Apoptotic effect of HIV-1 PR. (a) Cytotoxic effect of HIV-1 PR on HeLa cells. HeLa cells were transfected with c-myc-HIV-1 PR (1 μg/ml), encoding catalytically active HIV-1 PR and the number of dead cells at various post-transfection time points was assessed by PI staining by flow cytometry using BD LSR Fortessa. (b) HeLa cells were transfected with indicated amounts of c-myc-HIV-1 PR. Six hours post-transfection, the cells were labeled with FITC Alexa Fluor® 488 annexin V and propidium iodide (PI), and the apoptotic + dead cells were detected using BD LSR Fortessa. (c) Representative gating for control and HIV-1 PR expressing cells. (d) The HEK-293 cells were transfected with varying amounts of c-myc-HIV-1 PR. Changes in ΔΨm were determined six hours post-transfection by staining with JC-1 fluorochrome followed by flow cytometry using BD LSR Fortessa. (e) Representative gating for control and HIV-1 PR expressing cells. Healthy cells containing red JC-1 aggregates were detected in the FL 2 channel, whereas green JC-1 monomers in apoptotic cells were monitored in the FL1 channel (FITC). (f) HEK-293 cells were transfected with c-myc-HIV-1 PR, and 24 h post-transfection, fragmented DNA was isolated using the Suicide-TrackTM DNA Ladder Isolation Kit and separated by agarose gel. The mean percentage (±standard deviation) of the data pooled from three independent experiments is shown. Each data point was repeated at least twice.