Fig. 5

TBM inhibits autophagy flux in CRC cells. a. SW480 and HCT116 cells were treated with vehicle, TBM (10 μM), CQ (5 μM), or in combination for 24 h. Immunoblot analysis was used to detect the expression of LC3. b. The accumulation of LC3 puncta was examined by fluorescent analysis of cells transfected with GFP-LC3 plasmids and treated with CQ (5 μM) in the presence or absence of TBM (10 μM) for 24 h. Scale bar, 10 μm. c. The number of LC3 puncta was quantitated. d. Fluorescence analysis of cells transiently transfected with tandem mRFP-GFP-tagged LC3 and treated with vehicle or TBM (10 μM). Scale bar, 10 μm. e. The ratio of red puncta indicating autophagolysosome (GFP⁻/RFP⁺) versus yellow puncta indicating autophagosome (GFP⁺/RFP⁺) was quantitated. f. SW480 and HCT116 cells were treated with indicated concentrations of TBM. The expression of LAMP1 was examined by immunoblotting. g. Immunofluorescence analysis of the co-localization of endogenous LC3 and LAMP1 in SW480 and HCT116 cells treated with vehicle or TBM (10 μM) for 24 h. Scale bar, 10 μm. h. Immunoblot of ubiquitin in SW480 and HCT116 cells treated with TBM in indicated concentrations for 24 h. ***, p < 0.001