Fig. 6: nSMase2 induces autophagy via p38 MAPK activation and mTOR inhibition.

a Suppression of starvation-induced p38 MAPK phosphorylation and starvation-reduced mTOR phosphorylation by knockdown of nSMase2. PC12 cells were transfected with 50 nM nSMase2 siRNA (siSmpd3) or non-targeting negative control siRNA (siControl). At 48 h after transfection, the cells were incubated with HBSS for 2 h. Phospho-p38 MAPK, p38 MAPK, phosphor-mTOR, mTOR, phospho-Akt, Akt, phospho-JNK1/2, JNK1/2, phospho-AMPK, AMPK, phospho-ULK1 (Ser317), and ULK1 levels were analyzed by immunoblotting, and nSMase2 knockdown was confirmed simultaneously by immunoblotting. b Enhanced p38 MAPK phosphorylation and suppressed mTOR phosphorylation by nSMase2. PC12 cells were transfected with either plasmid-encoding V5-tagged WT nSMase2, catalytically inactive forms of nSMase2 (D428A and H639A), or empty vector. At 48 h after transfection, phospho-p38 MAPK, p38 MAPK, phospho-mTOR, and mTOR levels were analyzed by immunoblotting. c p38 MAPK inhibition with SB203580 attenuated the autophagy induction by nSMase2 overexpression. Cells overexpressing V5-tagged nSMase2 were pretreated with 10 μM SB203580 or the vehicle for 3 h. LC3 puncta were detected by immunofluorescence assay using anti-LC3 antibody and counted using ImageJ software in V5-positive cells. Scale bar = 5 μm. d mTOR inhibition with rapamycin partially reversed the effect of nSMase2 knockdown on LC3 puncta formation. PC12 cells transfected with siRNA (as described above) were pretreated with 750 nM rapamycin or the vehicle for 1 h and then starved for 2 h. LC3 puncta were detected by immunofluorescence assay using anti-LC3 antibody and counted using ImageJ software. Representative images of siRNA-transfected cells under starvation conditions are provided. Scale bar = 5 μm; Significant differences between the indicated groups, *p < 0.05, **p < 0.01, and ***p < 0.001