Figure 5.
Fibroblast Ligands and Signaling Pathway Inhibitors Promote Alveolosphere Growth In Vitro
(A, C, E, and G) qPCR of genes related to Fgf (A), Wnt (C), Tgf-β (E), and Bmp (G) signaling using cells obtained from direct and indirect epithelial-fibroblast co-culture assays before culture and day 7 and 14 (n = 3 wells for cultured cells and n = 3 animals for sorted cells). The data are representative of two independent experiments. For “FB; co-culture d7,” data for one sample were not available (n = 2). mRNA levels were normalized to that of Actb.
(B, D, F, and H) Alveolosphere size and colony-forming efficiency after adding mFgf7 or hFgf10 (B), CHIR99021 (D), hTGF-β1 or SB431542 (F), and mBmp4 or mNoggin (H). Sphere size was averaged for alveolospheres in each well. To calculate colony-forming efficiency, all colonies formed in each well were counted and the number of colonies was divided by 5 × 103 (epithelial cells plated). Data are shown for n = 3 wells and are representative of two independent experiments.
(I) Representative wells (epithelial cells; EGFP; entire Transwell stored in 24-well plates is shown) on day 14 after adding fibroblast ligand or signaling pathway inhibitor.
(J) Schematic for Fgf and Wnt signaling in fibroblast-epithelial interactions. Arrowheads indicate presumed activating signals.
Data represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA with Tukey's post hoc test or Student's t test). Scale bar: 1 mm. See also Figure S4.