Figure 4

Increased cellular ROS levels as a result of DEPP overexpression do not induce apoptosis. a) SH-EP/tetEGFP, SH-EP/tetDEPP and SH-EP/tetEYFP-DEPP cells were treated with 200 ng/ml doxy for 24 hours to induce DEPP expression. The protein expression of DEPP was determined by immunoblot. GAPDH served as loading control. b) SH-EP/tetEGFP, SH-EP/tetDEPP and SH-EP/tetEYFP-DEPP cells were treated with 200 ng/ml doxy for 24 hours and ROS levels were detected by live-cell imaging using MitoTrackerRed CM-H2XROS. Relative ROS staining was quantified using the Axiovert200M fluorescence microscope software. Each panel represents the mean ± s.e.m. of 60 to 70 cells; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05; **P < 0.025 compared to corresponding controls. c) SH-EP/tetDEPP and SH-EP/tetEYFP-DEPP cells were treated with 200 ng/ml doxy for 24 hours and SH-EP/FOXO3 cells with 50 nM 4OHT for 16 hours to induce FOXO3-mediated ROS accumulation. ROS levels were detected by live-cell imaging using MitoTrackerRed Red CM-H2XROS. Relative ROS staining was quantified using the Axiovert200M fluorescence microscope software. Each panel represents the mean ± s.e.m. of 10 to 20 cells. d) Cell lysates of SH-EP/tetEGFP, SH-EP/tetDEPP and SH-EP/tetEYFP-DEPP cells treated with 200 ng/ml doxy for 16 and 24 hours were subjected to immunoblot analyses using antibodies directed against DEPP, p66/SHC1 and phosphorylated pSer36-p66/SHC1. GAPDH served as loading control.