Figure 3. DHS permeabilizes mitochondrial membrane of IMR32 cells to induce apoptosis.

(A) Flow cytometry analyses of ΔΨ_m. The cells were incubated for different periods with DHS (0 and 20 μM), stained with JC-1 for 15 min, and ΔΨ_m loss quantified by flow cytometry from the increased green fluorescense. (B) ApaF1and cyt. c translocation. The cytoplasmic and mitochondrial cell extracts were respectively subjected to immunoblotting, using suitable antibodies against ApaF1 and cytochrome c. (C) Mitochondria depletion in cells. The IMR32-ρ^o cells were prepared and the mitochondrial DNA deficiency was assessed from the COX I expressions in the ρ⁺ and ρ^o cells. The protein bands in the immunoblots were detected using a Kodak Gel-doc software and the intensity ratios of the individual bands to that of IMR32-ρ^o control, taken as 1 (arbitrary unit) were quantified after normalizing with respective loading controls. (D) Increased sensitivity of the IMR32-ρ^o cells to DHS treatment. The ρ⁺ and ρ^o cells were incubated with different concentrations of DHS (0-40 μM) for 48 h, and the sub-G1 cell populations analyzed by flow cytometry. 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<0.01 compared to ρ⁺ cells. Representative dot plots, histograms and images are shown.