Fig. 8: Cholesterol depletion perturbs autophagic flux via ROS and JNK activity. | Cell Death Discovery

Fig. 8: Cholesterol depletion perturbs autophagic flux via ROS and JNK activity.

From: Autophagy is induced and modulated by cholesterol depletion through transcription of autophagy-related genes and attenuation of flux

Fig. 8

A, B Mv1Lu cells were subjected to CD by statin (16 h) with or without NAC or SP600125 as in Fig. 6. They were subjected to CHX-chase degradation assay as in Fig. 4E. A A representative experiment. B Quantification of p62 degradation (mean ± SEM of 6 independent experiments). Data were normalized to β-actin, taking the zero time (prior to CHX addition) for each condition as 1. The degradation rate of p62 was significantly enhanced following NAC or SP600125 treatment (*P < 0.05; **P < 0.01; one-way ANOVA and Dunnett’s multiple comparisons post hoc test). C Typical confocal 3-D renditions of LC3-EGFP-mRFP-expressing cells. Mv1Lu cells transiently transfected with the LC3-EGFP-mRFP expression vector ptfLC3 (“Materials and methods”) were subjected (or not) to CD with or without NAC or SP600125 as above, and imaged by spinning disk confocal microscopy. The EGFP signal in untreated cells was mainly diffuse, in accord with low autophagy induction. CD induced a shift to punctate labeling of EGFP and mRFP. Treatment of cholesterol-depleted cells with NAC or SP600125 reduced the EGFP signal, while the increase in mRFP LC3 puncta was retained. D Quantification of EGFP relative to mRFP levels in mRFP positive pixels, in single confocal midplanes (see “Materials and methods”). The intensities measured in the respective channels of the confocal microscope were analyzed using SlideBook. Bars are mean ± SEM of measurements on 34 (CD) or 30 (CD with NAC or SP600125) cells. Under the latter conditions, the EGFP/mRFP signal was significantly reduced (**P < 0.01; Student’s two-tailed t-test). E–G Chloroquine treatment does not induce autophagy-related genes and does not perturb the increase in their expression by CD. Cells were subjected to CD (16 h) with or without chloroquine (10 μM). The mRNA levels of autophagy-related genes were determined by RT-qPCR as in Fig. 7. No significant changes were induced by chloroquine (n.s., P > 0.05; Student’s two-tailed t-test).

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