Figure 3
Overexpression of HSP27 suppresses Vpr-induced cell death and apoptosis. (A) Suppression of Vpr-induced cell death by HSP27 in vpr-transfected HEK293-632 cells. Vpr and HSP27 expression was induced by muristerone A. Dead cells were detected by trypan blue straining 72 h after gene induction. (B) Anti-apoptotic activity of HSP27 in HEK293-632 cells. Apoptosis was detected by Annexin V assay using a BD ApoAlert Annexin V kit. Annexin V-positive cells that appear in the low right quadrant typically represent early apoptosis and cells in late apoptosis are found in the upper right quadrant (30). (C) HSP27 overexpression reduces cytopathic effect of HIV-1 infection. Dead cells (trypan blue-positive) were counted in HIV-infected H9 T-lymphocytes by trypan blue assay (left panel); cell viability was determined by an MTT assay (right panel). H9 cells were either mock-infected (H9 + mock), infected with HIV-1LAI without overexpressed HSP27 (H9-vector + HIV-1) or infected with HIV-1LAI with overexpressed HSP27 (H9-HSP27 + HIV-1). Cell death and cell viability of the infected cultures were examined three, five, seven, and ten days after infection. Percentage of cell death induced by HIV-1 infection was quantified by counting the number of blue cells over total cell population. Cell viability was quantified by measuring optical density at 630 nm using a commercial MTT assay kit. Average and standard errors represent a total of three independent experiments. *, statistical significance at the level of P < 0.001; **, statistical significance at the level of P < 0.0001. (D) Overexpression of HSP27 does not have an effect on cell viability. Cell death and cell viability were examined in uninfected H9 cells as in panel C.