Journal: Journal of Virology

Article Title: Herpes Simplex Virus Type 2 Glycoprotein H Interacts with Integrin αvβ3 To Facilitate Viral Entry and Calcium Signaling in Human Genital Tract Epithelial Cells

doi: 10.1128/JVI.00725-14

Figure Lengend Snippet: Glycoprotein H is not required for Akt or integrin αvβ3 relocalization. CaSki cells were synchronously infected with ΔgH2−/+ or ΔgH2−/− viruses (equivalent to 5 PFU/cell), and at 15 min post-temperature shift, the cells were fixed and stained with rabbit polyclonal Ab to Akt123 (green) and mouse MAb to integrin αvβ3 (red). Nuclei were stained with DAPI (blue). For comparison, mock-infected cells were also permeabilized (perm) with 1% Triton X-100. Representative XYZ images from 2 independent experiments are shown in panel a, and the mean (± standard error of the mean [SEM]) fluorescence intensity (MFI) per cell calculated from 100 cells for Akt (green) is shown in panel b and that for integrin (red) in panel c. The asterisks indicate significant increases in MFI relative to the nonpermeabilized mock-infected cells (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Article Snippet: Primary antibodies and dilutions for Western blots, confocal microscopy, and coimmunoprecipitation were as follows: mouse monoclonal antibody (MAb) anti-VP16, 1:500 (catalog no. sc-7545; Santa Cruz Biotechnology [sc], Santa Cruz, CA); anti-VP5 MAb, 1:200 (sc-56989); anti-gB MAb, 1:500 (sc-69799), goat anti-gB, 1:200 (sc-22090); anti-gD MAb, 1:1,000 (sc-56988); anti-gC MAb, 1:200 (sc-69801); anti-gH-gL MAb, 1:200 (CH31, gift from R. Eisenberg and G. Cohen, University of Pennsylvania); anti-β-actin MAb, 1:5,000 (A-5441; Sigma-Aldrich, St. Louis, MO); anti-phospho-Akt (ser 473) MAb, 1:500 (4051; Cell Signaling Technology Inc., Danvers, MA); anti-histone H1 MAb, 1:250 (sc-8030), rabbit anti-total Akt123, 1:1,000 (sc-8312); rabbit anti-pY 397 focal adhesion kinase (FAK), 1:1,000 (44-625G; Invitrogen), anti-total FAK MAb, 1:1,000 (610087; BD Bioscience), rabbit anti-goat immunoglobulin G (IgG), 1:500 (HAF017; R&D, Minneapolis, MN); goat polyclonal anti-integrin β8, 1:200 (sc-6638); goat polyclonal anti-integrin β6, 1:200 (sc-6633); rabbit polyclonal anti-integrin αv, 1:500 (sc6617-R); mouse anti-integrin αv, 1:200 (sc-9969); mouse anti-integrin αvβ3, 1:200 (sc7312); mouse anti-integrin β3, 1:50 (ab7167, Abcam, Cambridge, MA); rabbit anti-gH-gL, 1:500 (R137; gift from R Eisenberg and G. Cohen, University of Pennsylvania); rabbit polyclonal anti-PVRL1 (nectin-1), 1:100 (ab66985); and rabbit polyclonal anti-integrin β3, 1:500 (sc-14009).

Techniques: Infection, Staining, Fluorescence

Journal: Journal of Virology

Article Title: Herpes Simplex Virus Type 2 Glycoprotein H Interacts with Integrin αvβ3 To Facilitate Viral Entry and Calcium Signaling in Human Genital Tract Epithelial Cells

doi: 10.1128/JVI.00725-14

Figure Lengend Snippet: Integrin αvβ3 contributes to the virus-induced cytoplasmic Ca2+ response post-Akt phosphorylation. (a) CaSki cells transfected with siIntegrinαvβ3 RNA were mock infected (serum free medium) or infected with purified HSV-2(G) (10 PFU/cell), and cell lysates were prepared for Western blot analysis at the indicated times p.i. Blots were incubated with anti pS473-Akt123 and then stripped and probed with anti-total Akt123. A representative blot from 3 independent experiments is shown. (b) CaSki cells were loaded with Calcium Green 72 h posttransfection with the indicated siRNA and synchronously mock infected or infected with purified HSV-2(G) (5 PFU/cell). Live images were acquired 3 min after a temperature shift to 37°C. Cellular membranes were stained with CellTrace (red), nuclei were stained with Hoechst (blue), and Ca2+ is green. Representative XYZ images from 3 independent experiments are shown. Bars = 9.2 μm. (c) Transfected CaSki cells were loaded with fura-2, infected with purified HSV-2(G) (2 PFU/cell), or mock infected. To assess whether siRNA transfections impacted the intracellular Ca2+ stores, uninfected cells were treated with 1 μM ionomycin. The mean intracellular Ca2+ concentration (nM) over 1 h was calculated from 4 wells, each containing 5 × 104 cells; the asterisk indicates a significant increase in Ca2+ concentration relative to the mock-infected control (P < 0.05). (d) Parallel studies were conducted with cells treated with calcium-free buffer (control) or buffer supplemented with cilengitide (100 μM), and the Ca2+ response over 1 h p.i. was compared to that of mock-infected cells.

