Journal: bioRxiv

Article Title: SUMOylation Restricts Adaptive Thermogenesis by Suppressing Beiging Gene Networks

doi: 10.1101/2025.03.07.642034

Figure Lengend Snippet: (A) Hypothesis: SUMOylation inhibits the cAMP-PKA-p38 signaling pathway, limiting adaptive thermogenesis. (B) Heatmap showing the stable upregulation of most genes involved in adaptive thermogenesis and cAMP-PKA-p38 signaling in hASCs treated with transient SUMOylation inhibition and rosiglitazone, 22 days after adipogenic induction. (C) Western blot analysis of PKA substrates phosphorylation, assessed using a pan-PKA-target antibody. TBP was used as a loading control. The asterisk (*) indicates substrates significantly affected by TAK-981 and/or rosiglitazone treatment. (D) Quantification of PKA substrate signals from (C) . 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. (E) Western blot analysis of the phosphorylation of CREB, ATF1, p38, and ATF2. TBP was used as a loading control. (F-G-H) Quantification of the signals from (E) : p-CREB (F), p-p38 (G) and p-ATF2 (H). Signals were normalized to TBP for p-CREB and p-ATF2, and to p-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: The following antibodies were used: UCP1, abcam, ab209483; PKA phosphor-substrates, Cell Signaling 9624; p-CREB / p-ATF1, Cell Signaling 9198; p38 MAPK, Cell Signaling 9212; p-p38 MAPK, Cell Signaling 9211; p-ATF2, Cell Signaling 24329; SUMO2/3, abcam, ab81371 and TBP, Protein Tech, 22006-1-AP).

Techniques: Inhibition, Western Blot, Control, Software, Standard Deviation

Journal: bioRxiv

Article Title: SUMOylation Restricts Adaptive Thermogenesis by Suppressing Beiging Gene Networks

doi: 10.1101/2025.03.07.642034

Figure Lengend Snippet: (A) Hypothesis: SUMOylation inhibits the cAMP-PKA-p38 signaling pathway, limiting adaptive thermogenesis. (B) Heatmap showing the stable upregulation of most genes involved in adaptive thermogenesis and cAMP-PKA-p38 signaling in hASCs treated with transient SUMOylation inhibition and rosiglitazone, 22 days after adipogenic induction. (C) Western blot analysis of PKA substrates phosphorylation, assessed using a pan-PKA-target antibody. TBP was used as a loading control. The asterisk (*) indicates substrates significantly affected by TAK-981 and/or rosiglitazone treatment. (D) Quantification of PKA substrate signals from (C) . 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. (E) Western blot analysis of the phosphorylation of CREB, ATF1, p38, and ATF2. TBP was used as a loading control. (F-G-H) Quantification of the signals from (E) : p-CREB (F), p-p38 (G) and p-ATF2 (H). Signals were normalized to TBP for p-CREB and p-ATF2, and to p-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: The following antibodies were used: UCP1, abcam, ab209483; PKA phosphor-substrates, Cell Signaling 9624; p-CREB / p-ATF1, Cell Signaling 9198; p38 MAPK, Cell Signaling 9212; p-p38 MAPK, Cell Signaling 9211; p-ATF2, Cell Signaling 24329; SUMO2/3, abcam, ab81371 and TBP, Protein Tech, 22006-1-AP).

Techniques: Inhibition, Western Blot, Control, Software, Standard Deviation

Journal: bioRxiv

Article Title: SUMOylation Restricts Adaptive Thermogenesis by Suppressing Beiging Gene Networks

doi: 10.1101/2025.03.07.642034

Figure Lengend Snippet: (A) Hypothesis: SUMOylation inhibits the cAMP-PKA-p38 signaling pathway, limiting adaptive thermogenesis. (B) Heatmap showing the stable upregulation of most genes involved in adaptive thermogenesis and cAMP-PKA-p38 signaling in hASCs treated with transient SUMOylation inhibition and rosiglitazone, 22 days after adipogenic induction. (C) Western blot analysis of PKA substrates phosphorylation, assessed using a pan-PKA-target antibody. TBP was used as a loading control. The asterisk (*) indicates substrates significantly affected by TAK-981 and/or rosiglitazone treatment. (D) Quantification of PKA substrate signals from (C) . 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. (E) Western blot analysis of the phosphorylation of CREB, ATF1, p38, and ATF2. TBP was used as a loading control. (F-G-H) Quantification of the signals from (E) : p-CREB (F), p-p38 (G) and p-ATF2 (H). Signals were normalized to TBP for p-CREB and p-ATF2, and to p-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: The following antibodies were used: UCP1, abcam, ab209483; PKA phosphor-substrates, Cell Signaling 9624; p-CREB / p-ATF1, Cell Signaling 9198; p38 MAPK, Cell Signaling 9212; p-p38 MAPK, Cell Signaling 9211; p-ATF2, Cell Signaling 24329; SUMO2/3, abcam, ab81371 and TBP, Protein Tech, 22006-1-AP).

Techniques: Inhibition, Western Blot, 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: 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: The following antibodies were used: UCP1, abcam, ab209483; PKA phospho-substrates, Cell Signaling, 9624; p-CREB/p-ATF1, Cell Signaling, 9198; p38 MAPK, Cell Signaling, 9212; p-p38 MAPK, Cell Signaling, 9211; p-ATF2, Cell Signaling, 24329; SUMO2/3, abcam, ab81371; PPARG, Cell signaling, 2443; and TBP, Protein Tech, 22006-1-AP or abcam, 282715; γ-tubulin, Sigma, T5326: CEBPB, Santa Cruz, sc-7962 quantifications were performed using FiJi [ ].

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

Journal: bioRxiv

Article Title: SUMOylation Restricts Adaptive Thermogenesis by Suppressing Beiging Gene Networks

doi: 10.1101/2025.03.07.642034

Figure Lengend Snippet: (A) Hypothesis: SUMOylation inhibits the cAMP-PKA-p38 signaling pathway, limiting adaptive thermogenesis. (B) Heatmap showing the stable upregulation of most genes involved in adaptive thermogenesis and cAMP-PKA-p38 signaling in hASCs treated with transient SUMOylation inhibition and rosiglitazone, 22 days after adipogenic induction. (C) Western blot analysis of PKA substrates phosphorylation, assessed using a pan-PKA-target antibody. TBP was used as a loading control. The asterisk (*) indicates substrates significantly affected by TAK-981 and/or rosiglitazone treatment. (D) Quantification of PKA substrate signals from (C) . 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. (E) Western blot analysis of the phosphorylation of CREB, ATF1, p38, and ATF2. TBP was used as a loading control. (F-G-H) Quantification of the signals from (E) : p-CREB (F), p-p38 (G) and p-ATF2 (H). Signals were normalized to TBP for p-CREB and p-ATF2, and to p-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: The following antibodies were used: UCP1, abcam, ab209483; PKA phosphor-substrates, Cell Signaling 9624; p-CREB / p-ATF1, Cell Signaling 9198; p38 MAPK, Cell Signaling 9212; p-p38 MAPK, Cell Signaling 9211; p-ATF2, Cell Signaling 24329; SUMO2/3, abcam, ab81371 and TBP, Protein Tech, 22006-1-AP).

Techniques: Inhibition, Western Blot, Control, Software, Standard Deviation

Journal: bioRxiv

Article Title: SUMOylation Restricts Adaptive Thermogenesis by Suppressing Beiging Gene Networks

doi: 10.1101/2025.03.07.642034

Figure Lengend Snippet: (A) Hypothesis: SUMOylation restricts the transactivation activity of PPARA/G either directly or indirectly through the cAMP-PKA-p38 axis. (B) In silico prediction based on RNA-seq data, showing the transcription factors mobilized upon treatment with TAK-981 and rosiglitazone. (C-D) Volcano plots showing the PPARA-target genes (C) and PPARG-target genes (D) activated in response to SUMOylation inhibition and rosiglitazone. Differentially expressed genes were identified by comparing TAK-981 + Rosiglitazone-treated cells to Rosiglitazone-treated cells, as described in methodes. (E-I) Schematics of ChIP-qPCR regions and primer pairs used in the experiments depicted in J and K. (J-K) ChIP-qPCR assessment of H3K27ac (J ) and H3K27me3 (K) occurrences at beiging enhancers, as illustrated in (E-I). 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 (CTRL) was used as a negative control. Statistical significance was calculated using a one-sided, homoscedastic Student’s t-test, with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: The following antibodies were used: UCP1, abcam, ab209483; PKA phosphor-substrates, Cell Signaling 9624; p-CREB / p-ATF1, Cell Signaling 9198; p38 MAPK, Cell Signaling 9212; p-p38 MAPK, Cell Signaling 9211; p-ATF2, Cell Signaling 24329; SUMO2/3, abcam, ab81371 and TBP, Protein Tech, 22006-1-AP).

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