Fig. 3

PPT1 inhibitor DC661 inhibited autophagy by inhibiting lysosomes. A Confocal laser-scanning microscopy images of intracellular co-localization between PPT1 and lysosomes. LAMP-1 staining indicates the lysosomes, and Hoechst 33,258 indicates the nucleus. Co-localization is visualized by color and area overlap (red + green = yellow). Scale bar, 10 μm. B Fluorescence co-localization analysis according to (A) was performed by ImageJ software. C Immunofluorescence staining of PPT1 in HCC cells after treatment with DC661 (3 µM, 6 h). Scale bar, 20 μm. D Semiquantitative analysis of the mean fluorescence intensity (MFI) in the HCC cells according to (C) was performed by using ImageJ software (n = 6). Data represent mean ± SD; **P < 0.01. E Fluorescence images of LysoTracker Green (lysosome probe) in HCC cells after treatment with DC661 (3 µM, 6 h). Hoechst 33,258 was used to stain the nucleus. Scale bar, 50 μm. F Semiquantitative analysis of the MFI of LysoTracker Green in HCC cells according to (E) was performed by ImageJ software (n = 6). Data represent mean ± SD; **P < 0.01. G Western blot showing an increase in LC3-II and P62 in HCC cells treated with DC661 (3 µM, 6 h). H Western blot showing P62 degradation and LC3 lipidation in HCC cells treated with sorafenib and/or DC661. HCC cells were treated with or without 10 µM sorafenib in the presence or absence of 1 µM DC661 for 24 h