Figure 4

Pro-survival CPC secretome and second messengers inhibited by RTKIs. Real-time qPCR and Western blot analysis of RTKI effects on growth factor expression: (a) 10 µM SM for 24 h reduced HGF mRNA (n = 3), with (b) protein expression also assessed. HGF expression was reduced by IM or SM, PDGF-A and Wnt2 expression by 5 µM IM, 0.5 µM SM or 5 µM ST at 7 days, shown by (c) mRNA (n = 3) and (d) protein expression. Real-time qPCR and Western blot analysis of CPC second messenger expression following RTKI exposures: Akt1 expression levels reduced by 5 µM IM, 0.5 µM SM or 5 µM ST at 7 days, with p38 reduced by 0.5 µM SM or 5 µM ST at 7 days, with (e) mRNA (n = 3) and (f) level of phosphorylated protein/total protein normalized to the loading control (n = 3). All data are mean ± SEM; significance was determined by ANOVA with Holm-Šídák post hoc test to identify differences (*p < 0.05; **p < 0.01; ***p < 0.001). Blots cropped to relevant data, with optical density normalized to the loading control protein; full blot images presented in Supplementary Fig. 6. Panel (d) shows composite gels (for HGF and Wnt2), with all quantification using loading controls in the same gel and lane.