Fig. 3
UPF1 overexpression leads to enhanced reactivation of HIV-1 in J-Lat cells. J-Lat 10.6 cells were either mock transfected or transfected with Flag-UPF1 and were uninduced (DMSO) or reactivated (PMA). a Gating strategy to detect UPF1 overexpressing cells by detecting UPF1 mRNA levels by FISH-Flow. b Of the UPF1 overexpressing cells gated for in a, reactivation, monitored by GFP production, was quantified by flow cytometry and the percentages of reactivation were normalised to the mock-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 < 0.05). c Cell lysates were run on acrylamide gels and UPF1 and pr55Gag protein levels were detected by Western Blotting. d Example dot plot depicting vRNA expression in mock transfected and UPF1 overexpressing populations using FISH-Flow technique. e The % of vRNA expressing cells were quantified and normalised to the mock-PMA condition. Error bars represent the standard deviation from three independent experiments. Asterisks represent statistically significant difference between groups (One-way ANOVA; p < 0.01). f J-Lat cells were either mock transfected and uninduced (Mock DMSO), mock transfected and reactivated with PMA (Mock PMA) or transfected with FLAG-UPF1 and left uninduced (FLAG-UPF1 DMSO). The % of vRNA expressing cells were quantified. Error bars represent the standard deviation from three independent experiments. Asterisks represent statistically significant difference between groups (One-way ANOVA; p > 0.05). g J-Lat cells were mock transfected or transfected with FLAG-UPF1, FLAG-UPF1-Δ20-150 or FLAG-UPF1-DE and reactivated using PMA. Cell lysates were run on SDS-PAGE gels and UPF1 and pr55Gag protein levels were detected by Western Blotting. h Reactivation was quantified in FLAG-UPF1-Δ20-150 and FLAG-UPF1-DE expressing cells and the percentages of reactivation were normalised to the mock-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 (One-way ANOVA; p < 0.0001 and p < 0.05 respectively)