basal membranes divided by the signal value at inner / outer membranes ) of roots on ammonium ( n = 31 cells from 6 roots ) or nitrate ( n = 24 cells from 6 roots ) supplemented medium . The statistical significance was evaluated with ANOVA at p < 0 . 05 . The box chart components are defined as ; box ( 25 % - 75 % ) , central band ( median line ) , central box ( mean ) and the range is within 1 . 5IQR . d . Microscopic images showing PIN2 - Dendra fluorescent signal five hours after photoconversion of PIN2 - Dendra into its red form . Depletion of the red signal and recovery of the green signal over a 6 hours period was followed in parallel in 5 DAG old roots12 HAT to ammonium or nitrate supplemented media . Note the increase in the intensity of the green signal in roots transferred to nitrate . Graph represents the mean signal + SD ( n = 6 roots per condition , 20 cells per root analyzed ) . The experiment was repeated 3 times . Scale bar = 20 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4b. Higher magnification of pseudo-colored confocal images of 5 DAG old roots expressing PIN2-GFP 12 HAT to ammonium or nitrate supplemented media. "e" denotes epidermis and "c" cortex, respectively. Color code represents GFP intensity from low (blue) to high (white) values. Scale bar=12 µm. White stars mark PIN2-GFP protein localization on the lateral membranes. Box plots display lateralization index (fluorescent signal detected on apical/basal membranes divided by the signal value at inner/outer membranes) of roots on ammonium (n=31 cells from 6 roots) or nitrate (n=24 cells from 6 roots) supplemented medium. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.d. Microscopic images showing PIN2-Dendra fluorescent signal five hours after photoconversion of PIN2-Dendra into its red form. Depletion of the red signal and recovery of the green signal over a 6 hours period was followed in parallel in 5 DAG old roots12 HAT to ammonium or nitrate supplemented media. Note the increase in the intensity of the green signal in roots transferred to nitrate. Graph represents the mean signal + SD (n=6 roots per condition, 20 cells per root analyzed). The experiment was repeated 3 times. Scale bar=20 µm.


Figure 5d . Box plots display the distribution of the cell membrane derived PIN2 - GFP fluorescence intensity ( FI ) values ( in arbitrary units , a . u . ) in roots transferred to ammonium ( ( grey , epidermis ( ep ) and red , cortex ( co ) and to nitrate ( blue , epidermis ( ep ) and green , cortex ( co ) ) . 5 cells per roots were analyzed in at least 9 roots per genotype per treatment . The statistical significance was evaluated with ANOVA at p < 0 . 05 . The box chart components are defined as ; box ( 25 % - 75 % ) , central band ( median line ) , central box ( mean ) and the range is within 1 . 5IQR . g . Box plots display lateralization index ( fluorescent signal detected on apical / basal membranes vs inner / outer membranes ) of P2wt , P2D and P2A roots transferred to ammonium ( grey ) or nitrate ( red ) supplemented medium . At least 24 cells from 5 roots were analyzed per genotype per treatment . The statistical significance was evaluated with ANOVA at p < 0 . 05 . The box chart components are defined as ; box ( 25 % - 75 % ) , central band ( median line ) , central box ( mean ) and the range is within 1 . 5IQR .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5d. Box plots display the distribution of the cell membrane derived PIN2-GFP fluorescence intensity (FI) values (in arbitrary units, a.u.) in roots transferred to ammonium ((grey, epidermis (ep) and red, cortex (co) and to nitrate (blue, epidermis (ep) and green, cortex (co)). 5 cells per roots were analyzed in at least 9 roots per genotype per treatment. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.g. Box plots display lateralization index (fluorescent signal detected on apical/basal membranes vs inner/outer membranes) of P2wt, P2D and P2A roots transferred to ammonium (grey) or nitrate (red) supplemented medium. At least 24 cells from 5 roots were analyzed per genotype per treatment. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.


