Fig. 1

DEPP expression induces autophagy in human neuroblastoma cells. a SH-EP/tetCtr and SH-EP/tetDEPP 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 200 ng/ml doxy for 5 h to induce DEPP expression 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 untreated cells. Values are means ± s.e.m. b SH-EP/tetEGFP and SH-EP/tetDEPP cells were treated with 200 ng/ml doxy for 8 h and LC3-I/LC3-II, p62, and DEPP expression were assessed by immunoblot analyses. GAPDH served as loading control. Densitometric analyses were performed 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/tetEGFP and SH-EP/tetEYFP-DEPP cells were treated with 200 ng/ml doxy for 4 h. Expression of EGFP and the EYFP-DEPP fusion protein as well as cellular ROS steady state levels were detected by confocal live-cell imaging. d SH-EP/tetEYFP-DEPP cells were treated with 200 ng/ml doxy and 5 mM NAC alone and in combination for 8 h. The LC3-I/LC3-II and DEPP expression were determined by immunoblot analyses. GAPDH served as loading control. Densitometric analyses were performed with the ImageJ 1.48 software. Control cells (Ctr.) 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