pcr select cdna subtraction kit  (TaKaRa)

Journal: Science Advances

Article Title: The nuclear import receptor importin-7 targets HPV from the Golgi to the nucleus to promote infection

doi: 10.1126/sciadv.adz6792

Figure Lengend Snippet: ( A ) Number of peptides corresponding to importin β family proteins identified by MS. Immunoprecipitation and sample preparation from HeLa S3 cells expressing traptamer FA-JX4 or control FA were performed as described in fig. S1B. All MS data are in table S1. ( B ) HeLa cells were infected with HPV16.L2F (MOI, ~0.4; GFP reporter plasmid) and treated at infection with 5 to 10 μM importazole (IPZ) or DMSO. At 48 hpi, GFP-positive cells were quantified by flow cytometry and normalized to DMSO (0 μM IPZ) controls. Data ( n = 3) are shown as individual points, means, and SDs; statistical significance was determined versus DMSO-treated cells by two-tailed, unequal variance t test. ** P < 0.01 and *** P < 0.001. ( C ) HeLa cells were transfected with 10 nM of the indicated siRNA for 48 hours and then infected (MOI, ~0.2) with HPV16.L2F (GFP reporter plasmid). At 48 hpi, GFP-positive cells were measured by flow cytometry and normalized to Scr siRNA controls. Data ( n = 3) are analyzed and presented as in (B), with statistical significance determined versus Scr-treated cells. * P < 0.05 and ** P < 0.01. ( D ) HeLa cells were transfected with siRNA-resistant HA-IPO7-mCherry or control HA-mCherry for 24 hours and then with Scr or IPO7 siRNA #2 for 48 hours and infected (MOI, ~0.2) with HPV16.L2F (GFP reporter plasmid). At 48 hpi, GFP expression in mCherry-positive cells was quantified by flow cytometry and normalized to the Scr siRNA + HA-mCherry control. Data ( n = 3) are analyzed and presented as in (B), with statistical significance determined between samples. * P < 0.05 and *** P < 0.001. ( E ) As in (C), except HaCaT cells were analyzed. *** P < 0.001. ( F ) As in (C), except HPV5.L2F (MOI, ~0.4) was used. *** P < 0.001. ( G ) As in (C), except HPV18.L2F (MOI, ~0.4) was used. *** P < 0.001.

Article Snippet: The IPO7 coding sequence was amplified from pUC19-mIPO7 (MG5A1798-U, Sino Biological) and cloned into the pCMVTNT-HA-COPG1-mCherry plasmid ( ) to replace COPG1 .

Techniques: Immunoprecipitation, Sample Prep, Expressing, Control, Infection, Plasmid Preparation, Flow Cytometry, Two Tailed Test, Transfection

Journal: Science Advances

Article Title: The nuclear import receptor importin-7 targets HPV from the Golgi to the nucleus to promote infection

doi: 10.1126/sciadv.adz6792

Figure Lengend Snippet: ( 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.

Article Snippet: The IPO7 coding sequence was amplified from pUC19-mIPO7 (MG5A1798-U, Sino Biological) and cloned into the pCMVTNT-HA-COPG1-mCherry plasmid ( ) to replace COPG1 .

Techniques: Transfection, Infection, Staining, Fluorescence, Labeling, Control, Two Tailed Test

Journal: Science Advances

Article Title: The nuclear import receptor importin-7 targets HPV from the Golgi to the nucleus to promote infection