Figure 1Expression level of Shox2 , Tbx15 , HoxC8 , and HoxC5 , mRNA was compared using quantitative real - time PCR ( qPCR ) of RNA isolated from different fat depots . Two subcutaneous depots ( inguinal and scapular white ) , three intra - abdominal fat depots ( perigonadal , perirenal , and mesenteric ) , and interscapular brown adipose tissue of 8 week - old male C57BL / 6 mice . Data are shown as mean ± SEM of six samples . Expression level of Shox2 , Tbx15 , HoxC8 , and HoxC5 , mRNA was compared using qPCR of RNA isolated from cultured stromovascular cells derived from adipose tissue of the Immortomouse ™ . The depot - specific cell lines were made from inguinal , scapular white , perigonadal , perirenal , mesenteric , and interscapular brown adipose tissue of 8 week - old male mice . Data are shown as mean ± SEM of cell lines from three individual mice . Bright - field image of depot - derived cell lines after in vitro adipogenic differentiation and Oil Red O staining ( Lee et al . , 2017a ) . Original photographs were taken at 10x magnification ( left panel ) . Scale bar = 100 μm . qPCR analysis for adiponectin ( Adipoq ) , peroxisome proliferator - activated receptor gamma ( Pparγ2 ) , and fatty acid synthase ( FAS ) in RNA isolated from depot - derived cell lines after in vitro differentiation . Data are shown as mean ± SEM of 3 cell lines / group .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Expression level of Shox2, Tbx15, HoxC8, and HoxC5, mRNA was compared using quantitative real-time PCR (qPCR) of RNA isolated from different fat depots. Two subcutaneous depots (inguinal and scapular white), three intra-abdominal fat depots (perigonadal, perirenal, and mesenteric), and interscapular brown adipose tissue of 8 week-old male C57BL/6 mice. Data are shown as mean ± SEM of six samples. Expression level of Shox2, Tbx15, HoxC8, and HoxC5, mRNA was compared using qPCR of RNA isolated from cultured stromovascular cells derived from adipose tissue of the Immortomouse™. The depot-specific cell lines were made from inguinal, scapular white, perigonadal, perirenal, mesenteric, and interscapular brown adipose tissue of 8 week-old male mice. Data are shown as mean ± SEM of cell lines from three individual mice.Bright-field image of depot-derived cell lines after in vitro adipogenic differentiation and Oil Red O staining (Lee et al., 2017a). Original photographs were taken at 10x magnification (left panel). Scale bar = 100 μm.qPCR analysis for adiponectin (Adipoq), peroxisome proliferator-activated receptor gamma (Pparγ2), and fatty acid synthase (FAS) in RNA isolated from depot-derived cell lines after in vitro differentiation. Data are shown as mean ± SEM of 3 cell lines/group.


Figure 2Representative picture of preadipocyte clonal cell lines showing media color 48 hours after last media change . Cells in all wells were 100 % confluent when media was changed . Heatmap depicting the 50 most variable genes among all preadipocyte clonal cell lines . Red represents high expression of genes , while blue represents low expression of genes . Heatmap depicting the 50 most variable genes among Type 2 and 3 preadipocyte clonal cell lines . Red represents high expression of genes , while blue represents low expression of genes .

O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Representative picture of preadipocyte clonal cell lines showing media color 48 hours after last media change. Cells in all wells were 100% confluent when media was changed.Heatmap depicting the 50 most variable genes among all preadipocyte clonal cell lines. Red represents high expression of genes, while blue represents low expression of genes. Heatmap depicting the 50 most variable genes among Type 2 and 3 preadipocyte clonal cell lines. Red represents high expression of genes, while blue represents low expression of genes.


