Isolation and Characterization of ADSCs

ADSCs were isolated from VAT of 6-wk-old WT, K43M, R31C, and K43M;Runx1^fl/fl;CRE^- mice and grown in vitro (Hou et al., 2018 (link)). Epididymal white adipose tissue (eWAT) from male mice and gonadal white adipose tissue (gWAT) from female mice were used in this study. ADSCs were isolated from the adipose stromal vascular fraction (SVF) according to a published procedure (Permana et al., 2004 (link)). Briefly, freshly collected eWAT and gWAT from normal chow-fed animals were digested in 1 x HBSS containing 1 mg/mL type I collagenase (Millipore Sigma, United States) at 37°C for 1 h. The suspension was filtered through a sterile 100 µm nylon mesh (Thermofisher Scientific) and centrifuged at 1,000 rpm for 5 min. The pellet fraction containing pre-adipocytes was washed 2 times with PBS +2% FBS and then incubated with red blood cell lysis buffer to remove red blood cells. Cells were then filtered through a sterile 70 µm nylon mesh (Thermofisher Scientific) and centrifuged at 1,000 rpm for 5 min and counted. Fractions of 3×10⁵ cells were used for subsequent staining and analysis using flow cytometry.