Fig. 4: p62 expression level is increased following cholesterol depletion.

Mv1Lu cells were grown and treated (or not) by statin or by HPβCD to induce CD as in Fig. 3, or treated with rapamycin (150 nM, 16 h). They were then lysed and analyzed by immunoblotting for p62 and β-actin (loading control). A, C Representative blots of the effects of statin-mediated CD and of rapamycin (A), or of HPβCD-induced CD, on p62 levels (C). B, D Quantification of p62 levels relative to β-actin. The level of p62 in the control sample for each blot was taken as 1. Bars are mean ± SEM of 6 (B) or 3 (D) independent experiments. Both CD treatments induced a significant increase in the p62 level relative to untreated cells, while rapamycin reduced the p62 level (*P < 0.05; ***P < 0.001; Student’s two-tailed t-test). E A representative blot of p62 degradation. Cells were subjected (or not) to statin-mediated CD, serum-starved (2 h), and subjected to CHX-chase degradation assay (“Materials and methods”). Note that the zero time point of CHX addition is after 16 h of CD treatment. F Quantification of p62 degradation (mean ± SEM, n = 4 independent experiments). Data were normalized to β-actin, taking the zero time (prior to CHX addition) for each experimental condition as 1. The degradation rate of p62 was significantly enhanced following CD (*P < 0.05, Student’s two-tailed t-test, comparing between control and CD samples at the same time point).