Figure 3Expression of Wt1 , leucine rich repeat neuronal 4 ( Lrrn4 ) , Uroplakin 3b ( Upk3b ) , in Immortomouse clonal cell line clusters as preadipocytes ( d0 ) and during six days of adipogenic differentiation . Bright - field images of preadipocyte cell lines under standard culture conditions . The photographs were taken at 10x magnification . Expression of Tagln , connective tissue growth factor ( Ctgf ) , Keratin 19 ( Krt19 ) , Mx1 , caspase 1 ( Casp1 ) , and C - X - C Motif Chemokine Ligand 12 ( Cxcl12 ) in Immortomouse clonal cell line clusters as preadipocytes ( d0 ) and during six days of adipogenic differentiation .

O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Expression of Wt1, leucine rich repeat neuronal 4 (Lrrn4), Uroplakin 3b (Upk3b), in Immortomouse clonal cell line clusters as preadipocytes (d0) and during six days of adipogenic differentiation.Bright-field images of preadipocyte cell lines under standard culture conditions. The photographs were taken at 10x magnification.Expression of Tagln, connective tissue growth factor (Ctgf), Keratin 19 (Krt19), Mx1, caspase 1 (Casp1), and C-X-C Motif Chemokine Ligand 12 (Cxcl12) in Immortomouse clonal cell line clusters as preadipocytes (d0) and during six days of adipogenic differentiation.


Figure 4Triglyceride content of cell cultures after six days of differentiation using the protocol described in Methods . Data are shown as mean ± SEM of 3 - 7 samples , and the entire experiment was repeated twice . [ 3H ] - labeled 2 - deoxyglucose uptake in preadipocyte cell lines in the basal state and after pretreatment for 20 minutes with 100 nM insulin . Uptake was measured for one hour , and data are shown as mean ± SEM of 3 - 7 samples and repeated twice . [ 3H ] - labeled 2 - deoxyglucose uptake in adipocytes after six days of adipogenic differentiation in the basal state and after stimulation for 20 minutes with 100 nM insulin . Uptake was measured for one hour , and data are shown as mean ± SEM of 3 - 7 samples . The experiment was repeated twice . [ 14C ] D - glucose incorporation into lipids in adipocytes after six days of adipogenic differentiation in the basal state and after stimulation for 20 minutes with 100 nM insulin . [ 14C ] D - glucose incorporation was measured after three hours , and data are shown as mean ± SEM of 3 - 7 samples . The experiment was repeated twice . Lipolysis rates after stimulation with 1 nM isoproterenol as measured by glycerol release from adipocytes after six days of adipogenic differentiation . Data are shown as mean ± SEM of 3 - 7 replicates and normalized by lipid content of the cells . The entire experiment was repeated twice . Extracellular acidification rate ( ECAR ) of preadipocytes and adipocytes after 4 days of in vitro differentiation was determined by calculating the area under the curve ( AUC ) during measurements of basal respiration . The whole experiment was repeated three times . Data are shown as mean ± SEM of 3 - 7 cell lines per group . Basal respiration of preadipocytes and adipocytes after 4 days of in vitro differentiation was determined by calculating the area under the curve ( AUC ) during measurements of oxygen consumption rate ( OCR ) . The whole experiment was repeated three times . Data are shown as mean ± SEM of 3 - 7 cell lines per group . Ratio of oxygen consumption rate ( OCR ) to extracellular acidification rate ( ECAR ) of preadipocytes and adipocytes after 4 days of in vitro differentiation . Data are shown as mean ± SEM of 3 - 7 cell lines per group . Representative Western blot analysis of three independent adipocyte cell lines after 6 days of in vitro differentiation for : pJnk T183 / Y185 and total Jnk after treatment with 10 nM TNFα for 15 minutes ( Inflammation ) ; pAkt S473 and total Akt after treatment with 100 nM insulin for 15 minutes ( Insulin Signaling ) ; pStat5 Y694 and total Stat5 after treatment with 500 nM GH for 20 minutes ( Growth Hormone ) .

