Fig. 2

UPF1 knockdown attenuates reactivation of HIV-1 in J-Lat cells. J-Lat 10.6 cells were either transfected with siNS or siUPF1 and were uninduced (DMSO) or reactivated (PMA). a Reactivation, monitored by GFP production, was quantified by Flow cytometry and the percentages of reactivation were normalised to the siNS-PMA reactivated condition. Error bars represent the standard deviation from three independent experiments with at least 10,000 cells counted per treatment. Asterisks represent statistically significant difference between groups (Two-way ANOVA; p < 0.0001). b Cell lysates were run on SDS-PAGE gels and UPF1 and pr55^Gag protein levels were detected by Western Blotting. c Example dot plot depicting vRNA expression in siNS-PMA and siUPF1 PMA conditions using FISH-Flow technique and, d The % of vRNA expressing cells were quantified and normalised to the siNS-PMA condition. Error bars represent the standard deviation from three independent experiments with at least 10,000 cells counted per treatment. Asterisks represent statistically significant difference between groups (Two-way ANOVA; p < 0.0001). e Levels of vRNA were quantified using RT-qPCR and normalised to the siNS-PMA condition. Error bars represent the standard deviation from two independent experiments, each done in triplicate. Asterisks represent statistically significant difference between groups (Two-way ANOVA; p < 0.001). f Gating strategy of cells separated into UPF1 low or high by detecting UPF1 mRNA levels by FISH-Flow. g The % of vRNA expressing cells in each condition normalised to the siNS-PMA/UPF1-high condition. Error bars represent the standard deviation from three independent experiments. Asterisks represent statistically significant difference between groups (One-way ANOVA; p < 0.05). h MFI of the vRNA signal were quantified. Asterisks represent statistically significant difference between groups (student’s t-test; p < 0.01)