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. 2020 Dec 3;8:601953. doi: 10.3389/fcell.2020.601953

FIGURE 5.

FIGURE 5

Overexpression of TFEB accelerated fusion of autophagosome and lysosome, inhibited exosomes and autophagy component release, promoted cell apoptosis, regulated cell invasion. (A,B) A549 cells exposed to ASS (10 dyn/cm2) for 0, 15, 30, and 60 min, the expression of TFEB was detected by qRT-PCR and western blot, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C) The expression level of TFEB after exposure to ASS (10 dyn/cm2) for 60 min were confirmed by cell immunofluorescence. (D) Immunofluorescence of LAMP1 and LC3B was performed on A549 cells exposed to ASS (10 dyn/cm2) for 60 min, the co-localization of LAMP1 puncta and LC3B puncta was observed in the merge image (yellow merge area), magnification, 600×. (E,F) A549 cells, after stable overexpression of TFEB or GW4689 treatment, were exposed to ASS (10 dyn/cm2) for 60 min, then isolated exosomes and autophagic components were identified and quantified by western blot, ****P < 0.0001 compared to control group, $, $$$$ P < 0.05 and P < 0.0001 compared to ASS (10 dyn/cm2) group, &&&, &&&& denote P < 0.001 and P < 0.0001 compared to OE-NC group. (G,H) Cell apoptosis was determined by TUNEL assay and flow cytometry, each group was treated the same as panel E, ** and *** denote P < 0.01 and P < 0.001 compared to control group, ###P < 0.001 compared to ASS (10 dyn/cm2) group, $, $$$ denote P < 0.05 and P < 0.01 compared to OE-NC group. (I) A549 cells, after stable overexpression of TFEB or GW4689 treatment, were exposed to ASS (10 dyn/cm2) for 60 min, Tranwell assay was used to detect cell invasion.