Fig 2: RA promotes brown adipogenesis in vitro through activating VEGFA/VEGFR2 signaling. Adipose-derived SVCs were cultured on Matrigel-coated plates in endothelial basal medium medium supplemented with either DMSO or RA for 6 days, then switched into DMEM medium supplemented with insulin and T3 for 4 days, and further cultured with insulin for 8 days. (a) PDGFRa+ cells at D2. (b) Representative images of RA-treated cells at D12, D16 and D18. (c) Representative image of D18 control cells and Vegfr2 knockout cells treated with DMSO or RA (upper: bright field; lower: Oil-Red-O). (d) MTT assay of WT or Vegfr2 KO PDGFRa+ cells treated with or without 1 µm RA. (e) mRNA levels in D18 cells. (f) Oxygen consumption rate of cells. Data presented are mean±s.e.m., n=6, *P<0.05. Scale bar=200 µm.

Fig 3: RA promotes in vitro angiogenesis of SVCs via VEGFA/VEGFR2 signaling. (a) VEGFA protein content in the medium cultured with adipose-derived SVCs for 2 days, and cells were treated with different doses of RA. (b) Quantification of VEGFA protein, data presented were normalized according to the content of a non-specific band. (c) The expression of Vegfa in SVCs treated with RA for 2 days. (d) Representative images (scale bar=100 µm), and (e) Vegfa expression of adipose tissue derived control or Vegfr2 knockout SVCs cultured on matrigel-coated plates in endothelial basal medium for 2 days with or without 1 µm RA (same doses for other in vitro studies if unstated). (f) Putative retinoic acid response elements (pRARE) on the Vegfa promoter are indicated by letters, some of which share several nucleotides; closely located pRARE were analyzed using one pair of primers. (g) SVCs were treated with RA or DMSO for 4 h, and binding of RAR and retinoid X receptor (RXR) to pRAREs were analyzed by ChIP. (h) The pRARE sites with altered RAR/RXR binding due to RA were chosen. These sites on a Vegfa promoter luciferase reporter plasmid were mutated. The luciferase activity of WT and mutated Vegfa promoter reporter plasmids were analyzed after 4 h of RA treatment. Data presented are mean±s.e.m., n=6, *P<0.05.