doi: 10.1126/sciadv.adz6792

Figure Lengend Snippet: ( A ) HeLa cells were mechanically homogenized and fractionated through a 0.5 to 1.6 M sucrose gradient. Portions of each fraction (#1 to #11, top to bottom) were immunoblotted with the indicated antibodies to identify organelle markers. Fractions #3 containing TGN46 and GM130 represents the Golgi-enriched fraction. ( B ) Fraction #3 collected from (A) was subjected to IP with an anti-GM130 antibody or a control IgG and then immunoblotted with the indicated antibodies. ( C ) HeLa S3 cells were transfected with 10 nM Scr or IPO7 siRNA #2 for 48 hours and then uninfected or infected with HPV16.L2F (MOI, ~100). PLA (green) for IPO7 and GM130 was performed at 30 hpi; nuclei were stained with DAPI (blue). Similar results were obtained in two independent experiments. Scale bar, 10 μm. ( D ) PLA fluorescence intensity per cell (>80 cells) as in (C), shown as individual values, means, and SDs, with statistical significance determined by one-way ANOVA. *** P < 0.001 and **** P < 0.0001. ( E ) Uninfected HeLa cells were transfected with the mNG2-SREBP1 reporter construct for 48 hours and then with 10 nM Scr or IPO7 siRNA #2 for another 48 hours. Samples were fixed and permeabilized, and nuclei were stained with DAPI. The nuclear import of mNG2-SREBP1 was indicated by colocalization of mNG2-SREBP1 fluorescence (green) and DAPI (blue). Similar results were obtained in two independent experiments. Scale bar, 10 μm. ( F ) Ratio of nuclear to total mNG2-SREBP1 intensity per cell (>26 mNG-positive cells) as in (E) is plotted as individual values, means, and SDs, with statistical significance determined by two-tailed, unequal variance t test. **** P < 0.0001.

Article Snippet: The IPO7 coding sequence was amplified from pUC19-mIPO7 (MG5A1798-U, Sino Biological) and cloned into the pCMVTNT-HA-COPG1-mCherry plasmid ( ) to replace COPG1 .

Techniques: Control, Transfection, Infection, Staining, Fluorescence, Construct, Two Tailed Test

Journal: Science Advances

Article Title: The nuclear import receptor importin-7 targets HPV from the Golgi to the nucleus to promote infection

doi: 10.1126/sciadv.adz6792

Figure Lengend Snippet: ( 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).

Article Snippet: The IPO7 coding sequence was amplified from pUC19-mIPO7 (MG5A1798-U, Sino Biological) and cloned into the pCMVTNT-HA-COPG1-mCherry plasmid ( ) to replace COPG1 .

Techniques: Infection, Staining, Fluorescence, Western Blot, Control, Transfection

Journal: Science Advances

Article Title: The nuclear import receptor importin-7 targets HPV from the Golgi to the nucleus to promote infection

doi: 10.1126/sciadv.adz6792

Figure Lengend Snippet: ( A ) Diagram of the NLSs in HPV16 L2. Three postulated NLSs are indicated as red boxes, with basic amino acids in the NLSs highlighted in red. The predicted transmembrane domain (TMD) spanning amino acids 45 to 67 is denoted as a black box. The RBS is indicated as a blue box, the amino acids required for retromer binding are highlighted in blue, and the corresponding RBS mutation to alanines are underlined in the DM peptide. The cNLS mutation to alanines are underlined in the 6A peptide. ( B ) Whole-cell extracts of uninfected HeLa cells were incubated with N-terminal biotin-tagged peptides containing residues S296–S316 or S434–R461 of HPV16 L2. The pull-down experiment without peptide was used as a negative control. Samples captured with streptavidin beads were analyzed by immunoblotting for IPO7 or β-actin. ( C ) Coomassie stain of the purified proteins. EGFP-FLAG, C-terminal FLAG-tagged EGFP. FLAG-IPO7, N-terminal FLAG-tagged IPO7. ( D ) N-terminal biotin-tagged peptides containing residues S434–R461 of HPV16 L2 (WT) and the corresponding DM and 6A mutant peptides as indicated in (A) were incubated with purified EGFP-FLAG or FLAG-IPO7. Samples captured with streptavidin beads were analyzed by Western blotting (WB) for FLAG. ( E ) HeLa cells were transfected with indicated DNA constructs for 24 hours to express WT HA-(Δ1–67) L2-3xFLAG (WT) or 6A HA-(Δ1–67) L2-3xFLAG (6A). The cells were lysed, and the resulting extracts were analyzed as in . ( F ) HeLa cells treated with 20 nM Scr or KPNA2 siRNA were uninfected or infected with HPV16.L2F PsV (MOI, ~1). At 24 hpi, cells were lysed and analyzed as in , except antibodies against FLAG, IPO7, KPNA2, or HSP90 (as a loading control) were used to analyze the input.

Article Snippet: The IPO7 coding sequence was amplified from pUC19-mIPO7 (MG5A1798-U, Sino Biological) and cloned into the pCMVTNT-HA-COPG1-mCherry plasmid ( ) to replace COPG1 .

Techniques: Binding Assay, Mutagenesis, Incubation, Negative Control, Western Blot, Staining, Purification, Transfection, Construct, Infection, Control