Fig. 6

MAZ promotes bone metastasis of PCa cells dependent on KRas-mediated RalGEFs signaling a, b Western blot analysis of p-ERK, activated RalA, p-AKT(S473), p-AKT(T308) in the indicated cell lines. The total ERK, total RalA, and α-tubulin were used as a loading control. c The RalGEFs pathway inhibitor RBC8 reduced migration and invasive abilities of MAZ-overexpression PC-3 cells, but the AKT, PI3k and ERK pathway inhibitors API2, BMK120 and FR180204 did not. The dose and duration of use of these three inhibitors were 3 μM (RBC8) for 48 h, 1 uM (API2) for 24 h, 1 uM (BMK120) for 24 h and 50 uM (FR180204) for 72 h, respectively. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05.d Representative BLIs signal of bone metastasis of a mouse from the indicated groups of mice at 12 mins and 8 weeks respectively. RBC8 was dissolved in DMSO and injected i.p. daily at 50 mg/kg/day which initiated 24 h after inoculation of PC-3 cells in the left cardiac ventricle. Mice in the control group were injected with the appropriate amount of solvent per day. e Representative radiographic images of bone metastases in the indicated mice (arrows indicate osteolytic lesions). f Representative H&E-stained sections of tibias from the indicated mouse. g The sum of bone metastasis score for each mouse in tumor-bearing mice inoculated with MAZ-overexpression (n = 10) and MAZ-overexpression with RBC8 (n = 10) cells. *P < 0.05. h Quantification of the BLI signaling in the MAZ-overexpression and MAZ-overexpression with RBC8 groups at 25, 35, 45, 55 and 65 day respectively. *P < 0.05. T-test was not performed on days 55 and 65 owing to the death of a large number of mice. i Kaplan-Meyer analysis of mouse survival in the MAZ-overexpression and MAZ-overexpression with RBC8 groups (Gehan-Breslow-Wilcoxon Test). j Kaplan-Meyer analysis of mouse bone metastasis-free survival in the MAZ-overexpression and MAZ-overexpression with RBC8 groups (Gehan-Breslow-Wilcoxon Test)