Article Snippet: Primary antibodies and dilutions for Western blots, confocal microscopy, and coimmunoprecipitation were as follows: mouse monoclonal antibody (MAb) anti-VP16, 1:500 (catalog no. sc-7545; Santa Cruz Biotechnology [sc], Santa Cruz, CA); anti-VP5 MAb, 1:200 (sc-56989); anti-gB MAb, 1:500 (sc-69799), goat anti-gB, 1:200 (sc-22090); anti-gD MAb, 1:1,000 (sc-56988); anti-gC MAb, 1:200 (sc-69801); anti-gH-gL MAb, 1:200 (CH31, gift from R. Eisenberg and G. Cohen, University of Pennsylvania); anti-β-actin MAb, 1:5,000 (A-5441; Sigma-Aldrich, St. Louis, MO); anti-phospho-Akt (ser 473) MAb, 1:500 (4051; Cell Signaling Technology Inc., Danvers, MA); anti-histone H1 MAb, 1:250 (sc-8030), rabbit anti-total Akt123, 1:1,000 (sc-8312); rabbit anti-pY 397 focal adhesion kinase (FAK), 1:1,000 (44-625G; Invitrogen), anti-total FAK MAb, 1:1,000 (610087; BD Bioscience), rabbit anti-goat immunoglobulin G (IgG), 1:500 (HAF017; R&D, Minneapolis, MN); goat polyclonal anti-integrin β8, 1:200 (sc-6638); goat polyclonal anti-integrin β6, 1:200 (sc-6633); rabbit polyclonal anti-integrin αv, 1:500 (sc6617-R); mouse anti-integrin αv, 1:200 (sc-9969); mouse anti-integrin αvβ3, 1:200 (sc7312); mouse anti-integrin β3, 1:50 (ab7167, Abcam, Cambridge, MA); rabbit anti-gH-gL, 1:500 (R137; gift from R Eisenberg and G. Cohen, University of Pennsylvania); rabbit polyclonal anti-PVRL1 (nectin-1), 1:100 (ab66985); and rabbit polyclonal anti-integrin β3, 1:500 (sc-14009).

Techniques: Transfection, Infection, Purification, Western Blot, Incubation, Staining, Concentration Assay

Journal: Journal of Virology

Article Title: Herpes Simplex Virus Type 2 Glycoprotein H Interacts with Integrin αvβ3 To Facilitate Viral Entry and Calcium Signaling in Human Genital Tract Epithelial Cells

doi: 10.1128/JVI.00725-14

Figure Lengend Snippet: Glycoprotein H is not required for Akt or integrin αvβ3 relocalization. CaSki cells were synchronously infected with ΔgH2−/+ or ΔgH2−/− viruses (equivalent to 5 PFU/cell), and at 15 min post-temperature shift, the cells were fixed and stained with rabbit polyclonal Ab to Akt123 (green) and mouse MAb to integrin αvβ3 (red). Nuclei were stained with DAPI (blue). For comparison, mock-infected cells were also permeabilized (perm) with 1% Triton X-100. Representative XYZ images from 2 independent experiments are shown in panel a, and the mean (± standard error of the mean [SEM]) fluorescence intensity (MFI) per cell calculated from 100 cells for Akt (green) is shown in panel b and that for integrin (red) in panel c. The asterisks indicate significant increases in MFI relative to the nonpermeabilized mock-infected cells (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Article Snippet: Primary antibodies and dilutions for Western blots, confocal microscopy, and coimmunoprecipitation were as follows: mouse monoclonal antibody (MAb) anti-VP16, 1:500 (catalog no. sc-7545; Santa Cruz Biotechnology [sc], Santa Cruz, CA); anti-VP5 MAb, 1:200 (sc-56989); anti-gB MAb, 1:500 (sc-69799), goat anti-gB, 1:200 (sc-22090); anti-gD MAb, 1:1,000 (sc-56988); anti-gC MAb, 1:200 (sc-69801); anti-gH-gL MAb, 1:200 (CH31, gift from R. Eisenberg and G. Cohen, University of Pennsylvania); anti-β-actin MAb, 1:5,000 (A-5441; Sigma-Aldrich, St. Louis, MO); anti-phospho-Akt (ser 473) MAb, 1:500 (4051; Cell Signaling Technology Inc., Danvers, MA); anti-histone H1 MAb, 1:250 (sc-8030), rabbit anti-total Akt123, 1:1,000 (sc-8312); rabbit anti-pY 397 focal adhesion kinase (FAK), 1:1,000 (44-625G; Invitrogen), anti-total FAK MAb, 1:1,000 (610087; BD Bioscience), rabbit anti-goat immunoglobulin G (IgG), 1:500 (HAF017; R&D, Minneapolis, MN); goat polyclonal anti-integrin β8, 1:200 (sc-6638); goat polyclonal anti-integrin β6, 1:200 (sc-6633); rabbit polyclonal anti-integrin αv, 1:500 (sc6617-R); mouse anti-integrin αv, 1:200 (sc-9969); mouse anti-integrin αvβ3, 1:200 (sc7312); mouse anti-integrin β3, 1:50 (ab7167, Abcam, Cambridge, MA); rabbit anti-gH-gL, 1:500 (R137; gift from R Eisenberg and G. Cohen, University of Pennsylvania); rabbit polyclonal anti-PVRL1 (nectin-1), 1:100 (ab66985); and rabbit polyclonal anti-integrin β3, 1:500 (sc-14009).

