Figure 6.

Cell viability is decreased and apoptosis is potentiated in ATG4B silenced ES cells. (A) Following starvation for 48 h, cell viability of A673 cells transfected with shRNA-ATG4B plasmid or shRNA-control plasmid was measured by CCK8 assay in with or without 3-MA to block autophagy for 24 h. (B and C) After starvation for 48 h, the percentage of apoptotic cell and caspase-3 activity of A673 cells transfected with shRNA-ATG4B plasmid or shRNA-control plasmid was measured by flow cytometry or fluorometric assay with or without 3-MA to block autophagy for 24 h. (D) Western blot analysis of PUMA, cytosolic cytochrome c and GAPDH in A673 cells transfected with shRNA-ATG4B plasmid or shRNA-control plasmid after starvation for 2 h. Data are representative of three independent experiments (mean ± standard deviation). ^**P<0.01 as indicated. ES cells, Ewing sarcoma cells; shRNA, short hairpin RNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.