Human Nucleoporins Promote HIV-1 Docking at the Nuclear Pore, Nuclear Import and Integration (original) (raw)
Figure 1
Lentiviral vector-encoded shRNAs achieve efficient knock-down of human nucleoporins and have negligible cytotoxic or cytostatic effects.
Hela cells (4×106) were transduced with lentiviral vectors (MOI 50) encoding shRNAs specific for the indicated nucleoporins and used at 2 days p.t for Nup153 shRNA and 5 days p.t for all others. (A) Knock-down was assessed by Western blotting using specific antibodies against the targeted nucleoporins in non-transduced (WT), LV-transduced (C) and LV-shRNA transduced cells. ß-actin labelling serves as loading control. Results are representative of at least 3 independent experiments. Numbers to the left of Western blots indicate sizes in KDa. (B) Subcellular localisation of nuclear pore components upon nucleoporin knock-down was tested by confocal fluorescence microscopy of LV- (Control) and LV-shRNA transduced cells using specific anti-Nup antibodies. Images were acquired on the same day with the same conditions and are representative of two independent experiments. (C) Cell viability was determined by detecting mitochondrial activity in living cells using the MTT assay. Results show the mean of two experiments carried out in triplicates +/− SD. (D) Cell cycling was assessed by propidium iodide labelling followed by flow cytometry. x and y ordinates show propidium iodide fluorescence and cell counts, respectively.