ccug 43136  (ATCC)

Journal: JCI Insight

Article Title: C3a and suPAR drive versican V1 expression in tubular cells of focal segmental glomerulosclerosis

doi: 10.1172/jci.insight.122912

Figure Lengend Snippet: (A) Level of serum suPAR in FSGS patients (n = 20). (B) Level of urinary suPAR in FSGS patients (n = 20). (C) Immunohistochemical staining of uPAR in renal tissues of FSGS patients (n = 5). (D) RT-PCR analysis of total versican, versican V1, V0, and V3 in tubular cells treated with 20% PS and uPAR-blocking antibody (n = 5). (E) RT-PCR analysis of total versican, versican V1, V0, and V3 in tubular cells treated with C3a and suPAR (n = 5). (F) RT-PCR analysis of ITGB6 in tubulointerstitial tissues of FSGS patients (n = 20). (G) IP analysis of the binding between suPAR and ITGB6 in tubular cells treated with 20% PS (n = 3). (H) Immunofluorescence staining of uPAR (green) and ITGB6 (red) in tubular cells treated with 20% PS (n = 3). Scale bars: 20 μm (C and H). (I) RT-PCR analysis of total versican, versican V1, V0, and V3 in tubular cells treated with 20% PS and si-ITGB6 (n = 5). (J) Rac1 activation assay in tubular cells treated with 20% PS and si-ITGB6 (n = 3). (K) RT-PCR analysis of total versican, versican V1, V0, and V3 in tubular cells treated with 20% PS and si-Rac1 (n = 5). For statistical analysis, a 2-tailed Student’s t test was used for A, B, and F, and 1-way ANOVA with Tukey’s post hoc test was used for D, E, I, J, and K. *P < 0.05 compared with control; #P < 0.05 compared with PS- or C3a-treated cells.

Article Snippet: CTNNB1 siRNA (sc-29209), ITGB6 siRNA (sc-43135), SRp40 siRNA (sc-38342), and Rac1 siRNA (sc-36351) were purchased from Santa Cruz Biotechnology. pENTR-N90-β-catenin and pcDNA3-EGFP-Rac1-Q61L plasmids were gifts from Xin Chen (Department of Biopharmaceutical Sciences, University of California, San Francisco, CA, USA) (Addgene plasmid 31787) and Gary Bokoch (Departments of Cell Biology and Immunology, Scripps Research Institute, La Jolla, CA, USA) (Addgene plasmid 12981) ( 57 , 58 ). pGEMT-SRp40 plasmid was purchased from Sino Biological (HG16418) and His-tagged versican V3 expression plasmid was from Genecopoeia (Z6286).

Techniques: Immunohistochemical staining, Staining, Reverse Transcription Polymerase Chain Reaction, Blocking Assay, Binding Assay, Immunofluorescence, Activation 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: Silencing of integrin αvβ3 prevents HSV entry. (a) Primary genital tract cells generated from cervical tissue were transfected with siControl RNA or siRNA targeting integrin αvβ3 and were analyzed 72 h later for silencing by Western blotting, or (b) the silenced cells were inoculated with HSV-2(G) (MOI, 1 PFU/cell), and nuclear extracts were prepared 1 h p.i. and analyzed for the presence of the tegument protein VP16 and histone H1 (as a nuclear extract control). Blots shown are representative of results obtained in 2 independent experiments. (c) Transfected primary genital tract cells (siControl or siIntegrinαvβ3) were exposed to HSV-1(K26GFP) (MOI, 5 PFU/cell) and at 1 or 4 h p.i., cells were fixed and viewed by confocal microscopy; nuclei were stained with blue fluorescent Hoechst stain, and plasma membrane was stained with red fluorescent Alexa Fluor 594 wheat germ agglutinin stain. Top panels represent the XY image, bottom panels represent the XZ image. Bar =10 μm. (d) In parallel experiments, cells were infected in the presence of serum-free medium (control) or cilengitide (100 μM), and at 4 h p.i., the cells were fixed and viewed by confocal microscopy. (e) For both siRNA transfection and cilengitide treatment experiments, 100 cells from different fields were counted. Results are presented as the percentage of GFP-positive cells and are means (± SD) from 2 independent experiments; asterisks indicate significance (*, P < 0.05; **, P < 0.01) relative to the respective control.

