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Figure 5 | Retrovirology

Figure 5

From: GCN5-dependent acetylation of HIV-1 integrase enhances viral integration

Figure 5

GCN5 depletion in infected cells reduces HIV-1 integration. (A) Left panels: extracts from siRNA-treated Hela cells analyzed by Western blotting with anti-GCN5 antibody (top) or anti-α-tubulin antibody (bottom). Lane 1: cells transfected with non-targeting siRNA (Ctrl siRNA); lane 2: cells transfected with GCN5-targeting siRNA (siGCN5). Right panels: extracts from stable GCN5 knockdown HEK 293T cell clones or control cells immunoblotted with anti-GCN5 antibody (top panel) or anti-α-tubulin antibody (bottom panel). Lane 3: untransduced HEK 293T cells; lane 4: HEK 293T cells carrying empty pGIPZ vector; lane 5: HEK 293T cells expressing mutant, non-targeting GCN5 shRNAmir; lanes 6-8: HEK 293T clones (Cl8, Cl9 and Cl13) expressing GCN5 shRNAmir. (B) siRNA-treated Hela cells (left histogram) or HEK 293T cells stably transduced with pGIPZ lentiviral vectors (right histogram) were infected with NL4.3-Luc and analyzed for luciferase activity 48 hours after infection. The histograms represent percentages of luciferase activity relative to control, non-silenced cells. Means ± SEM from three independent experiments are reported. (C-E) Total DNA extracted from siRNA-treated HeLa cells (left histograms) or HEK 293T cells stably transduced with pGIPZ lentiviral vectors (right histograms) was analyzed by RT-Q-PCR for total HIV-1 DNA (C), integrated HIV-1 DNA (D), and two-LTR circles (E). In (C-E), results are presented as percentages relative to control, non-silenced cells. Reported values are means ± SEM from three independent experiments. Statistical significance values shown in (B-E) were calculated by using the Student's two-sided t test. Asterisks directly above bars indicate differences between knockdown and control, non-silenced cells. ***, P < 0,001; **, P < 0,01; *, P < 0,05. Conversely, where asterisks are not present, values obtained did not significantly differ (P > 0,05) from those obtained with control, non-silenced cells.

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