Figure 3

Knockdown of DEPP reduces FOXO3-mediated apoptosis. a) SH-EP/FOXO3 (SH-EP/FOXO3-shDEPP clone-10, -12 and -13; left panel) and NB15/FOXO3 (NB15/FOXO3-shDEPP bulk; right panel) cells were infected with vectors coding for DEPP-specific shRNA. Knockdown efficiency was verified by immunoblot (upper panel) and quantitative RT-PCR (lower panel). Cells were treated with 50 nM 4OHT to induce DEPP expression. b) SH-EP/FOXO-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 as well as NB15/FOXO3-shCtr and NB15/FOXO3-shDEPP (bulk) cells were treated with 50 nM 4OHT for the indicated time points. PI-FACS analyses were performed to detect apoptotic cells. Shown is the mean ± s.e.m. of three independent experiments; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05; **P < 0.025; ***P < 0.01 compared to corresponding controls. c) ROS accumulation was detected using MitoTrackerRed CM-H2XROS by live-cell imaging of SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 as well as of NB15/FOXO3-shCtr and NB15/FOXO3-shDEPP (bulk) cells treated with 50 nM 4OHT for 4 and 16 hours and for 12 and 48 hours respectively to induce FOXO3 dependent biphasic ROS accumulation. Relative ROS staining was quantified using the Axiovert200M fluorescence microscope software (Axiovision 4.6). Each panel represents the mean ± s.e.m. of 50 to 80 cells; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05; **P < 0.025 compared to corresponding controls. d) Immunoblot analysis of p66/SHC1 and phosphorylated pSer36-p66/SHC1 expression in SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 cells treated with 50 nM 4OHT for the indicated time points. GAPDH served as loading control.