Article Snippet: Integrin β 6 and integrin β 8 siRNA (sc-43135 and sc-43137, respectively) were purchased from Santa Cruz Biotechnology.

Techniques: Generated, Transfection, Western Blot, Confocal Microscopy, Staining, Infection

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: Silencing of integrin αvβ3, but not other integrins, reduces HSV plaque formation. (a) CaSki cells were transfected with siRNA targeting αv, α5, β1, β3, β6, or β8 alone or a combination of αvβ3 and were infected 72 h later with serial dilutions of HSV-2(G). Viral plaques were counted at 48 h p.i. Results are presented as PFU on siRNA-transfected cells as a percentage of PFU on nontransfected cells and are means (± standard deviations [SD]) from at least 3 independent experiments conducted in duplicate. Only wells in which the number of plaques ranged between 25 and 200 plaques were used to calculate the viral titer. (b) CaSki cells were transfected with siRNA targeting the indicated integrins, and gene expression was determined by RT-PCR at 72 h posttransfection. Results are presented as percent expression relative to that of nontransfected cells and are means (± SD) from at least 3 independent experiments. (c) CaSki cells were transfected with siControl (siCtrl) or siRNA targeting integrin αvβ3 (here termed siIntegrinαvβ3 or siInαvβ3), siIntegrin β3 (siIntβ3), siIntegrin β6 (siIntβ6), or siIntegrin β8 RNA (siIntβ8), and protein expression was evaluated by Western blotting at 72 h posttransfection; the blots are representative of at least 2 independent experiments. (d) Primary vaginal cells cultivated from CVL pellets were transfected with the indicated siRNA and were infected 72 h later with HSV-2(333)ZAG (MOI, 0.1 PFU/cell) and monitored for GFP expression; images are representative of 2 independent experiments. Asterisks indicate significant difference relative to the control (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Article Snippet: Integrin β 6 and integrin β 8 siRNA (sc-43135 and sc-43137, respectively) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

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: Integrin β 6 and integrin β 8 siRNA (sc-43135 and sc-43137, respectively) were purchased from Santa Cruz Biotechnology.

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: Integrin β 6 and integrin β 8 siRNA (sc-43135 and sc-43137, respectively) were purchased from Santa Cruz Biotechnology.

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: Integrin αvβ3 interacts with glycoprotein H. (a) CaSki cells were synchronously infected with purified HSV-2 (5 PFU/cell), and cell lysates were harvested 2 and 15 min post-temperature shift and incubated with monoclonal mouse anti-integrin αvβ3 (left panels) or an isotype control MAb (middle panel). Immune complexes were precipitated, and equivalent volumes of the whole-cell lysate (starting material), supernatant, and pellet were subjected to Western blotting with rabbit anti-gH-gL (upper left) or goat anti-gB (lower left) antibodies. In reciprocal experiments, lysates were precipitated with monoclonal antibodies to gH-gL and analyzed by Western blotting with rabbit polyclonal antibodies to integrin αv (right panel). Controls included uninfected cell lysates; blots shown are representative of 5 independent experiments. (b) CaSki cells were synchronously infected with purified HSV-2(G) (5 PFU/cell) (no siRNA) 72 h after being transfected with the indicated siRNA. The cells were subsequently fixed and probed with monoclonal mouse antibodies (MAb) to integrin αvβ3 or gD and rabbit sera (RIg) to gH-gL or nectin-1 and assessed in a proximity ligation assay. (c) Additional proximity ligation studies were conducted with nontransfected CaSki cells that were synchronously infected with HSV-2(G) as in panel b and fixed and probed with the indicated antibodies. Proximity ligation results are representative of 2 independent experiments. Bar = 10 μM.

Article Snippet: Integrin β 6 and integrin β 8 siRNA (sc-43135 and sc-43137, respectively) were purchased from Santa Cruz Biotechnology.

