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. 2019 Mar 7;8(3):223. doi: 10.3390/cells8030223

Figure 6.

Figure 6

Conditioned medium (CM) from estrogen-stimulated CAFs up-regulates CTGF levels through FGFR1 signaling pathway in MDA-MB-231 cells. (a) Pairwise linear regressions of FGFR1 versus CTGF mRNA levels were performed on METABRIC dataset of 1904 breast tumor samples. Scatter plot shows positive correlation between FGFR1 and CTGF expression. (b–d) CTGF mRNA and protein levels in FGFR1 (WT) and FGFR1 (KO) MDA-MB-231 cells exposed for 3 h to CM from CAFs treated for 18 h with vehicle [CM/CAFs (+vehicle)], 10 nM E2 [CM/CAFs (+E2)] or 100 nM G-1 [CM/CAFs (+G-1)], or exposed to 25 nM FGF2, as positive control, evaluated by qPCR and western blot. In RNA experiments, values were normalized to the expression of 18S and shown as fold changes of CTGF mRNA expression upon CM from CAFs treated with E2 and G-1 respect to cells exposed to CM from CAFs treated with vehicle. Each column represents the mean ±SD of three independent experiments performed in triplicate. (e–g) Up-regulation of CTGF protein expression in MDA-MB-231 cells exposed for 3 h to CM from CAFs treated for 18 h with vehicle [CM/CAFs (+vehicle)], 10 nM E2 [CM/CAFs (+E2)], or 100 nM G-1 [CM/CAFs (+G-1)] was no longer observed in the presence of 1 μM FGFR1 inhibitor PD173074, 10 μM MEK inhibitor PD98059 or 100 nM PI3K inhibitor Wortmannin (WM). β-actin served as loading control. Side panels show densitometric analysis of the blots normalized to the loading controls. Immunoblots shown are representative of three independent experiments. (**) indicates p < 0.01 and (*) indicates p < 0.05.