Figure 5

Loss of BRAT1 leads to inhibition of Akt activity and Akt activation by SC79 partially restores BRAT1 knockdown cells. (A) Control (NC) and BRAT1 knockdown (sh3) HeLa cells were cultured in DMEM media with 10% serum (M). To examine serum-induced activation, cells were cultured in DMEM media without serum (SF and SFS) for 24 h and then continuously cultured with 10% serum (SFS) or without serum (SF) for 1 h. Protein extracts were blotted using indicated antibodies for phospho- or whole proteins. Actin protein was used as internal control. (B) Cells were cultured with (media) or starved for 24 h and then serum was added into these media. SC79 (5 μg/ml) were treated at 2, 10 μg/ml for 30 min and then total extracts were analyzed for phosphorylation and expression of indicated protein by immunoblotting. (C) Cells were cultured with or without SC79 for indicated time and then cell proliferation was measured using MTT assay. More SC79 was added at D2 and D4 (down arrow). (D) Cells were cultured with or without SC79 (5 μg/ml) for 24 h and stained with mitosox for 10 min. Flow cytometry was conducted to detect mitosox positive cells (gated) from control and BRAT1 knockdown cells. Quantified flow cytometry data (left) were representative of two independent experiments (right). *Student’s t-test: p < 0.05.