integrin β 3 sirna  (Santa Cruz Biotechnology)

Journal: Nature Communications

Article Title: GABA-independent activation of GABA B receptor by mechanical forces

doi: 10.1038/s41467-025-64811-2

Figure Lengend Snippet: a IP 1 production in HEK293 cells transfected with either control siRNA or integrin β 3 siRNA, along with the expressing of vector and G qi9 , or GABA B receptor and G qi9 , under either adhesion or suspension conditions. Data are present as mean ± s.e.m. from five biologically independent experiments and analyzed using unpaired t test (two-tailed) to determine significance. *** P < 0.001, not significant (ns) > 0.05. b Co-immunoprecipitation of GABA B receptor and integrin β 3 in HEK293 cells transfected with GABA B receptor constructs (Snap-tagged GB1 and GB2) using anti-integrin β 3 antibody, under basal condition. Only GB1 in cell surface was labeled and visualized using an impermeable Snap fluorescent substrate. c –f Co-immunoprecipitation of GABA B receptor and integrin β 3 in HEK293 cells transfected with GABA B receptor constructs (HA-tagged GB1 and GB2) using anti-HA antibody, under conditions including suspension or adhesion ( c ), without (control) or with shear stress ( c ), Blebbistatin (50 μM, 30 min) treatment ( d ), RGDS (10 μM, 12 h) treatment ( e ), or CGP54626 (50 μM, 30 min) treatment ( f ). Blots are representative from at least three biologically independent experiments ( b , n = 4; c , suspension or adhesion, n = 3; control or with shear stress, n = 4; d , n = 3; e, n = 3; f , n = 4). The amount of integrin β 3 immunoprecipitated by IgG or HA antibody are present as mean ± s.e.m. in ( e ) and analyzed using unpaired t test (two-tailed) to determine significance. ** P < 0.01. g Schematic representation of the BRET assay detecting direct interaction between GABA B receptor and integrin β 3 . Rluc was fused in C-terminal of GB1 or GB2 subunit (GB1 Rluc or GB2 Rluc ) as luminescence donor. Venus was fused in C-terminal of integrin β 3 or integrin α V subunit (integrin β 3 Venus or integrin α V Venus ) as fluorescence acceptor. h , i Interaction of GABA B receptor and integrin β 3 interaction between GB1 and integrin β 3 or GB1 and integrin α V ( h ), or GB2 and integrin β 3 ( i ) detected using BRET titration assay. The BRET between mGlu2 with Rluc fused in the C-terminal and 5HT2a receptor with Venus fused in the C-terminal was measured as positive control. The BRET between GB1 Rluc and Venus was measured as negative control. Data were analyzed by nonlinear regression on a pooled data set from at least three biologically independent experiments (upper to lower, h , left, n = 16, 10, 12; right, n = 5, 5; i , n = 9, 9) each performed in triplicates, fitting with 1-site binding model in GraphPad Prism 8.

Article Snippet: The integrin β 3 siRNA (#sc-63292) and control siRNA (#sc-37007) were purchased from Santa Cruz (Shanghai, China) and the GB1 siRNAs were synthesized from GenePharma (Suzhou, China) and the sequences are: GCGGUUUCCAACGUUCUUUTT (Forward), AAAGAACGUUGGAAACCGCTT (Reverse); GCUACAAGAAGAUCGGCUATT (Forward), UAGCCGAUCUUCUUGUAGCTT (Reverse); GGGAGAAGCCAGUUCCCAUTT (Forward), AUGGGAACUGGCUUCUCCCTT (Reverse).

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Suspension, Two Tailed Test, Immunoprecipitation, Construct, Labeling, Shear, Bioluminescence Resonance Energy Transfer, Fluorescence, Titration, Positive Control, Negative Control, Binding Assay

Journal: Nature Communications

Article Title: GABA-independent activation of GABA B receptor by mechanical forces

doi: 10.1038/s41467-025-64811-2

Figure Lengend Snippet: a Schematic representation of GABA B -ΔVFT truncation, in which GB1 subunit lacks of the VFT domain (GB1-ΔVFT), but retains the ability to be activated by positive allosteric modulator Rac BHFF (yellow square). Co-immunoprecipitation experiments were performed using anti-HA antibody targeting to the HA tag, which is fused in the N-terminal of GB1 or GB1-ΔVFT. b Co-immunoprecipitation of GABA B receptor or GABA B -ΔVFT and integrin β 3 using anti-HA antibody. Blots are from one representative of five biologically independent experiments. c IP 1 production in cells transfected with GABA B receptor, or GABA B -ΔVFT, along with G qi9 under suspension or adhesion conditions. Data are present as mean ± s.e.m. from four biologically independent experiments each performed in triplicates and analyzed using unpaired t test (two-tailed) to determine significance. * P < 0.05, not significant (ns) > 0.05. d Real-time recording of intracellular Ca 2+ release in HEK293 cells expressing GABA B -ΔVFT and G qi9 . After recording the basal state of Ca 2+ release for 50 seconds, cells were subjected to shear stress for 100 seconds. Shear stress was then halted for 150 seconds, after which Rac BHFF was added for 200 seconds. Data are present as mean ± s.e.m. from 135 cells recorded.

