Journal: Nucleic Acids Research

Article Title: Transient SUMOylation inhibition in human pre-adipocytes stably imprints a transcriptional beiging fate

doi: 10.1093/nar/gkag232

Figure Lengend Snippet: Analysis of the impact of transient SUMOylation inhibition in pre-adipocytes and rosiglitazone on gene expression in mature adipocytes. ( A ) Schematic representation of the experimental setup. Confluent human white pre-adipocytes were treated transiently for 48 h with TAK-981 prior to adipogenic induction on day 0. Differentiation took place as described in the “Materials and methods” section with rosiglitazone administration for the initial seven days (CTRL and TAK) or for the complete period of differentiation (Rosi and TAK + Rosi). ( B ) Validation of transient SUMOylation inhibition by SUMO2/3 western blot at the indicated time points. Volcano plots displaying differentially expressed genes (DEGs) (log 2 FC > 1 or log 2 FC < −1; padj < 0.05) resulting from TAK-981 treatment of pre-adipocytes ( C ) rosiglitazone treatment ( D ) and cotreatment with TAK-981+ rosiglitazone ( E ) compared to control cells. Data were collected in mature adipocytes 21 days post adipogenic induction. BATLAS DEGs are highlighted. ( F ) Heatmap visualization of the expression patterns of BATLAS genes across all conditions described in Fig. . Biological replicates were ordered by experimental condition (CTRL, Rosi, TAK, TAK + Rosi) to facilitate comparisons. ( G ) Dot plot showing the differential expression of UCP1 (RNA-seq) across comparative analyses between conditions. ( H ) Enrichment of the BATLAS signature within individual samples and condition assessed using ssGSEA. Statistical differences between experimental groups were evaluated using a one-way analysis of variance.

Article Snippet: Human white pre-adipocytes (hTERT A41hWAT-SVF, ATCC, CRL-3386) were maintained and differentiated based on previously established protocols [ ].

Techniques: Inhibition, Gene Expression, Biomarker Discovery, Western Blot, Control, Expressing, Quantitative Proteomics, RNA Sequencing

Journal: Nucleic Acids Research

Article Title: Transient SUMOylation inhibition in human pre-adipocytes stably imprints a transcriptional beiging fate

doi: 10.1093/nar/gkag232

Figure Lengend Snippet: Analysis of TAK-981 and rosiglitazone effects on UCP1 expression in pre-adipocytes and human adipose stem cells. ( A ) Time-course analysis of PPARG2 messenger RNA (mRNA) expression in hTERT A4hWAT-SVF cells under the conditions described in Fig. . Data were normalized to the expression of 18S and represent the mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $ \pm $\end{document} standard deviation from at least three independent experiments. ( B ) Time-course analysis of UCP1 mRNA expression in hTERT A4hWAT-SVF cells under the same conditions as in panel (A). Data were normalized to the expression of 18S and represent the mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $ \pm $\end{document} standard deviation from at least three independent experiments. ( C ) Analysis of PPARG2 mRNA expression in hASCs at day 12 post adipogenic induction under the same conditions as in panel (A). Data were normalized to the expression of 18S and represent the mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $ \pm $\end{document} standard deviation from at least three independent experiments. ( D ) Analysis of UCP1 mRNA expression in hASCs at day 12 post adipogenic induction under the same conditions as in panel (A). Data were normalized to the expression of 18S and represent the mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $ \pm $\end{document} standard deviation from at least three independent experiments. ( E ) Western blot analysis of UCP1 protein levels in hTERT A41hWAT-SVF cells at indicated days after adipogenic induction. Differentiated brown cells (hTERT A4hBAT-SVF) lysates were used as a positive control. ( F ) Western blot analysis of UCP1 protein levels in hASCs in the same conditions as in panel (D). ( G ) Experimental conditions used in panels (H) and (I). The upper section shows the conditions of continuous rosiglitazone treatment, as previously detailed in Fig. . The lower section indicates the introduction of a six-day gap between differentiation induction with rosiglitazone and the prolongation of the treatment. ( H ) RT-qPCR analysis of UCP1 mRNA expression in hTERT A4hWAT-SVF cells under the conditions in panel (G). Data were normalized to the expression of 18S and represent the mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $ \pm $\end{document} standard deviation from at least three independent experiments. ( I ) Western blot analysis of UCP1 protein levels in conditions indicated in panel (G). Statistical analyses: ONE-WAY-ANOVA with Tukey’s multiple comparison test.