Techniques: Infection, Purification, Incubation, Western Blot, Transfection, Proximity Ligation Assay, Ligation

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: Construction and characterization of an HSV-2 gH-null virus. (a) Genotypic characterization of ΔgH-2 was performed by PCR using two primer sets to confirm appropriate replacement of gH. The left flank of UL22 was tested with primers gH2-L-check and sacB-Out, while the right flank of this gene was assessed with primers gH2-R-check and Hyg-Out (Table 1). (b) The gH-null virus was propagated on gH-1-expressing F6 cells to yield complemented virus (ΔgH−/+) or on Vero cells to yield noncomplemented virus (ΔgH−/−). The viruses were purified on sucrose gradients, and equivalent numbers of viral particles (estimated by comparing VP5 expression on Western blots) were analyzed for expression of viral proteins by immunoblotting with rabbit polyclonal Ab to gH-gL or murine MAbs to gB, gD, gC, and VP5. (c) Representative fluorescence microscopy image obtained 36 h p.i. of F6 or Vero cells with ΔgH−/+. (d) To evaluate whether deletion of gH impacted binding, CaSki cells were exposed to serial 2-fold dilutions of relatively equal numbers of purified complemented or noncomplemented gH-null viruses (starting with viral particle numbers equivalent to an MOI of 5 PFU/cell) at 4°C for 4 h. Binding was assessed by performing Western blots of cellular lysates with gD and β-actin, and results shown are representative of 3 independent experiments. (e) CaSki cells were inoculated with purified virus (relative particle numbers equivalent to an MOI of 1 PFU/cell on F6 cells), and nuclear extracts were prepared 1 h p.i. and probed for the tegument protein VP16 and histone 1 (H1). A blot representative of results from 3 independent experiments is shown. (f) CaSki cells were synchronously infected with purified ΔgH−/+ or ΔgH−/− (equivalent to 5 PFU/cell) and fixed and probed with monoclonal mouse antibodies to integrin αvβ3 and rabbit sera to gH-gL and assessed in a proximity ligation assay. Results are representative of 3 independent experiments.

Article Snippet: Integrin β 6 and integrin β 8 siRNA (sc-43135 and sc-43137, respectively) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Purification, Western Blot, Fluorescence, Microscopy, Binding Assay, Infection, Proximity Ligation 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: Integrin αvβ3-gH interactions trigger release of cytosolic Ca2+. (a) CaSki cells were loaded with Calcium Green and synchronously infected with ΔgH2−/+ or ΔgH2−/− viruses (equivalent to 5 PFU/cell) or mock infected (medium). Live images were acquired at the indicated time post-temperature shift. Nuclei were stained with Hoechst (blue), and plasma membrane was stained with red fluorescent Alexa Fluor 594 wheat germ agglutinin. Representative extended-focus images from 3 independent experiments are shown. (b) CaSki or primary genital tract cells were loaded with fura-2 and exposed to ΔgH2−/+ or ΔgH2−/− viruses (equivalent to 5 PFU/cell), and the mean intracellular Ca2+ concentration (nM) over 1 h was calculated from 4 wells, each containing 5 ×104 CaSki (upper panel) or 3 × 104 primary (lower panel) cells; the asterisk indicates significant increase in Ca2+ concentration relative to that seen with mock infection (P < 0.01). (c) CaSki cells were exposed to ΔgH2−/+ or ΔgH2−/− viruses (equivalent to 2 PFU/cell) for 4 h at 4°C, unbound virus was removed by washing, and cells were shifted to 37°C for 15 min (synchronous infection). The cells were then fixed and stained for a proximity ligation assay with mouse MAb to gB and rabbit polyclonal antibodies to Akt. Images are representative of results of 2 independent experiments.

Article Snippet: Integrin β 6 and integrin β 8 siRNA (sc-43135 and sc-43137, respectively) were purchased from Santa Cruz Biotechnology.

Techniques: Infection, Staining, Concentration Assay, Proximity Ligation 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: Integrin αvβ3-gH interactions are required for FAK phosphorylation. (a) CaSki cells transfected with the indicated siRNA and were exposed 72 h later to serum-free medium (mock-infected) or infected with HSV-2(G) (MOI, 10 PFU/cell) and cellular lysates prepared 5 min p.i. Western blots were performed and probed for phosphorylated FAK (pY397FAK), total FAK, integrin αv, and β-actin. A blot representative of 3 independent experiments is shown. The blots were scanned, and the mean percentage of phosphorylated FAK relative to total FAK from the 3 independent experiments is indicated. (b) To assess the role of gH, CaSki cells were infected with ΔgH2−/+ or ΔgH2−/− viruses (equivalent to 5 PFU/cell), and at the indicated times p.i., analyzed for phosphorylated and total FAK. Results are representative of 2 independent experiments.

