Fig. 1: Ablation of Cdkn2a maintains beige adipocyte characteristics after the rewarming period following cold exposure.

a Scheme of Cdkn2a^Ucp1 KO reporter mice used to conditionally knockout Cdkn2a in UCP1⁺ cells (Ucp1-Cre^ERT2; Rosa26R^RFP; Cdkn2a^fl/fl) and label with RFP. b Experimental procedure to track beige adipocytes in vivo. Two-month-old UCP1-RFP (control) and Cdkn2a^Ucp1 KO male mice were housed under cold conditions for 7 consecutive days to induce beige fat formation. Mice were then administered tamoxifen (TAM) and fed a chow diet at room temperature for 60 days. c Body weight of male control (n = 6) or Cdkn2a^Ucp1 KO mice (n = 8). d Immunofluorescence staining of UCP1 and RFP in IGW from control or Cdkn2a^Ucp1 KO male mice at day 0 and day 60 post withdrawal of cold stimulus. Scale bar, 50 μM. e Quantification of the percentage of RFP+ cells that express endogenous UCP1. f Representative H&E staining images of IGW from control or Cdkn2a^Ucp1 KO male mice at day 0 post withdrawal of cold stimulus. Scale bar, 100 μM. g Tile scan of IGW from control or Cdkn2a^Ucp1 KO male mice at day 60 post withdrawal of cold stimulus. Scale bar, 100 μM. h Quantification of adipocyte sizes in IGW from control or Cdkn2a^Ucp1 KO male mice at day 60 post withdrawal of cold stimulus (n = 3). i qPCR analysis of the mRNA expression of p16^Ink4a, p19^Arf and thermogenic genes in IGW from control or Cdkn2a^Ucp1 KO male mice at day 0 post withdrawal of cold stimulus (n = 6). j qPCR analysis of the mRNA expression of p16^Ink4a, p19^Arf and thermogenic genes in IGW from control or Cdkn2a^Ucp1 KO male mice at day 60 post withdrawal of cold stimulus (n = 6). k Western blot analysis of UCP1 in IGW from control or Cdkn2a^Ucp1 KO male mice at day 0 and 60 post withdrawal of cold stimulus (n  = 3). l Western blot analysis of TH in IGW from control or Cdkn2a^Ucp1 KO male mice at day 60 post withdrawal of cold stimulus (n = 3). p values were determined by two-tailed Student’s t test. Data are expressed as means ± SEM.