Fig. 3: Nutrient starvation upregulates nSMase2.

PC12 cells were starved with HBSS for the indicated times. a Induction of autophagy by nutrient starvation in PC12 cells. Degradation of p62 and LC3 turnover were detected by immunoblotting for assessing autophagic flux. For LC3 turnover assays, cells were starved with HBSS with or without 50 μM CQ for the indicated times. b Activation of nSMase2 by starvation. Specific activity of nSMase2 was analyzed using [¹⁴C]-labeled sphingomyelin. c Increase in nSMase2 expression by starvation. Protein expression levels of nSMase2 in starved cells were determined by immunoblotting and were normalized to β-actin levels. d No changes in nSMase2 mRNA levels were induced by starvation. The mRNA levels of nSMase2 were measured by quantitative real-time PCR and were normalized to Hprt1. e Starvation-induced phosphorylation of nSMase2 at a serine residue. PC12 cells were starved with HBSS for the indicated time, and cell lysates were incubated with biotin-conjugated sphingomyelin (the nSMase2 substrate) followed by pull-down with streptavidin-sepharose beads. The pellets were analyzed using immunoblots to detect serine phosphorylation of nSMase2. The data are presented as the mean ± SEM of three independent experiments. Significant differences, *p < 0.05, **p < 0.01, and ***p < 0.001 (one-way ANOVA followed by LSD test); NS, not significant