Fig. 3

UPF2 and SMG6 knockdown enhance HIV-1 vRNA expression in primary HIV-1-infected MDMs. Human monocytes were differentiated into MDMs and then transfected with control siRNA (siNS) or siRNAs directed against UPF1, UPF2 or SMG6. After 24 h, cells were infected with NL4-3-Bal-IRES-HSA virus (MOI: 1.0) and kept in culture for 6 days. a RT activity in cell supernatants was analysed and fold changes in the RT activity were normalized to the siNS condition and to the % of infected cells in each condition. Error bars represent the standard deviation from three independent experiments with cells from three different donors each (One-way ANOVA; ns: not significant). b Viral titre in cell supernatants was quantified using the X-gal staining assay in TZM-bl cells and fold changes in viral titre were normalized to the siNS condition and to the % of infected cells in each condition. Error bars represent the standard deviation from three independent experiments with cells from three different donors each (One-way ANOVA; ns: not significant). c Integrated proviral DNA was measured using a combined Alu-HIV-1 PCR and PCR products were visualized in a 1% agarose gel and DNA staining d Fold change in the levels of integrated proviral DNA visualized in C and normalized to the siNS condition. Error bars represent the standard deviation from three independent experiments with cells from three different donors each (One-way ANOVA; ns: not significant). e The NMD target Gas5 mRNA and vRNA levels were measured by RT-PCR and PCR products were visualized on a 1% agarose gel and DNA staining. f Fold change in the levels of vRNA visualized in E and normalized to the siNS HIV-1 + condition. Error bars represent the standard deviation from three independent experiments with cells from three different donors each (One-way ANOVA; ns: not significant, *p ≤ 0.05 and p **p ≤ 0.01)