Figure 2. DHS induces caspase-9/caspase-3-mediated apoptosis in IMR32 cells.

(A) and (B) Sub-G1 population analyses. The cells were incubated with different concentrations of DHS (A) or Resv (B) for 48 h, and the sub-G1 populations analyzed by flow cytometry. (C) Annexin V/PI assay. The cells were incubated with DHS (20 μM) for 0-12 h, co-stained with PI and Annexin-V FITC and analyzed by flow cytometry. (D) Nuclear chromatin condensation. The cells were incubated with DHS (0, 20 and 40 μM) for 48 h, stained with Hoechst 33342, and observed under an Axioskop II Mot plus (Zeiss) microscope (40 × optics). The scale bar represents 10 μm, while white arrows indicate fragmented and condensed nuclei. (E) Expressions of the caspases. The caspases expressions in the whole cell extracts of the cells, incubated with DHS (0 and 20 μM) for different time-points were assessed by immunoblots. 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. (F) Effect of the caspases inhibitors on apoptosis induction. The cells were treated with vehicle (0.1% DMSO) or pan-caspase or caspase-9/caspase-8/caspase-3-specific inhibitors (10 μM) for 1 h followed by incubation with DHS (20 μM) for 48 h, and the sub-G1 cell populations analyzed by flow cytometry. All determinations were made in duplicates for immunoblots and microscopy, and five replicates in flow cytometry analyses in 3-4 different experiments. The values are mean ± S. E. M. ^*p<0.05, ^**p<0.01 compared to vehicle control; ^$p<0.05, ^#p<0.01 compared to only DHS treatment. Representative dot plots, histograms and images are shown.