Fig 1: HPV binds to IPO7 during virus entry.(A) HeLa S3 cells were uninfected or infected with HPV16.L2F (MOI, ~100). PLA (green) for IPO7 and FLAG (to detect L2) was performed at 24 hpi; nuclei were stained with DAPI (blue). Similar results were obtained in two independent experiments. Scale bar, 10 μm. (B) PLA fluorescence intensity per cell (>100 cells) as in (A), shown as individual values, means, and SDs, with statistical significance determined by one-way ANOVA. ****P < 0.0001. (C) Whole-cell extracts from uninfected or HPV16.L2F-infected (MOI, ~1) HeLa cells were collected at the indicated time points. A portion of the resulting extracts (input) was analyzed by immunoblotting for FLAG (to detect L2) and IPO7. The remaining extracts were subjected to IP with an anti-IPO7 antibody or control IgG and analyzed by immunoblotting for FLAG and IPO7. (D) HeLa cells were pretreated with 9 μM RO3306 or DMSO as a control for 24 hours and then uninfected or infected with HPV16.L2F (MOI, ~1), with RO3306 or DMSO maintained in the medium. At 24 hpi, cells were lysed and analyzed in (C). (E) HeLa cells treated with 10 nM Scr or a mixture of 5 nM COPA and 5 nM COPG1 (COPA/G1) siRNA were uninfected or infected with HPV16.L2F PsV (MOI, ~1). At 24 hpi, cells were lysed, and a portion of the extracts (input) was immunoblotted for FLAG, IPO7, γ-COP, or β-actin (as a loading control). The remaining extracts were subjected to IP with an anti-IPO7 antibody or a control IgG and analyzed as in (C). (F) As in (E), except cells were transfected with 10 nM Scr or IPO7 siRNA #2 before infection. Samples were subjected to IP with an anti–γ-COP antibody or control IgG and analyzed as in (E).
Fig 2: IPO7 promotes Golgi-to-nucleus transport of HPV.(A) HeLa S3 cells were transfected with 10 nM Scr or IPO7 siRNA #2 for 48 hours and then left uninfected or infected with HPV16.L2F (MOI, ~100). At 32 hpi, PLA (signals shown in green) was performed with antibodies recognizing FLAG (to detect L2) and GM130. Nuclei were stained with 4ʹ,6-diamidino-2-phenylindole (DAPI; blue). Similar results were obtained in two independent experiments. Scale bar, 10 μm. (B) PLA fluorescence intensity per cell (>100 cells) as in (A), shown as individual values, means, and SDs, with statistical significance determined by one-way analysis of variance (ANOVA). ***P < 0.001 and ****P < 0.0001. (C) As in (A), except infection at MOI of ~150 with HPV16.L2F-containing EdU-labeled genome. Cells were fixed 32 hpi, subjected to Click-iT EdU detection (green), and immunostained for Nesprin-2 (magenta). Scale bar, 10 μm. (D) Nuclear, extranuclear, and total EdU intensity per cell in multiple images as in (C). Using a cutoff defined as the mean intensity per uninfected cell plus one SD, all 89 control cells and 86 of 89 IPO7 KD cells were EdU positive. One-way ANOVA was used to determine statistical significance. ns, not significant; ****P < 0.0001. (E) As in (A), except infection at MOI of ~15. Cells were fixed at 32 hpi and immunostained for FLAG (green) and GM130 (magenta); DNA was stained by DAPI (blue). Cells displaying condensed chromosomes in DAPI staining and fragmented Golgi (GM130 puncta) were identified as mitotic cells. Similar results were obtained in two independent experiments. Scale bars, 10 μm. (F) Ratio of FLAG intensity on condensed chromosomes to total per mitotic cell (>27 mitotic cells) as in (E), shown as individual values, means, and SDs, with statistical significance determined by two-tailed, unequal variance t test. ****P < 0.0001.
Supplier Page from Sino Biological, Inc. for Mouse IPO7 Gene ORF cDNA clone in cloning vector