FIG. 2.

TFEB accumulates in aggresome-bodies in smokers and COPD subjects with increasing severity of emphysema that results in autophagy flux impairment. (A–C) Immunostaining of human lung tissue sections collected from non-emphysema (GOLD 0) and COPD-emphysema subjects (GOLD I–IV) shows the colocalization of TFEB (green, white arrows) in perinuclear aggresome-bodies (red; aggresome-specific dye, insets). This TFEB-aggresome colocalization (yellow, insets) statistically correlates with the severity of emphysema (from GOLD I–IV; red arrows) and lung function (FEV1-% predicted) decline, r = −0.88. We also observed that the increased tissue destruction in GOLD IV emphysema subjects may be influencing further increase in TFEB-positive aggresome-bodies compared with severe (GOLD III) COPD-emphysema subjects. The Hoechst (blue) dye was used to stain the nuclei to visualize the tissue area used for immunostaining (Supplementary Fig. S2). Moreover, smokers (GOLD 0 to GOLD IV) with incrementing emphysema severity show a higher TFEB-aggresome colocalization, verifying TFEB-autophagy-impairment in smokers and COPD-emphysema subjects. Scale bar, 25 μM, *p < 0.05, **p < 0.01, ***p < 0.001; n = 5–10 per group. (D) Beas2b cells were incubated with BacMam reagent (with Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B) for 16 h, followed by treatment with CSE (5%), GEM (10 μM), and/or FIS (25 μM) for 12 h. Fluorescence microscopy was used to quantify the number of GFP/RFP-positive autophagosomes that suggests an impaired autophagy flux (Scale bars, 100 μm). The red arrows indicate yellow puncta bodies, while white arrows show GFP-positive cells. (E) The data analysis of GFP-RFP-positive puncta bodies is shown as mean ± SEM of n = 6 replicates (**p < 0.01, ***p < 0.001). Results indicate that CSE induces autophagy flux impairment, while autophagy-inducing drugs (GEM/FIS) can restore autophagy flux in the presence of CSE. Chloroquine (endosomal acidification and autophagy inhibitor) is used as a positive control to identify RFP-GFP-positive (yellow) autophagosomes indicative of autophagy flux inhibition. The insets (A, D) show the representative magnified image for positive or negative staining, marked by red or white arrows. FIS, fisetin; GEM, gemfibrozil. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars