Fig. 6: RasGRP1 affects the growth of liver cancer cells.

a Huh7 cells with or without overexpressing lentivirus-delivered RasGRPP1 were cultured with 20 ng/ml EGF, 20 ng/ml FGF, B27 (2%) and the indicated concentration of IL-6 on low-attachment plates. (A representative image of three independent experiments is shown, Scalebar, 100 μm). b Diameter of hepatocellular carcinoma (HCC) progenitor cell-like spheroids shown in (a). (means ± SEM, paired t test, two-sided, *p = 0.03, *p = 0.0462 in sequence, n = 3 HCC progenitor cell-like spheroids). c qPCR analysis of RasGRP1, SOS1, SOS2, EGFR and EGF expression in HepG2 and Huh7 cells (means ± SEM, unpaired t test, two-sided, *p = 0.0482, *p = 0.0001, *p = 0.0001 in sequence, n = three independent experiments). d Analysis of Huh7 cell migration by scratch wound assays. Images were acquired at the indicated times (hours) (A representative image of three independent experiments is shown). e Quantitative analysis of analysis shown in (d) (means ± SEM, paired t test, one-side, *p = 0.0313, n = three independent experiments). f Cell Counting Kit-8 (CCK-8) assays were used to detect the role of RasGRP1 in Huh7 cells treated with 10 ng/ml EGF (means ± SEM, paired t test, two-side, *p = 0.0172, *p = 0.0033 in sequence, n = three independent experiments). g Immunoblot analysis of the indicated molecules in the lysates of Huh7 cells treated with 10 ng/ml EGF for the indicated times (minutes) (the image shown in g is a representative of five independent experiments. h The activation of AKT signalling as shown in (e) was quantified by determining the band intensity and was calculated as the ratio of the phosphorylated AKT level (Ser473) to total AKT level and normalised to that in unstimulated samples. (means ± SEM, Wilcoxon signed-rank test, two-sided, *p = 0.028, *p = 0.028, *p = 0.028 in sequence, n = five independent experiments). ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.