Fig. 5
The CM derived from miR-16-transfected fibroblasts displays reduced pro-tumorigenic properties. a Serum-starved A549 cells were stimulated for the indicated periods with the CM collected from CAF154-hTERT fibroblasts and diluted 1:2 in medium without serum. Western blot was performed to detect the activation of cMet, AKT, and ERK pathways. Actin is shown as a loading control. b A549 (upper panel) and LT73 (bottom panel) cells were stimulated with CM derived from CAF154-hTERT fibroblasts transfected with control miR-C or miR-16, and cell proliferation was measured 72 h later by CTG (**p = 0.0066, n = 5; *p = 0.0386). c A549 cells stimulated as in b were employed in wound-healing experiments. d A549 cells were stimulated with CM collected from CAF154-hTERT fibroblasts transfected with a siRNA specific for HGF. e HGF concentration in the CM of a panel of primary patient-derived fibroblasts. Arrows indicate CM media used in f and i (black arrows for high-concentration and green arrows for low-concentration of HGF). f Wound-healing experiments performed as in c with A549 cells stimulated with CM shown in e, with or without an HGF-neutralizing antibody (HGFi). g Results of migration experiments (f) after 24 h of migration (*p = 0.0355; ***p = 0.0004). h Time necessary to close half of the gap (T1/2) was plotted together with the concentration of HGF in the CM (R2 = 0.5647, slope = − 269.0 ± 83.51). i Migration experiments were performed with Calu-1 cells stimulated with a fibroblast-derived CM containing high levels of HGF (CAF206) and one with low levels of the cytokine (CAF190). j Results of migration experiments (i) after 12 h of migration in three independent experiments (*p = 0.0241)