Article Snippet: Human white pre-adipocytes (hTERT A41hWAT-SVF, ATCC, CRL-3386) were maintained and differentiated based on previously established protocols [ ].

Techniques: Expressing, Standard Deviation, Western Blot, Positive Control, Quantitative RT-PCR, Comparison

Journal: Nucleic Acids Research

Article Title: Transient SUMOylation inhibition in human pre-adipocytes stably imprints a transcriptional beiging fate

doi: 10.1093/nar/gkag232

Figure Lengend Snippet: Transient SUMOylation inhibition enhances PPARA/G transactivation and positive epigenetic changes at beiging enhancers. ( A ) In silico prediction of mobilized TFs based on RNA-seq data, showing the TFs mobilized upon treatment with TAK-981 and rosiglitazone, 22 days after adipogenic induction. Volcano plots showing the PPARA-target genes ( B ) and PPARG-target genes ( C ) activated in response to SUMOylation inhibition and rosiglitazone. DEGs were identified by comparing TAK-981+ rosiglitazone-treated cells to control cells, as described in Fig. and the “Materials and methods” section. ChIP-qPCR assessment of H3K27ac ( D ) and H3K27me3 ( E ) occurrences at selected beiging enhancers, as illustrated in . Error bars represent the average to the mean of two to four independent experiments. ChIP signals were normalized to inputs, and a gene desert at chromosome 12 (Gene des.) was used as a negative control. Statistical significance was calculated using a two-way ANOVA with Tukey’s multiple comparisons test. ChIP-qPCR assessment of H3K27ac ( F ) and H3K27me3 ( G ) occurrences at UCP1 enhancers, as illustrated in . Error bars represent the average to the mean of two to four independent experiments. ChIP signals were normalized to inputs, and a gene desert at chromosome 12 (Gene des.) was used as a negative control. Statistical significance was calculated using a two-way ANOVA with Tukey’s multiple comparisons test. ( H ) ChIP-qPCR assessment of PPARG occurrences at UCP1 enhancers, as illustrated in . Error bars represent the average to the mean of two independent experiments. ChIP signals were normalized to inputs and a gene desert at chromosome 12. Statistical significance was calculated using a two-way ANOVA with Tukey’s multiple comparison test. ( I ) Model illustrating the effects of transient SUMOylation inhibition and rosiglitazone treatment on heterochromatin factors, HATs, chromatin structure, and PPARG transactivation during the beige identity specification. Transient SUMOylation inhibition in pre-adipocytes stably primes the chromatin to facilitate the positive effect of rosiglitazone on the transactivation activity of beiging TFs like PPARs. The stable effect of SUMOylation inhibition on the epigenome indicates that SUMOylation in adiposе stem cells restricts cellular identity shift toward beiging in mature adipocytes.

Article Snippet: Human white pre-adipocytes (hTERT A41hWAT-SVF, ATCC, CRL-3386) were maintained and differentiated based on previously established protocols [ ].

