Fig. 3

TSN elevated lysosome pH and impaired lysosomal proteolytic function. A MDA-MB-231, MDA-MB-436 and BT-549 cells were stained with LysoSensor™ Yellow/Blue DND-160 after treatment of 1 μM TSN, 100 nM Torin1, 30 μM CQ or DMSO for 24 h. The excited fluorescence signal at 440/535 nm was measured by 96-well plate reader. B, C MDA-MB-231 and MDA-MB-436 cells were treated with 1 μM TSN, 100 nM Torin1, 50 nM BAF or DMSO for 24 h. Total cellular extracts were prepared and subjected to western blot using antibodies against CTSB and CTSD. GAPDH was used as a loading control. D Lysotracker with a final concentration of 50 nM was used to label the acid compartment for 30 min. The cells were fixed and stained with Hoechst for further Confocal Scanning. E Cells were pre-incubated with 0.01 μg/μL DQ™ Red BSA for 30 min in serum-free DMEM, then treated with 1 μM TSN, 100 nM Torin1, 50 nM BAF or DMSO for 6 h. The images were collected by Incucyte S3 Live-Cell System. The results were the mean ± SD values obtained from three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001 vs. CTL)