Fig. 1: TGFB1 promotes stemness in the OSE.

a Primary (left; n = 3) and secondary (right; n = 5) sphere-forming capacity of mOSE cells treated with TGFB1 (10 ng/mL). Data points represent the average number of spheres per 4 fields of view for each replicate after 14 days of spheroid culture. b Left: Phase contrast images of control and TGFB1-treated spheroids (left). Scale bar = 100 μm. Right: Relative sizes of spheroids in control and TGFB1 conditions (primary spheroids n = 3; secondary spheroids n = 4). Each point on the plot represents the average size of spheroids from 4 fields of view for 4 separate wells. c Primary sphere-forming capacity of human OSE treated with TGFB1 (n = 4). hOSE spheroids were cultured in methylcellulose to prevent aggregation. Spheroids were cultured for 28 days. Scale bar = 100 μm. d Fold change values for a panel of putative stem cell markers in mOSE treated with TGFB1 for 4 days. e Relative protein quantifications of CD44 throughout a time course of TGFB1 treatment in mOSE (n = 3). Quantifications represent Western blot pixel densitometry, normalized to B-actin and scaled to the mean intensity in untreated samples. A representative blot is included in Supplemental Fig. 2b. f Immunofluorescence co-stain of CD44 and Ki67 in control and TGFB1-treated spheroids. White arrows highlight CD44 + cells in control spheroids. Scale bar = 100 μm, inlet scale bar = 15 μm. Separate channels are shown in Supplementary Fig 3g. Primary sphere-forming capacity of CD44- and CD44 + mOSE cells (n = 3). All boxplots show median value (horizontal black line), estimated 25th and 75th percentiles, and whiskers represent 1.5 times the interquartile range. Linear regression models were used for all statistical tests. *p < 0.05, **p < 0.01.