Fig. 2

FOXO3-induced autophagy depends on DEPP expression. a SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-10, −12 and −13 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 50 nM 4OHT for 5 h to activate FOXO3 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 < 0.025 compared to 4OHT-treated SH-EP/FOXO3-shCtr cells. Values are means ± s.e.m. b Immunoblot analyses of LC3-I/LC3-II, Gabarapl1, p62 and DEPP expression of SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-13 cells treated with 50 nM 4OHT for 8 h. GAPDH served as loading control. Densitometric analyses of LC3-II and p62 expression relative to GAPDH was done with the ImageJ 1.48 software. Untreated cells were set as 100%. 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 < 0.025. c SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-13 cells were treated with 50 nM 4OHT for 6 h and real time RT-PCR analyses of LC3 and Gabarapl1 expression were performed. 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