Fig. 4

Oxidative stress activates FOXO3/DEPP and thereby triggers autophagy. a SH-EP/shCtr, SH-EP/shDEPP, and SH-EP/shFOXO3-17 cells were treated with 1 μM H₂O₂ for 1.5 h and real time RT-PCR analyses of DEPP expression were performed. Statistical analysis was done with the Student’s unpaired t-test, *P < 0.05 compared to untreated cells. Shown are mean values ± s.e.m of three independent experiments, each performed in triplicates. b SH-EP/shCtr, SH-EP/shDEPP as well as SH-EP/shFOXO3-17 cells were grown on ibidi μ-slide 8 well™ slides and transiently transfected with the pLIB-EYFP-LC3-iresPuro plasmid. Twenty-four hours after transfection the cells were treated with 1 μM H₂O₂ for 2.5 h or cultured under low serum conditions (0.5% FCS) for 8 h and analyzed by live-cell fluorescence microscopy with an Axiovert200M fluorescence microscope. Autophagy was quantified by counting LC3 dots per cell using the ImageJ 1.48 software. Values are representative results of three independent experiments; statistical analysis was done with the Student’s unpaired t-test, ***P < 0.01 compared to untreated controls. Values are means ± s.e.m. c Immunoblot analyses of LC3-I/LC3-II and DEPP expression of SH-EP/shCtr, SH-EP/shDEPP, and SH-EP/-shFOXO3-17 as well as NB15/shCtr, NB15/shDEPP, and NB15/shFOXO3 cells treated with the indicated concentrations of H₂O₂ for 6 h (upper panel) or cultured at low serum conditions (0.5% FCS) for 8 h (lower panel). GAPDH served as loading control