Article Snippet: The integrin β 3 siRNA (#sc-63292) and control siRNA (#sc-37007) were purchased from Santa Cruz (Shanghai, China) and the GB1 siRNAs were synthesized from GenePharma (Suzhou, China) and the sequences are: GCGGUUUCCAACGUUCUUUTT (Forward), AAAGAACGUUGGAAACCGCTT (Reverse); GCUACAAGAAGAUCGGCUATT (Forward), UAGCCGAUCUUCUUGUAGCTT (Reverse); GGGAGAAGCCAGUUCCCAUTT (Forward), AUGGGAACUGGCUUCUCCCTT (Reverse).

Techniques: Immunoprecipitation, Transfection, Suspension, Two Tailed Test, Expressing, Shear

Journal: Nature Communications

Article Title: GABA-independent activation of GABA B receptor by mechanical forces

doi: 10.1038/s41467-025-64811-2

Figure Lengend Snippet: a Immunofluorescent staining of GFAP (red), GFP (green) and DAPI (cyan) in astrocytes transfected with control siRNA or GABA B receptor siRNA along with GFP, treatment with or without shear stress (15 dyn/cm 2 , 30 min). Images are representative from three biologically independent experiments. Scale bar: 10 μm. b , c Analysis of the cell size and GFAP expression of astrocytes with the same treatment in ( a ). Measurements are made on each cell by cell basis (ROI) from three biologically independent experiments (number of cells from left to right: n = 47, 37, 32, 40). Data are present as mean ± s.e.m and analyzed using ordinary one-way ANOVA with Tukey’s multiple comparisons test to determine significance. *** P < 0.001, ** P < 0.01, ns: non-significant. d RNA interference efficacy of the GB1 in astrocytes in ( b , c ) using qPCR detecting gbb1 expression. Data are present as mean ± s.e.m. from three biologically independent experiments and analyzed with unpaired t test (two-tailed) to determine significance. *** P < 0.001.

Article Snippet: The integrin β 3 siRNA (#sc-63292) and control siRNA (#sc-37007) were purchased from Santa Cruz (Shanghai, China) and the GB1 siRNAs were synthesized from GenePharma (Suzhou, China) and the sequences are: GCGGUUUCCAACGUUCUUUTT (Forward), AAAGAACGUUGGAAACCGCTT (Reverse); GCUACAAGAAGAUCGGCUATT (Forward), UAGCCGAUCUUCUUGUAGCTT (Reverse); GGGAGAAGCCAGUUCCCAUTT (Forward), AUGGGAACUGGCUUCUCCCTT (Reverse).

Techniques: Staining, Transfection, Control, Shear, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: GABA-independent activation of GABA B receptor by mechanical forces

doi: 10.1038/s41467-025-64811-2

Figure Lengend Snippet: a Real-time recording of intracellular Ca 2+ release in astrocytes under shear stress. Cells were transfected with control siRNA and treated with shear stress, baclofen (100 μM) or ATP (100 μM) in the indicated time point. Data are present as mean ± s.e.m. from 15 cells recorded and are representative from six biologically independent experiments. b Percentage of astrocytes in response to shear stress or baclofen in 236 recorded cells. c Real-time recording of intracellular Ca 2+ release in astrocytes under shear stress. Cells are transfected with siRNA knocking down the GABA B receptor and administered to shear stress, baclofen (100 μM), or ATP (100 μM) at the indicated time points. Data are present as mean ± s.e.m. from 45 cells recorded and are representative of six biologically independent experiments. d Percentage of astrocytes with the calcium response (sensitive) or without the calcium response (insensitive) to shear stress (control siRNA: 236 cells; GABA B siRNA: 314 cells). Data are analyzed with χ2 test (two-sided) to determine significance. **** P < 0.0001.

