Fig. 4: Excessive ROS results in an increase in LMP and leads to the cytosolic release of cathepsin B.
a Tub induced an increase in LMP. After exposure to Tub for 24 h, cells were stained with AO (5 μg/mL) for 20 min. The mean fluorescence intensity in the FL1 channel represents the extent of LMP. The bar chart was drawn from the flow cytometry results. b The level of cathepsins in the cytoplasm of lung cancer cells after Tub treatment. Cytosolic protein was extracted using a commercially available kit and subjected to western blot analysis. c The activity of cytosolic cathepsin B was significantly upregulated following Tub treatment. Cathepsin B activity is labelled by green fluorescence; lysosomes are labeled by red fluorescence (LysoBriteTM Red); and nuclei are labelled by blue staining with Hoechst 33342. The green fluorescence intensity in the cytosolic area represents the activity of cytosolic cathepsin B. Cathepsin B activity was quantified in more than 30 cells for each group with the help of the Image J software, and the data are presented in the right bar chart (scale bar = 5 µm). d Elimination of ROS by NAC (an ROS scavenger) reversed the increase in LMP induced by Tub. Lung cancer cells were treated with Tub (20 μM), NAC (ROS scavenger, 2 mM) or both Tub and NAC for 24 h. LMP was determined by AO staining for 20 min. A Q value of <0.85 indicates that NAC and Tub have an antagonistic effect on each other. e NAC treatment led to decreased activity of cytosolic cathepsin B in Tub-treated lung cancer cells (for details, see the description for panels c and d) (scale bar = 5 µm). f NAC partially reversed the lysosomal acidification in Tub-treated lung cancer cells. LysoTracker Red was used as a fluorescent probe for acid lysosomes (scale bar = 20 µm). *p < 0.05, **p < 0.01, ***p < 0.001 vs. the indicated groups.
