FIG. 5.

TFEB controls CSE-induced oxidative stress, aggresome-formation, cellular viability, and senescence. (A) Beas2b cells were treated with CSE (5%), GEM (10 μM), and/or FIS (25 μM) for 12 h and cell proliferation was assessed by MTS assay. The data show that CSE inhibits Beas2b cell survival, while TFEB-inducing drugs (GEM/FIS) can partially rescue CSE-impaired cell proliferation. (B) CM-DCFDA-based ROS assay shows that CSE-induced ROS activity can be alleviated by treatment with TFEB-inducing antioxidants, GEM or FIS (mean ± SEM, *p < 0.05, ***p < 0.001). (C, D) Beas2b cells were treated with CSE (5%), GEM (10 μM), and/or FIS (25 μM) for 12 h and the number of senescent cells were counted (blue, black arrows). The data summarized for n = 6 replicates (mean ± SEM, ***p < 0.001) indicate that TFEB-inducing drugs (GEM/FIS) can control CSE-induced senescence. (E) Beas2b cells were transfected with TFEB-shRNA for 24 h and/or treated with GEM (10 μM) for the final 12 h of transfection. Western blots show that TFEB-shRNA significantly inhibits the protein expression of TFEB that was restored by GEM treatment (TFEB/autophagy inducer, upper panel, soluble fraction). Knockdown of TFEB also shows significant accumulation of ubiquitinated proteins in the insoluble protein fraction as anticipated (similar to CSE exposure), which could be rescued by GEM treatment (lower panel). (F) β-Actin was used to normalize the expression of each protein and densitometry analysis of the blots is shown (n = 3, mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001). (G) TFEB inhibition by shRNA induces ROS activity, which could be inhibited by a parallel GEM treatment (n = 3, mean ± SEM, **p < 0.01, ***p < 0.001). (H) Beas2b cells were transfected with TFEB-shRNA for 24 h and/or treated with GEM (10 μM) for the final 12 h of transfection. The number of senescent cells (blue, black arrows) was quantified under the light microscope to quantify changes in senescence on TFEB inhibition and/or induction. (I) The analysis of the senescence data shows that TFEB knockdown increases the number of senescent cells and parallel treatment with TFEB/autophagy-inducing drug, GEM, controls this increase in senescence. The data confirm the functional role of TFEB-mediated autophagy in regulating CS-induced oxidative stress and resulting autophagy-impairment and cellular senescence. CM-DCFDA, chloromethyl derivative of 2′,7′-dichlorodihydrofluorescein diacetate; ROS, reactive oxygen species. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars