Fig. 12

Overexpression of caspase-3 and caspase-8, induction of apoptosis in HIV-1 latently infected cells and activation of HIV-1 replication. (a) U1 cells were transfected with pmaxGFP or pCasp3GFP or pCasp8GFP construct were subjected to Annexin V staining followed by flow cytometry, gating on GFP-positive cells, at 36 h post transfection (grey bars). HIV-1 activation was assessed by quantification of unspliced viral RNA using qRT-PCR and represented as fold change compared to DMSO treated cells (black bars). The data are shown as mean ± standard deviation (n = 3). (b) Whole cell lysates obtained from transfected cells were subjected to immunoblotting using anti-GFP, anti-HIV p24 and anti-GAPDH antibodies. Anti-GFP used in this experiment is raised against GFP from Aequorea victoria while GFP expressed from pmaxGFP® (Lonza) is from Pontellina plumata, therefore antibody could not recognize GFP in pmaxGFP® transfected cell lysate. (c) Transfected U1 cells were fixed and permeabilized using BDcCyto Fix/Perm solution and stained with HIV-1 p24 antibody (PE conjugated), samples were analyzed using flow cytometry. Upper panel represents untransfected and GFP alone transfected cells while lower panel shows data of caspase-GFP transfected cells