O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Triglyceride content of cell cultures after six days of differentiation using the protocol described in Methods. Data are shown as mean ± SEM of 3-7 samples, and the entire experiment was repeated twice.[3H]-labeled 2-deoxyglucose uptake in preadipocyte cell lines in the basal state and after pretreatment for 20 minutes with 100 nM insulin. Uptake was measured for one hour, and data are shown as mean ± SEM of 3-7 samples and repeated twice. [3H]-labeled 2-deoxyglucose uptake in adipocytes after six days of adipogenic differentiation in the basal state and after stimulation for 20 minutes with 100 nM insulin. Uptake was measured for one hour, and data are shown as mean ± SEM of 3-7 samples. The experiment was repeated twice.[14C] D-glucose incorporation into lipids in adipocytes after six days of adipogenic differentiation in the basal state and after stimulation for 20 minutes with 100 nM insulin. [14C] D-glucose incorporation was measured after three hours, and data are shown as mean ± SEM of 3-7 samples. The experiment was repeated twice.Lipolysis rates after stimulation with 1 nM isoproterenol as measured by glycerol release from adipocytes after six days of adipogenic differentiation. Data are shown as mean ± SEM of 3-7 replicates and normalized by lipid content of the cells. The entire experiment was repeated twice.Extracellular acidification rate (ECAR) of preadipocytes and adipocytes after 4 days of in vitro differentiation was determined by calculating the area under the curve (AUC) during measurements of basal respiration. The whole experiment was repeated three times. Data are shown as mean ± SEM of 3-7 cell lines per group.Basal respiration of preadipocytes and adipocytes after 4 days of in vitro differentiation was determined by calculating the area under the curve (AUC) during measurements of oxygen consumption rate (OCR). The whole experiment was repeated three times. Data are shown as mean ± SEM of 3-7 cell lines per group.Ratio of oxygen consumption rate (OCR) to extracellular acidification rate (ECAR) of preadipocytes and adipocytes after 4 days of in vitro differentiation. Data are shown as mean ± SEM of 3-7 cell lines per group.Representative Western blot analysis of three independent adipocyte cell lines after 6 days of in vitro differentiation for: pJnk T183/Y185 and total Jnk after treatment with 10 nM TNFα for 15 minutes (Inflammation); pAkt S473 and total Akt after treatment with 100 nM insulin for 15 minutes (Insulin Signaling); pStat5 Y694 and total Stat5 after treatment with 500 nM GH for 20 minutes (Growth Hormone).


