Fig. 6

mTOR is activated and interacts with the IKK complex upon genotoxic treatment. a Immunoblot analysis of ATM, TAK1, Akt and mTOR activation at different time points after bleomycin treatment of PSC27 cells. Total proteins of these factors and GAPDH were loading controls. b Representative IF images for mTOR phosphorylation (S2448) in PSC27 cells treated by bleomycin with or without RAD001 (50 nM) (blue, DAPI; red, γH2AX; green, p-mTOR). Cells were stained 7d after bleomycin treatment. Scale bars, 15 μm. c Immunoblot analysis of mTOR activation and downstream substrate (S6K1/4E-BP1) phosphorylation in cells treated by bleomycin with or without RAD001. Lysates were collected 7d after initiation of treatment, the same time point for sample acquirement of following assays. d DNA synthesis assay by BrdU incorporation for cells treated in different conditions, with positive ratios displayed in percentage. Right, representative images (green, BrdU; blue, DAPI). Scale bars, 15 μm. Data analyzed by Student’s t-test. e Stromal cells were stained in culture for SA-β-Gal, with results presented as positivity ratio. Right, representative images. Scale bars, 20 μm. Data analyzed by Student’s t-test. f Immunoblot examination of NF-κB pathway in stromal cells treated by bleomycin, with or without RAD001. Cell lysates were fractionated into cytoplasmic and nuclear extracts for comparative analysis. g Cells were transfected with a reporter construct that encodes NF-κB binding sites in the promoter region of a luciferase transgene. Signals were normalized as readings of firefly/renilla ratio. Data analyzed by Student’s t-test. h Co-IP assay to determine the interaction between mTOR and IKK complex. PSC27 cells were treated with bleomycin, and cell lysates were collected 7d later for IP analysis with anti-mTOR or anti-IKKα. IgG, control. Data in all bar plots are shown as mean ± SD and representative of 3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ^P > 0.05