Techniques: Inhibition, In Silico, RNA Sequencing, Control, ChIP-qPCR, Negative Control, Comparison, Stable Transfection, Activity Assay

Journal: Nucleic Acids Research

Article Title: Transient SUMOylation inhibition in human pre-adipocytes stably imprints a transcriptional beiging fate

doi: 10.1093/nar/gkag232

Figure Lengend Snippet: Stable mobilization of CEBPs and PPARG upon transient TAK-981 treatment in pre-adipocytes. ( A ) ATAC-seq experimental layout in hTERT A41hWAT-SVF pre-adipocytes. Time series analysis and hierarchical clustering of significant variations in chromatin accessibility at day 0 (D0), 12 h (12h), and day (D7) after TAK-981 treatment. ( C ) GOBP analysis of ATAC-seq clusters 2 and 6 revealed in panel (B). See and for an analysis of all clusters. ( D – F ) Volcano plots displaying the results of ATAC-seq inferred differential TF activity analyses performed at D0, 12h, and D7 after TAK-981 or DMSO treatment. Colored dots indicate significant differentially mobilized TFs (TAK-981 versus DMSO). Differential binding score >0.05 and pAdj <0.001. ( G ) Time series analysis inferred TF activity over time (TAK-981 versus DMSO) at D0, 12h, and D7 after TAK-981 treatment. ( H ) Western blot analysis of CEBPB SUMOylation in DMSO and TAK-981-treated cells. ( I ) Western blot analysis of CEBPB and PPARG in DMSO, rosiglitazone, TAK-981-treated cells and cotreated hTERT A41hWAT-SVF pre-adipocytes. ( J ) Western blot analysis of PPARG in DMSO, rosiglitazone, TAK-981-treated cells and cotreated hASCs.

Article Snippet: Human white pre-adipocytes (hTERT A41hWAT-SVF, ATCC, CRL-3386) were maintained and differentiated based on previously established protocols [ ].

Techniques: Activity Assay, Binding Assay, Western Blot

Journal: Nucleic Acids Research

Article Title: Transient SUMOylation inhibition in human pre-adipocytes stably imprints a transcriptional beiging fate

doi: 10.1093/nar/gkag232

Figure Lengend Snippet: Effect of transient SUMOylation inhibition in pre-adipocytes on cAMP-PKA-p38 signaling and adaptive thermogenesis in mature adipocytes. ( A ) Heatmap showing the stable upregulation of most genes involved in adaptive thermogenesis and cAMP-PKA-p38 signaling 22 days after adipogenic induction. Z -scores were calculated and plotted in GraphPad Prism. Treatments of pre-adipocytes were performed as shown in Fig. . ( B ) Western blot analysis of PKA substrates phosphorylation, assessed using a pan-PKA-target antibody, 22 days after adipogenic induction. TBP was used as a loading control. The asterisk (*) indicates substrates significantly affected by TAK-981 and/or rosiglitazone treatment. ( C ) Quantification of PKA substrate signals from panel (B). The signal for PKA substrates and TBP was quantified using Fiji software, with PKA substrate signals normalized to TBP. Error bars represent the standard deviation of four independent experiments. Student’s t -test was used to calculate P -values. ( D ) Western blot analysis of p-CREB, p-ATF1, p38, p-p38, and p-ATF2, 22 days after adipogenic induction. TBP was used as a loading control. ( E ) Quantification of p-CREB/ATF-1 signals from panel (D). Signals were normalized to TBP. Error bars represent the standard deviation of three independent experiments. Student’s t -test was used to calculate P -values. ( F ) Western blot analysis of p-CREB in the absence or presence of the PKA inhibitor RP-8-CPT-cAMPS. TBP was used as a loading control. Quantification of p-CREB, normalized to TBP, is showed in the lower panel. Error bars represent the standard deviation of two independent experiments. Quantification of p-p38 ( G ) and p-ATF2 ( H ) from experiment in panel (D). Signals were normalized to TBP for p-ATF2 and to p38 for p-p38. Error bars represent the standard deviation of three independent experiments. Student’s t -test was used to calculate P -values.

Article Snippet: Human white pre-adipocytes (hTERT A41hWAT-SVF, ATCC, CRL-3386) were maintained and differentiated based on previously established protocols [ ].

