ROR1 Is Expressed in Human Breast Cancer and Associated with Enhanced Tumor-Cell Growth (original) (raw)
Figure 5
ROR1 interacts with CK1ε to activate PI3K/AKT, leading to activation of CREB.
(A) MDA-MB-231tumor cells with or without ROR1 expression were examined for phosphorylated AKT at ser-473 (p-AKT) or total AKT (t-AKT). (B) MDA-MB-231 cells were cultured with (+) or without (−) LY294002 and examined for p-AKT, t-AKT, p-CREB, or t-CREB by immunobot analysis after 16 hours. (C) MDA-MB-231 cells (Ct-shRNA) or cells silenced for ROR1 (ROR1-shRNA) were cultured with different concentrations of LY294002 and monitored for cell growth after 48 hours using the WST-8 assay. The graphs depict the mean numbers of viable cells, ± S.E.M of triplicate samples, which are representative of three independent experiments. *P<0.05 and ***P<0.001). (D–E) MCF7 control cells (Ct-Vector) or cells made to express ROR1 (pCDNA3-ROR1) were examined for protein expression (D) or (E) monitored for growth over 4 days using the WST-8 assay. The graph depicts the mean numbers of viable cells ± S.E.M. over time in each of these cell lines, as indicated in the legend at the top of the figure, which are representative of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 by Student's t test. (F) Protein lysates from MDA-MB-231 cells or cells silenced for ROR1 were immunoprecipitated (IP) with ROR1 antibody. The bound immunoprecipitated products and whole cell lysate (WCL) were probed by the antibodies indicated in the Probe column. (G–H) MDA-MB-231 cells were treated without (−) or with CK1ε siRNA (CK1ε-siRNA) or control siRNA (Ct-siRNA) for 72 hours and examined for protein expression (G) or cell growth (H). The height of each bar in the graph F provides the mean number of viable cells ± S.E.M., which are representative of more than three independent experiments. P indicates the statistical significance.