Fig 1: Evaluation of AP contribution to adipogenesis in mgWAT. a, b Representative flow cytometry plots of CD31-/CD45- (Lin-), CD34+, CD29+, Sca-1+, CD24± populations from the stromal vascular fraction (SVF) of fixed murine inguinal/mgWAT at involution day 1 (a, left panel) or CD45-, CD34+, CD90+ cells within the SVF of human breast tissue (b, left panel). Representative images of unpurified cells in total SVF or FACS purified adipocyte precursors (APs) from mice (a, right panel) or humans (b, right panel) after culturing in adipogenic media for 8 days (a) or 13 days (b) and staining with Oil Red O. n = 3 biological repeats. c Schematic summarizing stages of adipogenesis. d Experimental strategy to measure EdU incorporation in adipocyte precursors by FACS (as in e and f), and in mature adipocytes (as in g, h). e Representative flow cytometry plots of EdU signal in APs (as defined in a) in virgin mice and at involution days 1 and 3. f The average percentage of EdU+ APs identified with flow cytometry as in e. n = 4 mice per time point, P = 0.51 for virgin vs. involution day 1 data. g, h Representative image (g) and quantification (h) of EdU immunofluorescence in Adipoq-Cre; mT/mG mouse injected with EdU according to the timeline in d. Note EdU+ (arrow) and EdU- nuclei (arrowhead) in mGFP+ adipocytes. White box indicates insets. n = 200–800 nuclei in each of four mice per group, P = 0.84. i Experimental strategy to inhibit adipogenesis during involution using GW9662, a pharmacological antagonist of PPAR?. j, k Representative image (j) and quantification (k) of MGs from GW9662- and vehicle-treated mice, stained with a perilipin antibody and DAPI. N = 8–17 fields in each of five mice per treatment group, P = 0.09. Scale bar is 50 µm in g and j. Data are mean ± SEM. Statistics were performed using a one-way ANOVA with Tukey’s multiple comparisons test (f) and a two-tailed unpaired t-test (h, k). ****P < 0.0001. Lin lineage, PPAR? peroxisome proliferator-activated receptor gamma, Inv involution, D day, DAPI 4',6-diamidino-2-phenylindole, EdU 5-ethynyl-2'-deoxyuridine, IF immunofluorescence, FACS fluorescence-activated cell sorting, SVF stromal vascular fraction