FIG. 3.

TFEB-inducing drugs rescue CSE-induced autophagy-impairment. (A, B) Immunoblotting of total protein lysates isolated from Beas2b cells treated with different autophagy-inducing drugs (cysteamine, CYS, 250 μM; GEM, 10 μM; and carbamazepine, CBZ, 20 μM; 6 h) indicates that GEM and CBZ significantly induce TFEB expression in Beas2b cells (n = 3, **p < 0.01). (C, D) The Beas2b cells were cotransfected with RFP-(Ub) Ubiquitin and GFP-(LC3B), the autophagy protein light chain-3, plasmids. After 24 h post-transfection, cells were treated with 5% CSE, GEM (10 μM, left panel), and/or FIS (25 μM, right panel) for 12 h, followed by fluorescence microscopy (Scale bars, 100 μm). The fluorescent images were used to count the cells positive for colocalization (yellow, red arrows,) of ubiquitinated (Ub) proteins (red) and LC3B-puncta bodies (green), where white arrows show single staining. Data represent mean ± SEM, n = 6, *p < 0.05, **p < 0.01, ***p < 0.001, and analysis is shown in (E) and (F). The data indicate that CSE treatment induces Ub-LC3B coexpression as yellow puncta bodies (aggresomes), which is alleviated by TFEB-autophagy inducers, GEM or FIS. Thus, GEM/FIS-mediated TFEB activation has the potential to rescue CSE-induced autophagy-impairment. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars