Figure 5

DEPP impairs catalase activity and sensitizes to H ₂ O ₂ -induced apoptosis. a + b) A catalase enzyme activity assay was performed in SH-EP/tetEGFP, SH-EP/tetDEPP, SH-EP/tetEYFP-DEPP cells treated with 200 ng/ml doxy (a) as well as of SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 cells treated with 50 nM 4OHT (b) for 24 hours. The catalase enzyme activity was calculated between treated and untreated cells (a). Shown are mean values ± s.e.m. of three independent experiments; statistical analysis was done with the Student’s unpaired t-test, ***P < 0.01. Cell lysates of SH-EP/tetEGFP, and SH-EP/tetDEPP cells treated with 200 ng/ml doxy for 24 hours as well as of 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 were subjected to immunoblot analyses using an antibody against catalase. GAPDH served as loading control. Quantification of catalase protein expression normalized to GAPDH (b) was done with the ImageJ 1.48 software. c) Immunoblot analysis of PPARG expression in SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 cells treated with 50 nM 4OHT for 24 hours was performed. GAPDH served as loading control. d) SH-EP/tetEGFP, SH-EP/tetDEPP and SH-EP/tetEYFP-DEPP cells (upper panel) were pre-treated with 200 ng/ml doxy for 24 hours and then incubated with 15 μM H₂O₂ for one hour. SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP clone-10, -12 and -13 (middle panel) and NB15/FOXO3-shCtr and NB15/FOXO3-shDEPP (bulk, lower panel) were treated with 25 μM H₂O₂ for 1 hour. PI-FACS analyses were performed to detect apoptotic cells. Shown are mean values ± s.e.m. of three independent experiments; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05, ***P < 0.01.