Figure 5Expression of Wt1 , Lrrn4 , Upk3b , Tagln , Ctgf , Krt19 , Mx1 , Casp1 , and Cxcl12 in primary preadipocytes isolated from white adipose tissue of 5 - 6 month old Wt1 - creERT2 ; Rosa26mT / mG , Tagln - cre ; Rosa26mT / mG , and Mx1 - cre ; Rosa26mT / mG male mice . mGFP and mTomato positive preadipocytes were isolated from the pooled visceral fat depots ( perigonadal , perirenal , mesenteric , and pericardial ) of Wt1 - creERT2 ; Rosa26mT / mG mice , the pooled subcutaneous fat depots ( subcutaneous and scapular white ) of Mx1 - cre ; Rosa26mT / mG , and both the pooled visceral ( Tagln - V ) and subcutaneous depots ( Tagln - S ) from Tagln - cre ; Rosa26mT / mG mice . Data are shown as mean ± SEM of 4 - 6 mice / group . Bright - field image of immortalized mGFP and mTomato positive preadipocytes after in vitro adipogenic differentiation . Neutral lipoids were stained with Oil Red O and original photographs were at 10x magnification ( left panel ) . Scale bar = 100 μm . Triglyceride content of immortalized mGFP and mTomato positive preadipocytes after six days of differentiation . Data are shown as mean ± SEM of 5 - 8 replicates , and the entire experiment was repeated with three independent cell lines . Basal extracellular acidification rate ( ECAR ) of immortalized mGFP and mTomato positive preadipocytes was determined by calculating the area under the curve ( AUC ) in basal conditions . The whole experiment was repeated twice . Data are shown as mean ± SEM of 3 cell lines per group . Basal respiration of preadipocytes was of immortalized mGFP and mTomato positive preadipocytes determined by calculating the area under the curve ( AUC ) during measurements of oxygen consumption rate ( OCR ) . The whole experiment was repeated twice . Data are shown as mean ± SEM of 3 cell lines per group . Ratio of basal oxygen consumption rate ( OCR ) to basal extracellular acidification rate ( ECAR ) of immortalized mGFP and mTomato positive preadipocytes . Data are shown as mean ± SEM of 3 cell lines per group .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Expression of Wt1, Lrrn4, Upk3b, Tagln, Ctgf, Krt19, Mx1, Casp1, and Cxcl12 in primary preadipocytes isolated from white adipose tissue of 5-6 month old Wt1-creERT2;Rosa26mT/mG, Tagln-cre;Rosa26mT/mG, and Mx1-cre;Rosa26mT/mG male mice. mGFP and mTomato positive preadipocytes were isolated from the pooled visceral fat depots (perigonadal, perirenal, mesenteric, and pericardial) of Wt1-creERT2;Rosa26mT/mG mice, the pooled subcutaneous fat depots (subcutaneous and scapular white) of Mx1-cre;Rosa26mT/mG, and both the pooled visceral (Tagln-V) and subcutaneous depots (Tagln-S) from Tagln-cre;Rosa26mT/mG mice. Data are shown as mean ± SEM of 4-6 mice/group.Bright-field image of immortalized mGFP and mTomato positive preadipocytes after in vitro adipogenic differentiation. Neutral lipoids were stained with Oil Red O and original photographs were at 10x magnification (left panel). Scale bar = 100 μm.Triglyceride content of immortalized mGFP and mTomato positive preadipocytes after six days of differentiation. Data are shown as mean ± SEM of 5-8 replicates, and the entire experiment was repeated with three independent cell lines.Basal extracellular acidification rate (ECAR) of immortalized mGFP and mTomato positive preadipocytes was determined by calculating the area under the curve (AUC) in basal conditions. The whole experiment was repeated twice. Data are shown as mean ± SEM of 3 cell lines per group.Basal respiration of preadipocytes was of immortalized mGFP and mTomato positive preadipocytes determined by calculating the area under the curve (AUC) during measurements of oxygen consumption rate (OCR). The whole experiment was repeated twice. Data are shown as mean ± SEM of 3 cell lines per group.Ratio of basal oxygen consumption rate (OCR) to basal extracellular acidification rate (ECAR) of immortalized mGFP and mTomato positive preadipocytes. Data are shown as mean ± SEM of 3 cell lines per group.


Figure 6Number of mGFP and mTomato positive preadipocytes isolated by FACS from the indicated white adipose depots from 5 - 6 month old male Wt1 - creERT2 ; Rosa26mT / mG , Tagln - cre ; Rosa26mT / mG , and Mx1 - cre ; Rosa26mT / mG mice . Data are shown as mean ± SEM of 4 - 9 mice .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Number of mGFP and mTomato positive preadipocytes isolated by FACS from the indicated white adipose depots from 5-6 month old male Wt1-creERT2;Rosa26mT/mG, Tagln-cre;Rosa26mT/mG, and Mx1-cre;Rosa26mT/mG mice. Data are shown as mean ± SEM of 4-9 mice.


Figure 7Representative images of whole - mount preparations of the indicated white adipose tissue depots of 5 - 6 month old Wt1 - creERT2 ; Rosa26mT / mG , Tagln - cre ; Rosa26mT / mG , and Mx1 - cre ; Rosa26mT / mG male mice . The photographs were taken at 10x magnification . Scale bar = 100 μm . Quantitation of mGFP and mTomato positive adipocytes from each of the indicated white adipose depots from 5 - 6 month old male Wt1 - creERT2 ; Rosa26mT / mG , Tagln - cre ; Rosa26mT / mG , and Mx1 - cre ; Rosa26mT / mG mice . Adipocytes were counted from four non - overlapping images per depot per mouse .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Representative images of whole-mount preparations of the indicated white adipose tissue depots of 5-6 month old Wt1-creERT2;Rosa26mT/mG, Tagln-cre;Rosa26mT/mG, and Mx1-cre;Rosa26mT/mG male mice. The photographs were taken at 10x magnification. Scale bar = 100 μm.Quantitation of mGFP and mTomato positive adipocytes from each of the indicated white adipose depots from 5-6 month old male Wt1-creERT2;Rosa26mT/mG, Tagln-cre;Rosa26mT/mG, and Mx1-cre;Rosa26mT/mG mice. Adipocytes were counted from four non-overlapping images per depot per mouse.


Figure 8Number of mGFP positive preadipocytes isolated by FACS from each of the indicated white adipose depots from 5 - 6 month - old Wt1 - creERT2 , Tagln - cre , and Mx1 - cre crossed to Rosa26mT / mG mice from the experiment shown in Figure 6B . Data are shown as mean ± SEM of 4 - 9 mice . Number of mGFP positive adipocytes from each of the indicated white adipose depots from 5 - 6 month - old Wt1 - creERT2 , Tagln - cre , and Mx1 - cre crossed to Rosa26mT / mG mice . Four separate non - overlapping images / depot / mouse from the experiment shown in Figure 6C . Data are shown as mean ± SEM of 4 - 9 mice .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8Number of mGFP positive preadipocytes isolated by FACS from each of the indicated white adipose depots from 5-6 month-old Wt1-creERT2, Tagln-cre, and Mx1-cre crossed to Rosa26mT/mG mice from the experiment shown in Figure 6B. Data are shown as mean ± SEM of 4-9 mice.Number of mGFP positive adipocytes from each of the indicated white adipose depots from 5-6 month-old Wt1-creERT2, Tagln-cre, and Mx1-cre crossed to Rosa26mT/mG mice. Four separate non-overlapping images/depot/mouse from the experiment shown in Figure 6C. Data are shown as mean ± SEM of 4-9 mice.


Figure 1HSATIII lncRNA - dependent localization of m6A - related factors . Control and HSATIII knockdown HeLa cells were exposed to thermal stress ( 42 ° C for 2 h and recovery for 1 h at 37 ° C ) and stained by HSATIII - FISH and immunofluorescence using an anti - WTAP antibody ( B ) or anti - YTHDC1 antibody ( C ) . The nuclei were stained with DAPI . Scale bar : 10 µm . YTHDC1 accumulation within nSBs was arrested by METTL3 knockdown . HeLa cells were stained as described in Figure 1C . Scale bar : 10 µm . Box plot of relative intensities of YTHDC1 to HSATIII within nSBs in individual nuclei ( n = 30 ) . The mean is indicated with X . The first and third quartiles are the ends of the box , the median is indicated with a vertical line in the box , and the minimum and maximum except for the outliers are the ends of the whiskers . The outliers are indicated with open circles . P - values ( Kruskal - Wallis test , followed by Dunn ' s multiple comparison test ) are shown above .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1HSATIII lncRNA-dependent localization of m6A-related factors. Control and HSATIII knockdown HeLa cells were exposed to thermal stress (42°C for 2 h and recovery for 1 h at 37°C) and stained by HSATIII-FISH and immunofluorescence using an anti-WTAP antibody (B) or anti-YTHDC1 antibody (C). The nuclei were stained with DAPI. Scale bar: 10 µm.YTHDC1 accumulation within nSBs was arrested by METTL3 knockdown. HeLa cells were stained as described in Figure 1C. Scale bar: 10 µm. Box plot of relative intensities of YTHDC1 to HSATIII within nSBs in individual nuclei (n=30). The mean is indicated with X. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. P-values (Kruskal-Wallis test, followed by Dunn's multiple comparison test) are shown above.


Figure 2RNA - MS analysis of HSATIII lncRNAs digested by RNase T1 . Base peak chromatogram ( BPC , top panel ) and extracted chromatograms ( XIC , lower panels ) for the RNase T1 - digested fragments of the isolated HSATIII lncRNAs . XICs detecting for the negatively - charged ions of AAUGp tetramer without modification ( second panel ) , with one methylation ( third panel ) , and two methylations ( bottom panel ) . The sequence , mass to charge ratio ( m / z ) and charge state are indicated . NL ; normalization level .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2RNA-MS analysis of HSATIII lncRNAs digested by RNase T1. Base peak chromatogram (BPC, top panel) and extracted chromatograms (XIC, lower panels) for the RNase T1-digested fragments of the isolated HSATIII lncRNAs. XICs detecting for the negatively-charged ions of AAUGp tetramer without modification (second panel), with one methylation (third panel), and two methylations (bottom panel) . The sequence, mass to charge ratio (m/z) and charge state are indicated. NL; normalization level.


Figure 3Changes of nSB localization of m6A - related factors at various time points during and after thermal stress exposure . HeLa cells were exposed to thermal stress ( 42 ° C for 2 h ) and cultured at 37 ° C for the indicated periods . m6A - related factors were visualized by HSATIII - FISH and IF using anti - WTAP antibody ( D ) or anti - YTHDC1 antibody ( F ) . Nuclei were stained with DAPI . Scale bar : 10 µm . ( E , G ) Box plot of relative intensities of WTAP ( D ) and YTHDC1 ( F ) to HSATIII within nSB areas in individual nuclei . nSB areas were defined by binarized images of HSATIII - FISH . The mean is indicated with X . The first and third quartiles are the ends of the box , the median is indicated with a vertical line in the box , and the minimum and maximum except for the outliers are the ends of the whiskers . The outliers are indicated with open circles . P - values ( Kruskal - Wallis test , followed by Dunn ' s multiple comparison test ) are shown above ( n = 30 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Changes of nSB localization of m6A-related factors at various time points during and after thermal stress exposure. HeLa cells were exposed to thermal stress (42°C for 2 h) and cultured at 37°C for the indicated periods. m6A-related factors were visualized by HSATIII-FISH and IF using anti-WTAP antibody (D) or anti-YTHDC1 antibody (F). Nuclei were stained with DAPI. Scale bar: 10 µm. (E, G) Box plot of relative intensities of WTAP (D) and YTHDC1 (F) to HSATIII within nSB areas in individual nuclei. nSB areas were defined by binarized images of HSATIII-FISH. The mean is indicated with X. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. P-values (Kruskal-Wallis test, followed by Dunn's multiple comparison test) are shown above (n=30).


Figure 4HSATIII dependent sequestration of nucleoplasmic YTHDC1 ( G ) and WTAP ( H ) at four time points during thermal stress and recovery . The experiments same as A and C were performed at the indicated timepoints . The mean is indicated with X . The first and third quartiles are the ends of the box , the median is indicated with a vertical line in the box , and the minimum and maximum except for the outliers are the ends of the whiskers . The outliers are indicated with open circles . P - values ( Kruskal - Wallis test , followed by Dunn ' s multiple comparison test ) are shown above ( n = 40 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4HSATIII dependent sequestration of nucleoplasmic YTHDC1 (G) and WTAP (H) at four time points during thermal stress and recovery. The experiments same as A and C were performed at the indicated timepoints. The mean is indicated with X. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. P-values (Kruskal-Wallis test, followed by Dunn's multiple comparison test) are shown above (n=40).


Figure 5Scatter plot of LogFC of whole introns ( grey ) and HSATIII - target introns ( blue ) upon SRSF9 KD ( X - axis ) and HSATIII KD ( Y - axis ) . 51 , 469 and 548 introns are plotted as whole introns and previously - reported HSATIII - target introns , respectively . Pearson ' s correlation ( r ) is indicated above .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Scatter plot of LogFC of whole introns (grey) and HSATIII-target introns (blue) upon SRSF9 KD (X-axis) and HSATIII KD (Y-axis). 51,469 and 548 introns are plotted as whole introns and previously-reported HSATIII-target introns, respectively. Pearson's correlation (r) is indicated above.


Figure 6RT - qPCR validation of splicing of nascent RNAs . Splicing efficiency of nascent RNAs was quantified as the ratio of the spliced form to the intron retaining isoform ( IR ) form and normalized to the control . Control siRNA and HSATIII control oligonucleotide are indicated with ( - ) . Data are shown as the mean ± SD ( n = 3 ) ; p - values ( Tukey ' s multiple comparison test ) are shown above . Read maps represent RNA - seq data of nuclear poly ( A ) + RNAs ( upper panel , biological triplicate ) and m6A - RIP RNAs ( lower panel , biological duplicate ) in control and HSATIII KD cells . The exon numbers of the FAM214A gene are indicated below . The retaining intron and the m6A peak are marked by a red line and arrow , respectively . For the entire FAM214A region , see also Figure EV4B .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6RT-qPCR validation of splicing of nascent RNAs. Splicing efficiency of nascent RNAs was quantified as the ratio of the spliced form to the intron retaining isoform (IR) form and normalized to the control. Control siRNA and HSATIII control oligonucleotide are indicated with (-). Data are shown as the mean ± SD (n=3); p-values (Tukey's multiple comparison test) are shown above.Read maps represent RNA-seq data of nuclear poly (A)+ RNAs (upper panel, biological triplicate) and m6A-RIP RNAs (lower panel, biological duplicate) in control and HSATIII KD cells. The exon numbers of the FAM214A gene are indicated below. The retaining intron and the m6A peak are marked by a red line and arrow, respectively. For the entire FAM214A region, see also Figure EV4B.


Figure 7In vitro pulldown assay of RNP complexes with various repeat RNAs . In vitro RNA pulldown was performed using biotinylated ( GGAAU ) × 20 and other three repeat RNAs incubated in HeLa nuclear extract . The co - precipitated proteins were detected by western blotting using specific antibodies . ALYREF , a non - specific RNA - binding protein , was used as a control . Quantification of data in A . Relative co - precipitation efficiency of each protein was normalized by the maximum value . Data are shown as the mean ± SD ( n = 3 ) . In vitro pulldown assay with methylated GGAAU repeat RNA . In vitro RNA pulldown assay was performed using biotinylated GGAAU and GGm6Am6AU repeat RNAs as in A . Quantification of data in C . Relative co - precipitation efficiency of each protein was normalized by the maximum value . Data are shown as the mean ± SD ( n = 3 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7In vitro pulldown assay of RNP complexes with various repeat RNAs. In vitro RNA pulldown was performed using biotinylated (GGAAU)×20 and other three repeat RNAs incubated in HeLa nuclear extract. The co-precipitated proteins were detected by western blotting using specific antibodies. ALYREF, a non-specific RNA-binding protein, was used as a control. Quantification of data in A. Relative co-precipitation efficiency of each protein was normalized by the maximum value. Data are shown as the mean ± SD (n=3).In vitro pulldown assay with methylated GGAAU repeat RNA. In vitro RNA pulldown assay was performed using biotinylated GGAAU and GGm6Am6AU repeat RNAs as in A. Quantification of data in C. Relative co-precipitation efficiency of each protein was normalized by the maximum value. Data are shown as the mean ± SD (n=3).