FIGURE 5.

SC75741 enhances autophagy via ATG5 pathway. (A). H4GT25 cells were pretreated with 1 μg/ml DOX for 24 h, wash out DOX, treated with DMSO or 5 μM SC75741 for 6,12, and 24 h. Cell lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (GFP/Tubulin). (B). H4GT25 cells were pretreated with 1 μg/ml DOX for 24 h, treated with 5 μM SC75741 with or without, Bafilomycin A1 (100 nM), NH4Cl (20 mM), Leupeptin (100 nM), E-64D (10 μM) for 8 h. Cell lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (GFP/Tubulin). (C). Schematic model of the detection of autophagy with Keima-TDP25 overexpressed in SH-SY5Y cells. The graphical was created using BioRender.com. (D). SH-SY5YKT25 cells were pretreated with 1 μg/ml DOX for 24 h, treated with or without 5 μM SC75741 for 3 h and the co-localization between Keima-red and LysoTracker green was done using live cell imaging and confocal microscopy. Scale bar, 20 μm; insets: scale bar, 5 μm. (E). SH-SY5YKT25 cells were pretreated with 1 μg/ml DOX for 24 h, treated with or without 5 μM SC75741 for 3 h and the co-localization between Keima-red and LysoTracker green was analyzed by two-tailed t test. (data represent mean ± SD; n = 9, ^**** p < 0.0001). (F). HEK293 WT cells were transfected with Keima-TDP25, pretreated with 1 μg/ml DOX for 24 h, treated with DMSO or 5 μM SC75741 for 6 and 12 h, and the Keima-TDP25 fluorescence was analyzed by fluorescence microscopy and compared with cells treated with DMSO. (data represent mean ± SD; n = 8, ^**** p < 0.0001, ^*** p < 0.001). (G). HEK293 ATG5-/- cells were transfected with Keima-TDP25, pretreated with 1 μg/ml DOX for 24 h, treated with DMSO or 5 μM SC75741 for 6 and 12 h, and the Keima-TDP25 fluorescence was analyzed by fluorescence microscopy and compared with cells treated with DMSO. (data represent mean ± SD; n = 8, ns, not significant, two-tailed t test).