Fig. 2: nSMase2 induces autophagy. | Cell Death & Disease

Fig. 2: nSMase2 induces autophagy.

From: Activation of neutral sphingomyelinase 2 by starvation induces cell-protective autophagy via an increase in Golgi-localized ceramide

Fig. 2

a Enhanced autophagic flux by overexpression of nSMase2. PC12 cells were transfected with 1 μg plasmid-encoding V5-tagged nSMase2 or empty vector. b Reduced autophagic flux by knockdown of nSMase2. PC12 cells were transfected with 50 nM nSMase2 siRNA (siSmpd3) or non-targeting negative control siRNA (siControl). c Catalytically inactive mutants of nSMase2 were unable to induce autophagy. 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, the cells were incubated with HBSS or growth medium for 2 h. Autophagic flux was determined by the levels of LC3 puncta formation and p62 degradation. For the LC3 puncta counting assay of the overexpression group, LC3 puncta were counted in only V5-positive cells. For the LC3 turnover assay, transfected cells were starved with HBSS with or without 50 μM CQ for 2 h, and LC3 and GAPDH were analyzed by immunoblotting. LC3-II levels were normalized to GAPDH levels, and the quantified immunoblot assay data are presented as the mean ± SEM of at least three independent experiments. Scale bar = 5 μm; Significant differences between the indicated groups, *p < 0.05, **p < 0.01 and ***p < 0.001; NS, not significant

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