Article Snippet: The integrin β 3 siRNA (#sc-63292) and control siRNA (#sc-37007) were purchased from Santa Cruz (Shanghai, China) and the GB1 siRNAs were synthesized from GenePharma (Suzhou, China) and the sequences are: GCGGUUUCCAACGUUCUUUTT (Forward), AAAGAACGUUGGAAACCGCTT (Reverse); GCUACAAGAAGAUCGGCUATT (Forward), UAGCCGAUCUUCUUGUAGCTT (Reverse); GGGAGAAGCCAGUUCCCAUTT (Forward), AUGGGAACUGGCUUCUCCCTT (Reverse).

Techniques: Shear, Transfection, Control

Journal: Nature Communications

Article Title: GABA-independent activation of GABA B receptor by mechanical forces

doi: 10.1038/s41467-025-64811-2

Figure Lengend Snippet: Antagonist CGP54626 -bound GABA B receptor fails to interact with integrin β 3 , whereas integrin β 3 interacts with GB1 VFT of GABA B receptor with constitutive activity. Mechanical force promotes GB1 VFT and integrin β 3 interaction to stabilize the closure of GB1 VFT -induced LB2 GB1 and LB2 GB2 in contact, further inducing an allosteric re-arrangement of the GABA B receptor 7TM towards an TM6-TM6 active conformation, culminating in the asymmetric activation of the receptor with G protein under GB2. Mechanical force acts as a positive allosteric modulator to boost up GABA-induced GABA B receptor activation through interaction between GB1 VFT and integrin β 3 .

Article Snippet: The integrin β 3 siRNA (#sc-63292) and control siRNA (#sc-37007) were purchased from Santa Cruz (Shanghai, China) and the GB1 siRNAs were synthesized from GenePharma (Suzhou, China) and the sequences are: GCGGUUUCCAACGUUCUUUTT (Forward), AAAGAACGUUGGAAACCGCTT (Reverse); GCUACAAGAAGAUCGGCUATT (Forward), UAGCCGAUCUUCUUGUAGCTT (Reverse); GGGAGAAGCCAGUUCCCAUTT (Forward), AUGGGAACUGGCUUCUCCCTT (Reverse).

Techniques: Activity Assay, 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: The human integrin α v siRNA (identification no. s7568, s7569, s7570), integrin β 3 siRNA (s7580, s7581, s7582), integrin α 5 siRNA (s7547, s7548, s7549), integrin β 5 siRNA (s7589, s7590, s7591), integrin β 1 siRNA (s7574, s7575, s7576), control siRNA (Silencer negative-control siRNA no. 1, catalog no. AM4636) were purchased from Applied Biosystems (Applied Biosystems, Foster City, CA).

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: The human integrin α v siRNA (identification no. s7568, s7569, s7570), integrin β 3 siRNA (s7580, s7581, s7582), integrin α 5 siRNA (s7547, s7548, s7549), integrin β 5 siRNA (s7589, s7590, s7591), integrin β 1 siRNA (s7574, s7575, s7576), control siRNA (Silencer negative-control siRNA no. 1, catalog no. AM4636) were purchased from Applied Biosystems (Applied Biosystems, Foster City, CA).

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: The human integrin α v siRNA (identification no. s7568, s7569, s7570), integrin β 3 siRNA (s7580, s7581, s7582), integrin α 5 siRNA (s7547, s7548, s7549), integrin β 5 siRNA (s7589, s7590, s7591), integrin β 1 siRNA (s7574, s7575, s7576), control siRNA (Silencer negative-control siRNA no. 1, catalog no. AM4636) were purchased from Applied Biosystems (Applied Biosystems, Foster City, CA).

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: The human integrin α v siRNA (identification no. s7568, s7569, s7570), integrin β 3 siRNA (s7580, s7581, s7582), integrin α 5 siRNA (s7547, s7548, s7549), integrin β 5 siRNA (s7589, s7590, s7591), integrin β 1 siRNA (s7574, s7575, s7576), control siRNA (Silencer negative-control siRNA no. 1, catalog no. AM4636) were purchased from Applied Biosystems (Applied Biosystems, Foster City, CA).

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: The human integrin α v siRNA (identification no. s7568, s7569, s7570), integrin β 3 siRNA (s7580, s7581, s7582), integrin α 5 siRNA (s7547, s7548, s7549), integrin β 5 siRNA (s7589, s7590, s7591), integrin β 1 siRNA (s7574, s7575, s7576), control siRNA (Silencer negative-control siRNA no. 1, catalog no. AM4636) were purchased from Applied Biosystems (Applied Biosystems, Foster City, CA).

