Figure 9. DHS-induced BAX and BID activation leads to LMP that partially controls MMP in IMR32 cells.

(A) and (B) LMP induction by DHS in the SCR, and BAX/BID/ BAX-BID-silenced cells and its quantification. (C) Effect of mitochondrial deficiency on DHS-induced LMP. (D) Effect of cathepsins inhibitors on mitochondrial accumulation of t-BID and cyt. c release and caspase-3 activation by DHS. The SCR controls and KD cells as well as the ρ⁺ and ρ^o cells were incubated with DHS (20 μM), and the induced LMP was quantified by flow cytometry after staining with AO. The t-BID and cyt. c levels in the mitochondrial extracts and active caspase-3 expressions in the whole cell extracts of the untreated, DHS-, cathepsins inhibitors- and DHS + cathepsins inhibitors-treated cells were analysed by western blotting. Cells were preincubated with cathepsin inhbitors for 1 h, prior to DHs treatment for these experiments. The protein bands were detected using a Kodak Gel-doc software and the intensity ratios of the individual bands to that of vehicle control, taken as 1 (arbitrary unit) were quantified after normalizing with respective loading controls. All determinations were made in duplicates for immunoblots and five replicates for flow cytometry analyses in 3-4 different experiments. The values are mean ± S. E. M. ^*p<0.05, ^**p<0.01 compared to respective vehicle controls; ^$p<0.05 compared to DHS treatment; ^#p<0.05 compared to DHS-treated SCR-cells. Representative images and histograms are shown.