Article Snippet: Integrin β 6 and integrin β 8 siRNA (sc-43135 and sc-43137, respectively) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Infection, Western Blot

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 is important for cell-to-cell spread. (a) CaSki cells were transfected with the indicated siRNA and 72 h later were infected with HSV-2(G). Plaques were visualized by immunostaining and representative images are shown. (b) To further assess the impact of integrin αvβ3 on cell-to-cell spread, a modified infectious center assay was performed in which “donor” cells were labeled with Mitotracker Orange, infected with HSV-2(333)ZAG, treated with a low-pH citrate buffer at 1 h p.i. to inactivate any extracellular virus. At 4 h p.i., the donor cells were trypsinized and plated at a ratio of 1:5 on “receiver” cells that had been transfected 72 h earlier with the indicated siRNA and then cocultured in medium containing pooled human immunoglobulin. The cocultures were incubated for 12 h, washed, fixed, and mounted in anti-fade reagent with DAPI to stain nuclei (blue). Representative images from 10 different fields and a minimum of 2 independent experiments are shown. Upper left, DAPI channel; upper right, GFP channel; lower left, Mitotracker Orange; and lower right, merge.

Article Snippet: Integrin β 6 and integrin β 8 siRNA (sc-43135 and sc-43137, respectively) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Infection, Immunostaining, Modification, Labeling, Incubation, Staining

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: Silencing of integrin αvβ3 prevents HSV entry. (a) Primary genital tract cells generated from cervical tissue were transfected with siControl RNA or siRNA targeting integrin αvβ3 and were analyzed 72 h later for silencing by Western blotting, or (b) the silenced cells were inoculated with HSV-2(G) (MOI, 1 PFU/cell), and nuclear extracts were prepared 1 h p.i. and analyzed for the presence of the tegument protein VP16 and histone H1 (as a nuclear extract control). Blots shown are representative of results obtained in 2 independent experiments. (c) Transfected primary genital tract cells (siControl or siIntegrinαvβ3) were exposed to HSV-1(K26GFP) (MOI, 5 PFU/cell) and at 1 or 4 h p.i., cells were fixed and viewed by confocal microscopy; nuclei were stained with blue fluorescent Hoechst stain, and plasma membrane was stained with red fluorescent Alexa Fluor 594 wheat germ agglutinin stain. Top panels represent the XY image, bottom panels represent the XZ image. Bar =10 μm. (d) In parallel experiments, cells were infected in the presence of serum-free medium (control) or cilengitide (100 μM), and at 4 h p.i., the cells were fixed and viewed by confocal microscopy. (e) For both siRNA transfection and cilengitide treatment experiments, 100 cells from different fields were counted. Results are presented as the percentage of GFP-positive cells and are means (± SD) from 2 independent experiments; asterisks indicate significance (*, P < 0.05; **, P < 0.01) relative to the respective control.

Article Snippet: Integrin β 6 and integrin β 8 siRNA (sc-43135 and sc-43137, respectively) were purchased from Santa Cruz Biotechnology.

Techniques: Generated, Transfection, Western Blot, Confocal Microscopy, Staining, Infection

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: Silencing of integrin αvβ3, but not other integrins, reduces HSV plaque formation. (a) CaSki cells were transfected with siRNA targeting αv, α5, β1, β3, β6, or β8 alone or a combination of αvβ3 and were infected 72 h later with serial dilutions of HSV-2(G). Viral plaques were counted at 48 h p.i. Results are presented as PFU on siRNA-transfected cells as a percentage of PFU on nontransfected cells and are means (± standard deviations [SD]) from at least 3 independent experiments conducted in duplicate. Only wells in which the number of plaques ranged between 25 and 200 plaques were used to calculate the viral titer. (b) CaSki cells were transfected with siRNA targeting the indicated integrins, and gene expression was determined by RT-PCR at 72 h posttransfection. Results are presented as percent expression relative to that of nontransfected cells and are means (± SD) from at least 3 independent experiments. (c) CaSki cells were transfected with siControl (siCtrl) or siRNA targeting integrin αvβ3 (here termed siIntegrinαvβ3 or siInαvβ3), siIntegrin β3 (siIntβ3), siIntegrin β6 (siIntβ6), or siIntegrin β8 RNA (siIntβ8), and protein expression was evaluated by Western blotting at 72 h posttransfection; the blots are representative of at least 2 independent experiments. (d) Primary vaginal cells cultivated from CVL pellets were transfected with the indicated siRNA and were infected 72 h later with HSV-2(333)ZAG (MOI, 0.1 PFU/cell) and monitored for GFP expression; images are representative of 2 independent experiments. Asterisks indicate significant difference relative to the control (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Article Snippet: Integrin β 6 and integrin β 8 siRNA (sc-43135 and sc-43137, respectively) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