Techniques: Infection, Staining, Fluorescence

Journal: Journal of Virology

Article Title: Herpes Simplex Virus Type 2 Glycoprotein H Interacts with Integrin αvβ3 To Facilitate Viral Entry and Calcium Signaling in Human Genital Tract Epithelial Cells

doi: 10.1128/JVI.00725-14

Figure Lengend Snippet: Integrin αvβ3 contributes to the virus-induced cytoplasmic Ca2+ response post-Akt phosphorylation. (a) CaSki cells transfected with siIntegrinαvβ3 RNA were mock infected (serum free medium) or infected with purified HSV-2(G) (10 PFU/cell), and cell lysates were prepared for Western blot analysis at the indicated times p.i. Blots were incubated with anti pS473-Akt123 and then stripped and probed with anti-total Akt123. A representative blot from 3 independent experiments is shown. (b) CaSki cells were loaded with Calcium Green 72 h posttransfection with the indicated siRNA and synchronously mock infected or infected with purified HSV-2(G) (5 PFU/cell). Live images were acquired 3 min after a temperature shift to 37°C. Cellular membranes were stained with CellTrace (red), nuclei were stained with Hoechst (blue), and Ca2+ is green. Representative XYZ images from 3 independent experiments are shown. Bars = 9.2 μm. (c) Transfected CaSki cells were loaded with fura-2, infected with purified HSV-2(G) (2 PFU/cell), or mock infected. To assess whether siRNA transfections impacted the intracellular Ca2+ stores, uninfected cells were treated with 1 μM ionomycin. The mean intracellular Ca2+ concentration (nM) over 1 h was calculated from 4 wells, each containing 5 × 104 cells; the asterisk indicates a significant increase in Ca2+ concentration relative to the mock-infected control (P < 0.05). (d) Parallel studies were conducted with cells treated with calcium-free buffer (control) or buffer supplemented with cilengitide (100 μM), and the Ca2+ response over 1 h p.i. was compared to that of mock-infected cells.

Article Snippet: Primary antibodies and dilutions for Western blots, confocal microscopy, and coimmunoprecipitation were as follows: mouse monoclonal antibody (MAb) anti-VP16, 1:500 (catalog no. sc-7545; Santa Cruz Biotechnology [sc], Santa Cruz, CA); anti-VP5 MAb, 1:200 (sc-56989); anti-gB MAb, 1:500 (sc-69799), goat anti-gB, 1:200 (sc-22090); anti-gD MAb, 1:1,000 (sc-56988); anti-gC MAb, 1:200 (sc-69801); anti-gH-gL MAb, 1:200 (CH31, gift from R. Eisenberg and G. Cohen, University of Pennsylvania); anti-β-actin MAb, 1:5,000 (A-5441; Sigma-Aldrich, St. Louis, MO); anti-phospho-Akt (ser 473) MAb, 1:500 (4051; Cell Signaling Technology Inc., Danvers, MA); anti-histone H1 MAb, 1:250 (sc-8030), rabbit anti-total Akt123, 1:1,000 (sc-8312); rabbit anti-pY 397 focal adhesion kinase (FAK), 1:1,000 (44-625G; Invitrogen), anti-total FAK MAb, 1:1,000 (610087; BD Bioscience), rabbit anti-goat immunoglobulin G (IgG), 1:500 (HAF017; R&D, Minneapolis, MN); goat polyclonal anti-integrin β8, 1:200 (sc-6638); goat polyclonal anti-integrin β6, 1:200 (sc-6633); rabbit polyclonal anti-integrin αv, 1:500 (sc6617-R); mouse anti-integrin αv, 1:200 (sc-9969); mouse anti-integrin αvβ3, 1:200 (sc7312); mouse anti-integrin β3, 1:50 (ab7167, Abcam, Cambridge, MA); rabbit anti-gH-gL, 1:500 (R137; gift from R Eisenberg and G. Cohen, University of Pennsylvania); rabbit polyclonal anti-PVRL1 (nectin-1), 1:100 (ab66985); and rabbit polyclonal anti-integrin β3, 1:500 (sc-14009).

Techniques: Transfection, Infection, Purification, Western Blot, Incubation, Staining, Concentration Assay