Techniques: Inhibition, Western Blot, Phospho-proteomics, Control, Software, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Transient SUMOylation inhibition in human pre-adipocytes stably imprints a transcriptional beiging fate

doi: 10.1093/nar/gkag232

Figure Lengend Snippet: Analysis of the impact of transient SUMOylation inhibition in pre-adipocytes and rosiglitazone on gene expression in mature adipocytes. ( A ) Schematic representation of the experimental setup. Confluent human white pre-adipocytes were treated transiently for 48 h with TAK-981 prior to adipogenic induction on day 0. Differentiation took place as described in the “Materials and methods” section with rosiglitazone administration for the initial seven days (CTRL and TAK) or for the complete period of differentiation (Rosi and TAK + Rosi). ( B ) Validation of transient SUMOylation inhibition by SUMO2/3 western blot at the indicated time points. Volcano plots displaying differentially expressed genes (DEGs) (log 2 FC > 1 or log 2 FC < −1; padj < 0.05) resulting from TAK-981 treatment of pre-adipocytes ( C ) rosiglitazone treatment ( D ) and cotreatment with TAK-981+ rosiglitazone ( E ) compared to control cells. Data were collected in mature adipocytes 21 days post adipogenic induction. BATLAS DEGs are highlighted. ( F ) Heatmap visualization of the expression patterns of BATLAS genes across all conditions described in Fig. . Biological replicates were ordered by experimental condition (CTRL, Rosi, TAK, TAK + Rosi) to facilitate comparisons. ( G ) Dot plot showing the differential expression of UCP1 (RNA-seq) across comparative analyses between conditions. ( H ) Enrichment of the BATLAS signature within individual samples and condition assessed using ssGSEA. Statistical differences between experimental groups were evaluated using a one-way analysis of variance.

Article Snippet: Human brown pre-adipocytes (hTERT-A41hBAT-SVF, ATCC, CRL-3385) were differentiated based on previously established protocols [ ].

Techniques: Inhibition, Gene Expression, Biomarker Discovery, Western Blot, Control, Expressing, Quantitative Proteomics, RNA Sequencing

Journal: Nucleic Acids Research

Article Title: Transient SUMOylation inhibition in human pre-adipocytes stably imprints a transcriptional beiging fate

doi: 10.1093/nar/gkag232

Figure Lengend Snippet: Analysis of TAK-981 and rosiglitazone effects on UCP1 expression in pre-adipocytes and human adipose stem cells. ( A ) Time-course analysis of PPARG2 messenger RNA (mRNA) expression in hTERT A4hWAT-SVF cells under the conditions described in Fig. . Data were normalized to the expression of 18S and represent the mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $ \pm $\end{document} standard deviation from at least three independent experiments. ( B ) Time-course analysis of UCP1 mRNA expression in hTERT A4hWAT-SVF cells under the same conditions as in panel (A). Data were normalized to the expression of 18S and represent the mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $ \pm $\end{document} standard deviation from at least three independent experiments. ( C ) Analysis of PPARG2 mRNA expression in hASCs at day 12 post adipogenic induction under the same conditions as in panel (A). Data were normalized to the expression of 18S and represent the mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $ \pm $\end{document} standard deviation from at least three independent experiments. ( D ) Analysis of UCP1 mRNA expression in hASCs at day 12 post adipogenic induction under the same conditions as in panel (A). Data were normalized to the expression of 18S and represent the mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $ \pm $\end{document} standard deviation from at least three independent experiments. ( E ) Western blot analysis of UCP1 protein levels in hTERT A41hWAT-SVF cells at indicated days after adipogenic induction. Differentiated brown cells (hTERT A4hBAT-SVF) lysates were used as a positive control. ( F ) Western blot analysis of UCP1 protein levels in hASCs in the same conditions as in panel (D). ( G ) Experimental conditions used in panels (H) and (I). The upper section shows the conditions of continuous rosiglitazone treatment, as previously detailed in Fig. . The lower section indicates the introduction of a six-day gap between differentiation induction with rosiglitazone and the prolongation of the treatment. ( H ) RT-qPCR analysis of UCP1 mRNA expression in hTERT A4hWAT-SVF cells under the conditions in panel (G). Data were normalized to the expression of 18S and represent the mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $ \pm $\end{document} standard deviation from at least three independent experiments. ( I ) Western blot analysis of UCP1 protein levels in conditions indicated in panel (G). Statistical analyses: ONE-WAY-ANOVA with Tukey’s multiple comparison test.

