Fig. 4

The effect of ROR2 in proliferation is mediated by inhibition of Akt phosphorylation. A Expression of ROR2 in both A375 and UACC903 cells inhibits Akt-T308 and -S473 phosphorylation. The graphs show the mean ± S.D. of p-Akt levels normalized to total Akt and expressed as the fold change (FC) relative to the corresponding control cell line (empty or scramble). Statistical significance was tested by a one-tailed Student’s T-Test (n = 3). B ROR2 expression in A375 prevents Akt phosphorylation by Wnt5a. The cells were incubated with Wnt5a conditioned media for 30 min and Akt-S473 phosphorylation was assessed as in A. Statistical significance was tested by ANOVA (n = 3). C Silencing of ROR2 increased Akt phosphorylation in M2 and MeWo cells. Akt phosphorylation upon ROR2 silencing in M2 and MeWo cells was assessed as in A. Statistical significance was tested by a one-tailed Student’s T-Test (M2) or ANOVA (MeWo) (n = 3). D ROR2 regulates phosphorylation of S6K. The graphs show the mean ± S.D. of the p-S6K levels normalized to total S6K and expressed as the fold change (FC) relative to the corresponding control cell line (Empty or scramble). Statistical significance was tested by a one-tailed Student’s T-Test (n = 3). E ROR2 increases PTEN expression. Western blot analysis of PTEN expression in A375-Empty, A375-ROR2, M2-scramble and M2-shROR2 cells. Bar graph shows the mean ± S.D. (from three independent experiments) of PTEN levels normalized to the loading control (GAPDH) and expressed as the fold change (FC) relative to control cells. The blots displayed are representative of three independent experiments. Statistical significance was tested by one-tailed Student’s T-Test, n = 3. F LY294002 (LY) inhibited the effects of ROR2 in cell proliferation. The cells were grown for two days in the presence or absence of 20 μM LY294002. Graphs show the mean ± S.D. of the number of cells. Statistical significance was tested by a one-tailed Student’s T-Test (n = 3). G LY294002 prevented the accumulation of cells in G2/M in M2-shROR2 cells. M2-scramble and M2-shROR2 cells were incubated with 20 μM LY294002 for 48 h and analyzed by flow cytometry. Bar graph shows the mean ± S.D. of the percentage of cells in each phase of the cell-cycle. Statistical significance was tested by a one-tailed Student’s T-Test. H LY294002 counteracts the increase in cell-cycle markers induced by ROR2 silencing. The cells were treated with 20 μM LY for 12 h. The graphs show the mean ± S.D. of each protein’s levels in cells transduced with ROR2 shRNA normalized to the corresponding loading control and expressed as the fold change (FC) relative to scramble cells. Statistical significance was tested by ANOVA (n = 3). *: p < 0.01, **: p < 0.001, ***: p < 0.0001, n = 3. n.s.: no significant