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: Integrin β 6 and integrin β 8 siRNA (sc-43135 and sc-43137, respectively) were purchased from Santa Cruz Biotechnology.

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: Integrin β 6 and integrin β 8 siRNA (sc-43135 and sc-43137, respectively) were purchased from Santa Cruz Biotechnology.

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: Integrin αvβ3 interacts with glycoprotein H. (a) CaSki cells were synchronously infected with purified HSV-2 (5 PFU/cell), and cell lysates were harvested 2 and 15 min post-temperature shift and incubated with monoclonal mouse anti-integrin αvβ3 (left panels) or an isotype control MAb (middle panel). Immune complexes were precipitated, and equivalent volumes of the whole-cell lysate (starting material), supernatant, and pellet were subjected to Western blotting with rabbit anti-gH-gL (upper left) or goat anti-gB (lower left) antibodies. In reciprocal experiments, lysates were precipitated with monoclonal antibodies to gH-gL and analyzed by Western blotting with rabbit polyclonal antibodies to integrin αv (right panel). Controls included uninfected cell lysates; blots shown are representative of 5 independent experiments. (b) CaSki cells were synchronously infected with purified HSV-2(G) (5 PFU/cell) (no siRNA) 72 h after being transfected with the indicated siRNA. The cells were subsequently fixed and probed with monoclonal mouse antibodies (MAb) to integrin αvβ3 or gD and rabbit sera (RIg) to gH-gL or nectin-1 and assessed in a proximity ligation assay. (c) Additional proximity ligation studies were conducted with nontransfected CaSki cells that were synchronously infected with HSV-2(G) as in panel b and fixed and probed with the indicated antibodies. Proximity ligation results are representative of 2 independent experiments. Bar = 10 μM.

Article Snippet: Integrin β 6 and integrin β 8 siRNA (sc-43135 and sc-43137, respectively) were purchased from Santa Cruz Biotechnology.

Techniques: Infection, Purification, Incubation, Western Blot, Transfection, Proximity Ligation Assay, Ligation

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: Construction and characterization of an HSV-2 gH-null virus. (a) Genotypic characterization of ΔgH-2 was performed by PCR using two primer sets to confirm appropriate replacement of gH. The left flank of UL22 was tested with primers gH2-L-check and sacB-Out, while the right flank of this gene was assessed with primers gH2-R-check and Hyg-Out (Table 1). (b) The gH-null virus was propagated on gH-1-expressing F6 cells to yield complemented virus (ΔgH−/+) or on Vero cells to yield noncomplemented virus (ΔgH−/−). The viruses were purified on sucrose gradients, and equivalent numbers of viral particles (estimated by comparing VP5 expression on Western blots) were analyzed for expression of viral proteins by immunoblotting with rabbit polyclonal Ab to gH-gL or murine MAbs to gB, gD, gC, and VP5. (c) Representative fluorescence microscopy image obtained 36 h p.i. of F6 or Vero cells with ΔgH−/+. (d) To evaluate whether deletion of gH impacted binding, CaSki cells were exposed to serial 2-fold dilutions of relatively equal numbers of purified complemented or noncomplemented gH-null viruses (starting with viral particle numbers equivalent to an MOI of 5 PFU/cell) at 4°C for 4 h. Binding was assessed by performing Western blots of cellular lysates with gD and β-actin, and results shown are representative of 3 independent experiments. (e) CaSki cells were inoculated with purified virus (relative particle numbers equivalent to an MOI of 1 PFU/cell on F6 cells), and nuclear extracts were prepared 1 h p.i. and probed for the tegument protein VP16 and histone 1 (H1). A blot representative of results from 3 independent experiments is shown. (f) CaSki cells were synchronously infected with purified ΔgH−/+ or ΔgH−/− (equivalent to 5 PFU/cell) and fixed and probed with monoclonal mouse antibodies to integrin αvβ3 and rabbit sera to gH-gL and assessed in a proximity ligation assay. Results are representative of 3 independent experiments.

