SMURF1 knockdown promotes differentiation in SDCs and decreased colony formation by sphere cells. (A) Adipogenesis culture as a measure for cellular differentiation. SDCs were grown in normal growth medium (top) or adipogenesis differentiation medium (bottom) for 7 days. Lipid droplet staining by Oil Red O was observed in SDCs with shSMURF1 knockdown grown in adipogenesis medium compared to shNeg or parental controls. Representative Oil Red O staining images shown are from the TR146 cell line. (B) Quantitation of Oil Red O staining in shNeg and shSMURF1 expressing SDCs cultured in adipogenesis medium from two representative cell lines. Data shown as mean ± SD, performed in triplicate experiments with multiple replicate wells. (C) Representative images of colony formation by SDCs in Matrigel anchorage-independent three-dimensional cultures supplemented with 20% FBS. (D) Summary of colony formation shown as mean number of colonies ± SE per 2,500 cells plated performed with three different cell lines as independent biological replicates (TR146, n = 2; SCC-58, n = 3; UMSCC-17B, n = 4). Statistical analysis was performed using two-tailed Student’s t-test with equal variance.