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. 2017 Jan 31;7:41862. doi: 10.1038/srep41862

Figure 4. IMB-6G blocks autophagic flux through attenuation of cathepsin activity in pancreatic cancer cells.

Figure 4

(a) MiaPaCa-2 cells were incubated with IMB-6G (5 μM) for 12 h, followed by staining with LysoTracker Red (100 nM) for 30 min, cells were incubated with a LysoSensor Green dye (2 μM) for 10 min. Merged (yellow) images are indicative of an acidic pH (scale bars, 20 μm). (b) MiaPaCa-2 cells were transfected with the EGFP-LC3 plasmid. After 24 h, the cells were treated with IMB-6G (5 μM) for 12 h before being incubated with DQ-Red BSA (10 μg/ml) for 30 min. The cells were fixed and analyzed for fluorescence microscopy (scale bars, 20 μm). (c) Enzymatic activity of CTSB and CTSL in IMB-6G treated MiaPaCa-2 and HupT-3 cells. Cells were treated with DMSO, IMB-6G (5 μM), CA-074Me (2 μM, CTSB inhibitor) or E-64 (10 μM, CTSB and CTSL inhibitor) for 24 h. Enzymatic activity was analyzed using fluorogenic kits. Data are presented as the mean ± SD from 3 independent experiments. (d) MiaPaCa-2 cells were treated with 5 μM IMB-6G for 12 or 24 h, the precursor and the mature form of CTSB, CTSD and CTSL were determined by immunoblotting. Bafilomycin (Baf, 200 nM) was used as a positive control.