Article Snippet: Integrin β 6 and integrin β 8 siRNA (sc-43135 and sc-43137, respectively) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Purification, Western Blot, Fluorescence, Microscopy, Binding Assay, Infection, Proximity Ligation 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: Integrin αvβ3-gH interactions trigger release of cytosolic Ca2+. (a) CaSki cells were loaded with Calcium Green and synchronously infected with ΔgH2−/+ or ΔgH2−/− viruses (equivalent to 5 PFU/cell) or mock infected (medium). Live images were acquired at the indicated time post-temperature shift. Nuclei were stained with Hoechst (blue), and plasma membrane was stained with red fluorescent Alexa Fluor 594 wheat germ agglutinin. Representative extended-focus images from 3 independent experiments are shown. (b) CaSki or primary genital tract cells were loaded with fura-2 and exposed to ΔgH2−/+ or ΔgH2−/− viruses (equivalent to 5 PFU/cell), and the mean intracellular Ca2+ concentration (nM) over 1 h was calculated from 4 wells, each containing 5 ×104 CaSki (upper panel) or 3 × 104 primary (lower panel) cells; the asterisk indicates significant increase in Ca2+ concentration relative to that seen with mock infection (P < 0.01). (c) CaSki cells were exposed to ΔgH2−/+ or ΔgH2−/− viruses (equivalent to 2 PFU/cell) for 4 h at 4°C, unbound virus was removed by washing, and cells were shifted to 37°C for 15 min (synchronous infection). The cells were then fixed and stained for a proximity ligation assay with mouse MAb to gB and rabbit polyclonal antibodies to Akt. Images are representative of results of 2 independent experiments.

Article Snippet: Integrin β 6 and integrin β 8 siRNA (sc-43135 and sc-43137, respectively) were purchased from Santa Cruz Biotechnology.

Techniques: Infection, Staining, Concentration Assay, Proximity Ligation 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: Integrin αvβ3-gH interactions are required for FAK phosphorylation. (a) CaSki cells transfected with the indicated siRNA and were exposed 72 h later to serum-free medium (mock-infected) or infected with HSV-2(G) (MOI, 10 PFU/cell) and cellular lysates prepared 5 min p.i. Western blots were performed and probed for phosphorylated FAK (pY397FAK), total FAK, integrin αv, and β-actin. A blot representative of 3 independent experiments is shown. The blots were scanned, and the mean percentage of phosphorylated FAK relative to total FAK from the 3 independent experiments is indicated. (b) To assess the role of gH, CaSki cells were infected with ΔgH2−/+ or ΔgH2−/− viruses (equivalent to 5 PFU/cell), and at the indicated times p.i., analyzed for phosphorylated and total FAK. Results are representative of 2 independent experiments.

Article Snippet: Integrin β 6 and integrin β 8 siRNA (sc-43135 and sc-43137, respectively) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Infection, Western Blot

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 is important for cell-to-cell spread. (a) CaSki cells were transfected with the indicated siRNA and 72 h later were infected with HSV-2(G). Plaques were visualized by immunostaining and representative images are shown. (b) To further assess the impact of integrin αvβ3 on cell-to-cell spread, a modified infectious center assay was performed in which “donor” cells were labeled with Mitotracker Orange, infected with HSV-2(333)ZAG, treated with a low-pH citrate buffer at 1 h p.i. to inactivate any extracellular virus. At 4 h p.i., the donor cells were trypsinized and plated at a ratio of 1:5 on “receiver” cells that had been transfected 72 h earlier with the indicated siRNA and then cocultured in medium containing pooled human immunoglobulin. The cocultures were incubated for 12 h, washed, fixed, and mounted in anti-fade reagent with DAPI to stain nuclei (blue). Representative images from 10 different fields and a minimum of 2 independent experiments are shown. Upper left, DAPI channel; upper right, GFP channel; lower left, Mitotracker Orange; and lower right, merge.

Article Snippet: Integrin β 6 and integrin β 8 siRNA (sc-43135 and sc-43137, respectively) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Infection, Immunostaining, Modification, Labeling, Incubation, Staining