Fig. 4: Silencing and pharmacological inhibition of HIFs restores HIV-LTR promoter activity under low oxygen.
From: Hypoxic microenvironment shapes HIV-1 replication and latency
a Jurkat cells were co-transfected with HIV-LTR-Luc or HRE-Luc along with siRNAs targeting HIF-1α and HIF-2α. 24 h later, transfected cells were cultured under 20% or 1% O2 conditions for 24 h and LTR or HRE activity assessed by luciferase assay. HIF expression was assessed by western blotting. The middle and right panels show Luc expression (Relative light units (RLU) and the red the dashed line represents the mean RLU of nontransfected cells (n = 4, mean ± SEM, Two-way ANOVA analysis). b Jurkat cells were cultured under 1% O2 for 24 h in the presence of increasing concentrations of the HIF pathway inhibitor NSC-134754 and assessed for HIF-1α and HIF-2α expression by western blotting. c HIV-LTR sequences were aligned and neighbor joining trees constructed (bootstrap = 1000) using ClustalX. Jurkat cells were transfected with selected HIV subtype LTR-Luc reporters and treated with NSC-134754 under 1% O2 for 24 h. LTR activity was determined by luciferase assay and the data expressed relative to the normoxic conditions (n = 3, mean ± SEM, Two-way ANOVA analysis). d Activated CD4 T cells isolated from human PBMCs were infected with VSV-G-pseudotyped HIV NL4.3-Luc and treated with NSC-134754 under 1% O2 for 24 h. LTR activity was determined by luciferase assay and the data expressed relative to the normoxic conditions. Each symbol represents data from an individual donor (n = 7; mean ± SEM, Wilcoxon matched-pairs signed rank test). e VSV-G-pseudotyped HIV NL4.3-Luc infected Jurkat cells were treated with DMOG under 20% O2 conditions for 24 h and luciferase activity measured. Data are expressed relative to the control untreated cells (n = 3, mean ± SEM, One-way ANOVA analysis). Cell lysates were collected to probe for HIF-1α and HIF-2α expression by western blotting. f VSV-G-pseudotyped HIV NL4.3-Luc infected activated CD4 T cells were treated with DMOG under 20% O2 conditions for 24 h and luciferase activity measured. Data are expressed relative to the control untreated cells. Each symbol represents data from an individual donor (n = 7; mean ± SEM, Wilcoxon matched-pairs signed rank test).
