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. 2022 Feb 2;30(4):1432–1450. doi: 10.1016/j.ymthe.2022.01.037

Figure 2.

Figure 2

FLX induces autophagy

(A) Representative confocal images of endogenous WIPI2+ puncta in MPS-IIIA MEFs untreated and treated with Torin-1 or FLX for 3 h. The plot shows the means of WIPI2 spots per cell ±SEMs of n = 102 cells from 3 independent experiments. ANOVA test, ∗∗p < 0.01 versus untreated; ∗∗∗p < 0.001 versus untreated. (B) Representative image of immunoblot analysis of endogenous p62 and LC3I/LC3II in MPS-IIIA MEFs upon 24 h of FLX treatment alone or in the presence of BafA1 for the last 3 h. The plot shows the densitometry of p62 (at left) and the LC3-II band (on right) normalized to actin as mean values ±SEMs of n = 6 lysates per condition pooled from 2 independent experiments. ANOVA test, ∗∗∗p < 0.001 versus untreated; ⋅⋅p < 0.01 versus FLX; ⋅⋅⋅p < 0.001 versus FLX. (C) Representative images from the high-content assay of ARPE-19 cells stably overexpressing mRFP-EGFP-LC3 plasmid starved with HBSS or untreated and treated with bafilomycin (BafA1) or FLX. The plot shows the quantification of autophagosomes (EGFP+ + mRFP+ spots per cell) compared to the autolysosomes (RFP+/GFP− spots per cell). Values are means ± SEMs of n > 1,000 cells pooled from 3 independent experiments. ANOVA test, ∗∗∗p < 0.001 versus untreated/autolysosomes; ⋅⋅⋅p < 0.001 versus untreated/autophagosomes.