Article Snippet: Human brown pre-adipocytes (hTERT-A41hBAT-SVF, ATCC, CRL-3385) were differentiated based on previously established protocols [ ].

Techniques: Expressing, Standard Deviation, Western Blot, Positive Control, Quantitative RT-PCR, Comparison

Journal: Nucleic Acids Research

Article Title: Transient SUMOylation inhibition in human pre-adipocytes stably imprints a transcriptional beiging fate

doi: 10.1093/nar/gkag232

Figure Lengend Snippet: Transient SUMOylation inhibition enhances PPARA/G transactivation and positive epigenetic changes at beiging enhancers. ( A ) In silico prediction of mobilized TFs based on RNA-seq data, showing the TFs mobilized upon treatment with TAK-981 and rosiglitazone, 22 days after adipogenic induction. Volcano plots showing the PPARA-target genes ( B ) and PPARG-target genes ( C ) activated in response to SUMOylation inhibition and rosiglitazone. DEGs were identified by comparing TAK-981+ rosiglitazone-treated cells to control cells, as described in Fig. and the “Materials and methods” section. ChIP-qPCR assessment of H3K27ac ( D ) and H3K27me3 ( E ) occurrences at selected beiging enhancers, as illustrated in . Error bars represent the average to the mean of two to four independent experiments. ChIP signals were normalized to inputs, and a gene desert at chromosome 12 (Gene des.) was used as a negative control. Statistical significance was calculated using a two-way ANOVA with Tukey’s multiple comparisons test. ChIP-qPCR assessment of H3K27ac ( F ) and H3K27me3 ( G ) occurrences at UCP1 enhancers, as illustrated in . Error bars represent the average to the mean of two to four independent experiments. ChIP signals were normalized to inputs, and a gene desert at chromosome 12 (Gene des.) was used as a negative control. Statistical significance was calculated using a two-way ANOVA with Tukey’s multiple comparisons test. ( H ) ChIP-qPCR assessment of PPARG occurrences at UCP1 enhancers, as illustrated in . Error bars represent the average to the mean of two independent experiments. ChIP signals were normalized to inputs and a gene desert at chromosome 12. Statistical significance was calculated using a two-way ANOVA with Tukey’s multiple comparison test. ( I ) Model illustrating the effects of transient SUMOylation inhibition and rosiglitazone treatment on heterochromatin factors, HATs, chromatin structure, and PPARG transactivation during the beige identity specification. Transient SUMOylation inhibition in pre-adipocytes stably primes the chromatin to facilitate the positive effect of rosiglitazone on the transactivation activity of beiging TFs like PPARs. The stable effect of SUMOylation inhibition on the epigenome indicates that SUMOylation in adiposе stem cells restricts cellular identity shift toward beiging in mature adipocytes.

Article Snippet: Human brown pre-adipocytes (hTERT-A41hBAT-SVF, ATCC, CRL-3385) were differentiated based on previously established protocols [ ].

Techniques: Inhibition, In Silico, RNA Sequencing, Control, ChIP-qPCR, Negative Control, Comparison, Stable Transfection, Activity Assay

Journal: Nucleic Acids Research

Article Title: Transient SUMOylation inhibition in human pre-adipocytes stably imprints a transcriptional beiging fate

doi: 10.1093/nar/gkag232

Figure Lengend Snippet: Stable mobilization of CEBPs and PPARG upon transient TAK-981 treatment in pre-adipocytes. ( A ) ATAC-seq experimental layout in hTERT A41hWAT-SVF pre-adipocytes. Time series analysis and hierarchical clustering of significant variations in chromatin accessibility at day 0 (D0), 12 h (12h), and day (D7) after TAK-981 treatment. ( C ) GOBP analysis of ATAC-seq clusters 2 and 6 revealed in panel (B). See and for an analysis of all clusters. ( D – F ) Volcano plots displaying the results of ATAC-seq inferred differential TF activity analyses performed at D0, 12h, and D7 after TAK-981 or DMSO treatment. Colored dots indicate significant differentially mobilized TFs (TAK-981 versus DMSO). Differential binding score >0.05 and pAdj <0.001. ( G ) Time series analysis inferred TF activity over time (TAK-981 versus DMSO) at D0, 12h, and D7 after TAK-981 treatment. ( H ) Western blot analysis of CEBPB SUMOylation in DMSO and TAK-981-treated cells. ( I ) Western blot analysis of CEBPB and PPARG in DMSO, rosiglitazone, TAK-981-treated cells and cotreated hTERT A41hWAT-SVF pre-adipocytes. ( J ) Western blot analysis of PPARG in DMSO, rosiglitazone, TAK-981-treated cells and cotreated hASCs.

Article Snippet: Human brown pre-adipocytes (hTERT-A41hBAT-SVF, ATCC, CRL-3385) were differentiated based on previously established protocols [ ].

Techniques: Activity Assay, Binding Assay, Western Blot

Journal: Nucleic Acids Research

Article Title: Transient SUMOylation inhibition in human pre-adipocytes stably imprints a transcriptional beiging fate

doi: 10.1093/nar/gkag232

Figure Lengend Snippet: Effect of transient SUMOylation inhibition in pre-adipocytes on cAMP-PKA-p38 signaling and adaptive thermogenesis in mature adipocytes. ( A ) Heatmap showing the stable upregulation of most genes involved in adaptive thermogenesis and cAMP-PKA-p38 signaling 22 days after adipogenic induction. Z -scores were calculated and plotted in GraphPad Prism. Treatments of pre-adipocytes were performed as shown in Fig. . ( B ) Western blot analysis of PKA substrates phosphorylation, assessed using a pan-PKA-target antibody, 22 days after adipogenic induction. TBP was used as a loading control. The asterisk (*) indicates substrates significantly affected by TAK-981 and/or rosiglitazone treatment. ( C ) Quantification of PKA substrate signals from panel (B). The signal for PKA substrates and TBP was quantified using Fiji software, with PKA substrate signals normalized to TBP. Error bars represent the standard deviation of four independent experiments. Student’s t -test was used to calculate P -values. ( D ) Western blot analysis of p-CREB, p-ATF1, p38, p-p38, and p-ATF2, 22 days after adipogenic induction. TBP was used as a loading control. ( E ) Quantification of p-CREB/ATF-1 signals from panel (D). Signals were normalized to TBP. Error bars represent the standard deviation of three independent experiments. Student’s t -test was used to calculate P -values. ( F ) Western blot analysis of p-CREB in the absence or presence of the PKA inhibitor RP-8-CPT-cAMPS. TBP was used as a loading control. Quantification of p-CREB, normalized to TBP, is showed in the lower panel. Error bars represent the standard deviation of two independent experiments. Quantification of p-p38 ( G ) and p-ATF2 ( H ) from experiment in panel (D). Signals were normalized to TBP for p-ATF2 and to p38 for p-p38. Error bars represent the standard deviation of three independent experiments. Student’s t -test was used to calculate P -values.

Article Snippet: Human brown pre-adipocytes (hTERT-A41hBAT-SVF, ATCC, CRL-3385) were differentiated based on previously established protocols [ ].

Techniques: Inhibition, Western Blot, Phospho-proteomics, Control, Software, Standard Deviation