Figure 5

HIV-1-induced neuronal death is mediated in part through IL-1β and IL-8 and not TNF-α present in supernatants of HIV-1 ^wt  virus-infected MDMs. Primary neurons exposed to HIV-1^wt, HIV-1∆Vpr and mock-infected (NT) MDM supernatants (10% v/v) as well as recombinant IL-1β (10 ng/ml), IL-8 (10 ng/ml) and TNF-α (100 ng/ml) for 24 – 48 hours were analyzed for neuronal apoptosis by (a) Annexin-V FITC staining, nuclei of the cells were stained with Hoechst 33342 and measured the neuronal death (%); (b) MTT assay, absorbance was measured and cell viability was normalized with negative controls. HIV-1^wt-infected MDM supernatants were more neurotoxic compared to HIV-1∆Vpr culture. (c) The cells were lysed in Caspase-Glo® 3/7 substrate and protease activity was measured as relative light units (RLU). (d) Neurons were lysed and 50 μg of protein for each sample was analyzed by western blot using specific antibodies for cleaved and total PARP. (e) Neurons exposed to supernatants from HIV-1^wt, HIV-1∆Vpr and mock-infected MDM (NT) were treated with and without anti-IL-1β, anti-IL-8 and anti-TNF-α and neurotoxicity was analyzed after 24–48 hours by Annexin-V staining. Results are the ± SEM of three individual experiments; *P < 0.05, **P < 0.01 in HIV-1^wt compared with control or HIV-1 ∆Vpr treated.