FIG. 6.

GEM rescues TFEB protein from aggresome-bodies as a mechanism to control sc-CS-induced autophagy-impairment. (A) Western blot analysis illustrating the levels of ubiquitinated proteins (Ub), p62 (impaired autophagy marker), TFEB (master autophagy regulator), and GRP94 (ER stress marker) in the soluble and insoluble protein fractions of murine lung protein lysates collected from C57BL/6 mice exposed to room air or subchronic cigarette smoke (sc-CS, 2 months, 5 h/day, 5 days/week) and/or treated with GEM [i.p., 40 mg/kg bw, one dose alternate days for 10 days]. Expression of each protein was normalized to β-actin loading control. (B) The data (mean ± SEM, n = 4, *p < 0.05, **p < 0.01, ***p < 0.001) confirm our findings that GEM treatment significantly diminishes CS-induced autophagy-impairment as evident by rescue of Ub, p62, VCP, and TFEB proteins from the insoluble protein fractions (aggresome-bodies). As anticipated, we only see a mild induction of ER stress marker, GRP94, levels (Fig. 4B, soluble fraction) compared with impaired-autophagy markers (Fig. 4B, Ub, p62, VCP, insoluble protein fractions), suggesting that misfolded proteins are accumulating mainly in aggresomes over ER. Our data suggest that sc-CS-induced autophagy-impairment is mediated by TFEB accumulation in aggresomes that can be rescued by GEM treatment. i.p., intraperitoneal; sc-CS, subchronic cigarette smoke. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars