Fig. 3

Characterization of Car4-positive preadipocytes. (a) Representative FACS plots (left panes) and quantification (right panel) of Car4-positive cells among APCs in the SVF from inguinal WAT of young (top) or aged (bottom) mice housed under ambient temperature (23℃, left) or cold exposure (10℃, middle). For the quantification, each dot represents an individual biological replicate. Data are presented as mean ± SEM (n = 3 per group). *p < 0.05 by one-tailed Student’s t-test. (b) Representative images of RNA in situ hybridization of the inguinal WAT from young (top) or aged (bottom) mice housed at 23℃ (left) or 10℃ (right). (c) Principal component analysis (PCA) score plots of primary Car4-positive cells, Dpp4-positive cells, and Car4⁻Dpp4⁻ cells derived from inguinal WAT of pooled young or aged mice housed at 23℃ or 10℃. (d) Representative FACS plots showing EdU incorporation in Car4-positive preadipocytes from aged mice housed at 23 °C (left) or 10 °C (right). SVF cells were labeled with EdU as described in Methods. Due to the need to pool cells from multiple mice per sample and technical variability in the assay, robust statistical analysis was precluded. This observation, however, is consistent with the Mki67 upregulation shown in Suppl. Fig. S3a. Numbers in plots indicate the percentage of cells in the respective gates. (e) Quantification of APCs proliferation with or without Car4-positive cells. n = 5–8 per groups.