Fig. 7: Loperamide, pimozide, and STF-62247 enhance the autophagic flux in MZ-54 cells.

a MZ-54 cells were treated with 20 µM IM/100 µM TIC, 17.5 µM loperamide, 15 µM pimozide, and 40 µM STF-62247 for 8, 2, 4 and 3 h, respectively. BafA1 was added 4 h before cell harvesting at a final concentration of 40 nM. Western blotting was performed with the indicated antibodies and vinculin as loading control. For quantification, LC3-II band intensities were normalized to vinculin band intensities. b Schematic representation of the GFP-LC3B-RFP-LC3BΔG autophagy flux sensor. Upon expression, the GFP-LC3B-RFP-LC3BΔG fusion protein is cleaved by the ATG4 proteases after which GFP-LC3B becomes conjugated to PE and localizes to autophagosomes which eventually fuse with lysosomes, inducing degradation of GFP-LC3B. RFP-LC3BΔG remains in the cytosol, where it serves as internal control. Scheme adapted from Kaizuka et al.⁴⁸ c Stable GFP-LC3B-RFP-LC3BΔG-expressing MZ-54 WT or ATG7 KO cells were treated as indicated in a for 16 h followed by Western blotting with vinculin as loading control. d, e Stable GFP-LC3B-RFP-LC3BΔG-expressing MZ-54 WT (d) or ATG7 KO cells (e) were treated with 20 µM IM/100 µM TIC, 15 µM loperamide, 15 µM pimozide or 40 µM STF-62247 for the indicated time points followed by flow cytometry. BafA1 was added 4 h before the measurement at a final concentration of 40 nM. Mean and SEM of three independent experiments performed in triplicate are shown. f Fluorescence microscopy of stable GFP-LC3B-RFP-LC3BΔG-expressing MZ-54 WT, ATG5 KO and ATG7 KO cells was performed after 16 h of treatment with 20 µM IM and 100 µM TIC, 17.5 µM loperamide, 15 µM pimozide, and 40 µM STF-62247. Scale bar = 50 µm. Significances are calculated versus untreated cells of the same cell line. *p < 0.05, **p < 0.01, ***p < 0.001. UT untreated, IM imipramine hydrochloride, TIC ticlopidine, LOP loperamide, PIMO pimozide, STF STF-62247