TNF-α/ActD+ZVAD does not induce autophagic cell death in hepatocytes. Primary mouse hepatocytes were either untreated (control) or treated with 10 ng/mL TNF-α + 0.1 μg/mL ActD + 50 μmol/L ZVAD in the presence or absence of 0.2 μmol/L Wort or 20 μmol/L CQ for 48 hours. A: Representative phase-contrast and PI staining overlaid images of hepatocytes. B: Quantification of the PI-positive necrotic cells. C: Primary hepatocytes were isolated from Atg5 F/F, Alb Cre+ mice and were treated as in panel A. Representative phase-contrast and PI staining overlaid images of hepatocytes are shown. D: Quantification of the PI-positive necrotic cells. Data are expressed as means ± SEM. n = 3 independent experiments, and more than 100 cells counted in each experiment. ∗∗∗P < 0.001 versus control. One-way analysis of variance analysis with Scheffé's post hoc test. Scale bar = 20 μm. ActD, actinomycin D; Alb, Albumin; Atg5 F/F, Atg5 Flox/Flox; CQ, chloroquine; PI, propidium iodide; TNF-α, tumor necrosis factor-α; Wort, wortmannin; WT, wild-type.