Figure 8.

Loss of functional ATM leads to the proliferation defect in CAFs partially through down-regulating β-catenin and cell cycle regulation genes. (A) CAFs were treated with KU60019 (5 μM) for the indicated time. Immunoblotting analyses were performed to detect activity of GSK3β and β-Catenin. Total histone 3 functions as a loading control for nuclear proteins. (B) CAFs were treated separately or jointly with LY294002 (20 μM), U0126 (25 μM), and XAV939 (10 μM) for 24 h. Activated GSK3β and β-Catenin were determined using Immunoblotting analysis. Total histone 3 functions as a loading control for nuclear proteins. (C) CAFs were separately treated with or without inhibitors including LY294002 (20 μM), U0126 (25 μM), and XAV939 (10 μM) for 24 h. ChIP assay was done with an anti-β-catenin antibody for immunoprecipitation followed by PCR with c-MYC promoter-specific primers. (D) CAFs were separately treated with or without inhibitors including LY294002 (20 μM), U0126 (25 μM), and XAV939 (10 μM) for 24 h. Immunoblotting analysis was performed with the indicated antibodies. (E) The c-Myc gene in CAFs was knocked down by the shRNA, and the expression of c-Myc related genes was tested by Immunoblotting analysis with the indicated antibodies. β-Actin worked as loading control. (F) The growth curves of CAFs with or without c-Myc depletion was measured with the MTT assay (*, P < 0.05).