Fig. 5: RPV induces accumulation of p62 and LC3 in late autophagic structures.

Representative confocal microscopy images of LX-2 cells that express mCherry-eGFP-LC3 (A) or mCherry-GFP-p62 (B) and had been treated with vehicle, TGF-β or TGF-β plus RPV (1, 2, and 4 µM) for 24 h. Cells were stained with Hoechst to detect nuclei. Red signal indicates the presence of the protein of interest in late autophagic structures (autophagolysosomes), while yellow signal (red plus green fluorescence) indicates its presence in early autophagic structures (autophagosomes). C, D Quantification of the fluorescence signal data. Images were analyzed with a custom made CellProfiler pipeline to detect red (red boxplots) and green fluorescent signal and to relate the two fluorescent signals (red/green double positive—orange boxplots). Bars represent median ± interquartile range (IQR, n = 3); all values were analyzed by two-way ANOVA with Bonferroni post-test for RPV + TGF-β vs TGF-β treatment (*, ** and **** represent a p value < 0.05, < 0.01 and < 0.0001 respectively) or paired Student´s t test of TGF-β vs vehicle and rapamycin (Rapa) vs vehicle (^##### represents a p value < 0.0001). E Analysis of the autophagic flux in LX-2 cells treated with vehicle, TGF-β or TGF-β plus RPV (2 and 4 µM) for 48 h under conditions of normal autophagy or those of inhibited autophagosome-lysosome fusion (cotreatment with chloroquine (CQ)). WB images of LC3 and GAPDH as a reference protein are representative of three independent experiments. Rapamycin (Rapa) was used a positive control of autophagy induction.