Fig. 5

DEPP impairs cellular proliferation through ROS/LC3-mediated ERK1/2 phosphorylation and p21 upregulation. a SH-EP/tetEGFP and SH-EP/tetDEPP cells were cultured with 200 ng/ml doxy alone or in combination with 5 μM PD98059 for 24 h and the levels of DEPP, phosphorylated pThr202/Tyr204-ERK1/2, ERK1/2, p21, p27, Cyclin D1, CDK4 and CDK6 were determined by immunoblot analyses. GAPDH served as loading control. Densitometric analysis of pThr202/Tyr204-ERK1/2 relative to ERK1/2 expression and p21 relative to GAPDH was done with the ImageJ 1.48 software. Untreated cells were set as 100%. b DEPP, LC3-I/LC3-II, phosphorylated pThr202/Tyr204-ERK1/2, ERK1/2, and p21 expression were assessed by immunoblot analyses of SH-EP/tetDEPP cells transiently transfected with siCtr and siLC3 oligonucleotides and afterwards treated with 200 ng/ml doxy and 5 mM NAC alone or in combination for 24 h. Densitometric analysis of pThr202/Tyr204-ERK1/2 relative to ERK1/2 expression and p21 relative to GAPDH was done with the ImageJ 1.48 software. Untreated cells were set as 100%. c The AlamarBlue viability assay was used to quantify the number of living cells. SH-EP/tetEGFP and SH-EP/tetDEPP cells were analyzed after treatment with 200 ng/ml doxy alone or in combination with 5 μM PD98059 for 24 h. Shown are mean values ± s.e.m. of five independent experiments, each performed in triplicates; statistical analysis was done with the Student’s unpaired t-test, ***P < 0.001 compared to corresponding controls