Fig. 3

Protective effect of PDI overexpression and quercetin treatment on ER stress activation, apoptotic cell death, and cell survival. a C2C12 cells (1 × 10³ cells/96-well plates) underwent PDI gene transfer 24 h before treatment either with tunicamycin (5 μg/ml) or MG132 (10 μM) for 6 h. Protein lysates (30 μg/lane) analyzed by immunoblot analysis using specific antibodies against PARP (full-length and cleaved form) to assess apoptotic cell death induction. GRP78/BiP and ERO1α assessed as ER stress markers and p21 as cellular senescence marker; antibodies against Turbo GFP used to verify GFP/PDI overexpression. GAPDH used as protein loading normalization. Densitometry performed using ImageJ software and relative band intensities of tunicamycin, and MG132-treated cells normalized to GAPDH and finally quantified with respect to untreated control, arbitrarily set to 1.0. b Mock transfected and PDI-overexpressing C2C12 cells preconditioned with quercetin (75 μM) for 24 h and then challenged with ER stress-inducer tunicamycin (Tuni). Immunoblot and densitometric analysis performed as already described, while (c) cell viability evaluated using WST-1 cell proliferation assay kit. Results reported as mean ± standard deviation of three independent experiments performed in duplicate. *p < 0.005. DMSO dimethylsulfoxide, ERO1α endoplasmic reticulum oxidoreductin-1α, GAPDH glyceraldehyde phosphate dehydrogenase, GFP green fluorescent protein, GRP78/BiP anti-binding immunoglobulin protein, PARP anti-poly(ADP-ribose) polymerase, PDI protein disulfide isomerase