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: The human integrin α v siRNA (identification no. s7568, s7569, s7570), integrin β 3 siRNA (s7580, s7581, s7582), integrin α 5 siRNA (s7547, s7548, s7549), integrin β 5 siRNA (s7589, s7590, s7591), integrin β 1 siRNA (s7574, s7575, s7576), control siRNA (Silencer negative-control siRNA no. 1, catalog no. AM4636) were purchased from Applied Biosystems (Applied Biosystems, Foster City, CA).

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: The human integrin α v siRNA (identification no. s7568, s7569, s7570), integrin β 3 siRNA (s7580, s7581, s7582), integrin α 5 siRNA (s7547, s7548, s7549), integrin β 5 siRNA (s7589, s7590, s7591), integrin β 1 siRNA (s7574, s7575, s7576), control siRNA (Silencer negative-control siRNA no. 1, catalog no. AM4636) were purchased from Applied Biosystems (Applied Biosystems, Foster City, CA).

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: The human integrin α v siRNA (identification no. s7568, s7569, s7570), integrin β 3 siRNA (s7580, s7581, s7582), integrin α 5 siRNA (s7547, s7548, s7549), integrin β 5 siRNA (s7589, s7590, s7591), integrin β 1 siRNA (s7574, s7575, s7576), control siRNA (Silencer negative-control siRNA no. 1, catalog no. AM4636) were purchased from Applied Biosystems (Applied Biosystems, Foster City, CA).

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: The human integrin α v siRNA (identification no. s7568, s7569, s7570), integrin β 3 siRNA (s7580, s7581, s7582), integrin α 5 siRNA (s7547, s7548, s7549), integrin β 5 siRNA (s7589, s7590, s7591), integrin β 1 siRNA (s7574, s7575, s7576), control siRNA (Silencer negative-control siRNA no. 1, catalog no. AM4636) were purchased from Applied Biosystems (Applied Biosystems, Foster City, CA).

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

Journal: Acta biomaterialia

Article Title: Effect of RGD-functionalization and stiffness modulation of polyelectrolyte multilayer films on muscle cell differentiation

doi: 10.1016/j.actbio.2012.12.015

Figure Lengend Snippet: (A) Staining of actin cytoskeleton (red) after 4 h of culture. (B) Staining of phosphorylated focal adhesion kinase (pFAK Y397, green) after 4 h of culture. (C) Myoblast migration measured over 5 h after seeding. (D) Effect of blocking β1 and/or β3 integrins using siRNA: quantification of the cell area after 4 h of adhesion (* p < 0.05 compared to scrambled siRNA). Focal adhesions/complexes are indicated by white arrowheads.

Article Snippet: Transfection by siRNA Cells were transfected with siRNA against β 1 and β 3 integrins (ON-TARGET plus SMARTpool, respectively Mouse ITGB1 and Mouse ITGB3, Thermo Scientific Dharmacon) individually or at the same time, a scrambled siRNA (All Stars negative Control siRNA, Qiagen) being taken as control.

Techniques: Staining, Migration, Blocking Assay

Journal: Acta biomaterialia

Article Title: Effect of RGD-functionalization and stiffness modulation of polyelectrolyte multilayer films on muscle cell differentiation

doi: 10.1016/j.actbio.2012.12.015

Figure Lengend Snippet: Adhesion involving β3 integrins on NCL-RGD films (soft films with covalently attached RGD peptide) provides favorable conditions for myogenic differentiation of C2C12 cells: when the medium is changed to DM, the rate of proliferating cells decreases and that of myogenin increases. Adhesion on CL and CL-RGD films (stiff films) involving β1 and β3 integrins promotes ROCK activation leading to a high proliferative state even in DM and to low myogenin expression. When the cells on stiff films are treated with ROCK inhibitor during differentiation, the rate of proliferating cells decreases significantly on both CL and CL-RGD films. Additionally, on CL films, myogenin expression increases, allowing the cells to undergo myogenic differentiation. However, on CL-RGD films, ROCK inhibition was not sufficient to induce myogenin expression and to allow cell to differentiate. We suggest that mechanical signals (stiffness) on CL-RGD films may affect cell interaction with biochemical signals (RGD peptide), resulting in the inhibition of β3 integrins by RGD peptide or by β1 integrins.

Article Snippet: Transfection by siRNA Cells were transfected with siRNA against β 1 and β 3 integrins (ON-TARGET plus SMARTpool, respectively Mouse ITGB1 and Mouse ITGB3, Thermo Scientific Dharmacon) individually or at the same time, a scrambled siRNA (All Stars negative Control siRNA, Qiagen) being taken as control.

Techniques: Activation Assay, Expressing, Inhibition