NAS inhibits autophagic cell death in vitro. Atg5^−/− MEFs and Atg5^+/+ MEFs (A), and PCN (B) cell death were induced by 2 h or 18 h exposure as indicated to 1000 μmol/L H₂O_2, with or without a series of indicated concentrations of NAS as well as 20 μmol/L 3-MA. Cells were preincubated with NAS or 3-MA for 2 h. Cell viability was evaluated by MTS assay (A) and cell death was quantified by LDH assay (B). Statistically significant differences (n = 6–8) are indicated with *p < 0.05 and **p < 0.01. The data are presented as mean ± SEM. Autophagy induced by subjecting PCNs to 2 h (C, E) or 18 h (D, E) H₂O₂ (1000 μmol/L) with or without 10 μmol/L NAS. PCNs were treated by the administration of 10 μmol/L NAS for 2 and 18 h. Whole-cell lysates were extracted, and samples were analyzed by Western blot (each of which contained 50 μg protein) using antibodies to LC3, Beclin-1, and p62. β-Actin was used as a loading control. The blots are representative of three independent experiments (C, D). Densitometry was performed to quantify the intensity of the bands from the three independent experiments; *p < 0.05, **p < 0.01. Autophagy of PCNs was assessed by 20 μm MDC stain. Autophagic vacuoles were labeled by MDC staining (E).