Fig. 4

Myofibroblast differentiation requires CD271/NGF activation. a Gene expression in whole SCAT progenitor cells treated for the indicated times with 5 ng/mL of TGFβ1 determined by RT-qPCR. The results are expressed as log2 gene expression ratio of treated to non-treated cells and are means ± s.e.m. of experiments performed on n = 4 (day 0 to day 3) to n = 12 (day 4) independent donors. Two-way ANOVA followed by Tukey’s multiple comparison test, *P < 0.05, **P < 0.01. b NGF mRNA levels in matched native progenitor cell subsets from n = 6 independent donors. One-way ANOVA, *P < 0.05. c Representative αSMA immunostaining (green) with DAPI (blue) on AT progenitor cells non-treated (control), treated with TGFβ1 in the presence or absence of either control antibody or anti-NGF antibody for 4 days. Scale bars: 50 µm. d Transcript levels of genes of interest were measured by RT-qPCR. The results are expressed as percentages of TGFβ-treated cells and are means ± s.e.m. of experiments performed on n = 6 independent donors. Two-way ANOVA followed by Bonferroni’s multiple comparison test, *P < 0.05, **P < 0.01. e Representative western blot analysis performed on progenitor cells treated with 5 ng/mL of TGFβ1 for 4 days in the presence or absence of gamma-secretase inhibitor (γSI, 25 µmol/L). Left panel: CD271 isoform (full length, the carboxy-terminal domain and intracellular domain) detection, right panel: GLI1, αSMA, and GAPDH detection. The CD271 intracellular domain protein, αSMA, and GLI1 expressions were quantified and normalized to GAPDH. The results are expressed as fold changes compared with control cells and are means ± s.e.m. of n = 3 (TGFβ1 + γSI) and n = 5 (control and TGFβ1) independent experiments. One-way ANOVA followed by Dunnett’s multiple comparison test, *P < 0.05. f Representative αSMA (red) and COLLAGEN 1 (red) immunostaining with DAPI (blue) in non-treated (control), TGFβ-treated cells with or without gamma-secretase inhibitor. Scale bars: 50 µm