Journal: Molecular Cancer Research

Article Title: β8 Integrin Mediates Pancreatic Cancer Cell Radiochemoresistance

doi: 10.1158/1541-7786.mcr-18-1352

Figure Lengend Snippet: Figure 5. b8 integrin interactome and subcellular colocalization. A, Sequential immunoprecipitation-mass spectrometry–based analysis of b8 integrin interactome was performed in unirradiated and 6-Gy X-rays irradiated Patu8902 cells. Time point of analysis after irradiation was 2 hours. B, Comparative changes in the b8 integrin interactome between control and irradiation upon categorization into different molecular functions using the PANTHER classification system. C, Immunofluorescence costaining of b8 integrin with GM130 (Golgi), mitochondria, aV integrin, APPL2, and Caveolin 1. Scale bars, 10 mm. D, Pearson correlation analysis of C. Data show mean SD (n ¼ 3).

Article Snippet: The antibodies against b1 integrin (WB 1:1,000, Abcam, ab179471), b8 integrin (IF 1:200, WB 1:1,000; Abcam, ab80673, Rb, recognizes aa 614-663 (this antibody was generally used in this study); Abnova, H00003696-M01, Mo, recognizes aa 392–503 (this antibody was used only where specifically indicated), GM130 (IF 1:100, WB 1:1,000, BD, 610822), MEK1/2 (WB 1:1,000, Cell Signaling Technology, 4694s), gH2AX (WB 1:1,000, Cell Signaling Technology, 9718s), aV integrin (IF 1:250, Novus, NB100-2618), APPL2 (IF 1:100, Sigma-Aldrich, SAB1400605), Caveolin 1 (IF 1:100, BD, 610407), LC3B (IF 1:500, Sigma-Aldrich, SAB4200361), b-actin (WB 1:10,000, Sigma-Aldrich, A5441), HRP-conjugated secondary antibody (GE, Rb NXA931, Mo NXA931), Alexa Fluor 488– or Alexa Fluor 594–conjugated secondary antibodies (1:500, Life Technologies) were purchased and used as indicated.

Techniques: Immunoprecipitation, Mass Spectrometry, Irradiation, Control

Journal: bioRxiv

Article Title: A Kaposi’s Sarcoma-Associated Herpesvirus Infection Mechanism is Independent of Integrins α3β1, αVβ3, and αVβ5

doi: 10.1101/270108

Figure Lengend Snippet: Canonical KSHV integrin receptors are not required for infection of Caki-1 and HeLa cells. (A, H) WT and indicated integrin subunit KO Caki-1 cells were immunostained for surface expression of the indicated integrins. Grey histograms represent isotype controls. (B) WT and integrin KO Caki-1 pools were infected with KSHV in duplicate and infection percentage was measured by flow cytometry two days post infection. The infection rates of the KO pools were normalized to the average WT infection rate and data was pooled from multiple experiments. (C) ITGA3/ITGAV double KO Caki-1 cells were immunostained for surface integrin α3 and αV expression. Grey histograms represent the isotype controls. (D) WT and ITGA3/ITGAV double KO Caki-1 cells were infected with KSHV in triplicate and infection rate was quantified by flow cytometry. The infection rate of the DKO pool was normalized to the average WT infection rate and data was pooled from multiple experiments. (E) Mixed WT / ITGAV KO and WT/ITGB1 KO HeLa populations were immunostained for surface integrin αV and β1 expression. Grey histograms represent the isotype controls. (F) Mixed integrin KO HeLa pools were infected with KSHV in triplicate and infection percentages were measured by flow cytometry two days post infection. The pools were also immunostained for the corresponding integrins and gated on integrin-high or -low populations as indicated in (E). The infection rates of integrin-low cells were normalized to integrin-high cells in each well and a representative experiment is shown. *, p < 0.05. (G) Schematic integrin pairing diagram (adapted from 79) showing expression data measured by surface immunostaining and flow cytometry and subunits targeted by our CRISPR-Cas9 KO approach. Bold connections denote heterodimers previously implicated in KSHV infection.

Article Snippet: Heparan sulfate antibody (F58-10E4) was purchased from Amsbio, integrin α3 antibody (P1B5 from Calbiochem, integrin αV, integrin β7, EphA2, and EphA5 antibodies (MAB12191, MAB4669, AF3035, and MAB541, respectively) from R...D Systems, integrin β1 and integrin β3 antibodies (T2S/16 and PM6/13, respectively) from Novus Biologicals, integrin β5 and EphA2 antibodies (AST-3T and SHM16, respectively) from BioLegend, xct and GAPDH antibodies (ab37185 and ab181602, respectively) from Abcam, DC-SIGN antibody (DCN47.5) from Miltenyi Biotec, EphA4 antibody (4C8H5) from ThermoFisher, EphB2 antibody (2D12C6) from Santa Cruz Biotech, and Flag antibody (M2) from Sigma-Aldrich.

Techniques: Infection, Expressing, Flow Cytometry, Immunostaining, CRISPR

Journal: bioRxiv

Article Title: A Kaposi’s Sarcoma-Associated Herpesvirus Infection Mechanism is Independent of Integrins α3β1, αVβ3, and αVβ5

doi: 10.1101/270108

Figure Lengend Snippet: KSHV infection of PGKs depends on HS interactions but is not inhibited by integrin- or Eph-blocking agents. (A) PGKs were immunostained for surface expression of known KSHV receptors and analyzed by flow cytometry. Grey histograms represent isotype controls. (B) PGK cells were preincubated with fibronectin or laminin at 50 μg/L, GRGDSP or GRGESP peptides at 2 mM or an appropriate volume control of DMSO, and ephrin-A4-Fc or EGFR-Fc as a control at 5 μg/mL for one hour at 4°C. For the no treatment and heparin condition, cells were pre-incubated in normal media at 4°C. For the heparin block condition, virus was blocked with heparin at 500 μg/mL for one hour at 37°C. Cells were then washed and infected in triplicate with KSHV, or heparin-blocked KSHV for two hours at 37°C. Ephrin-A4-Fc and EGFR-Fc concentrations were maintained during the infection. Infection percentage was quantified by flow cytometry two days post infection. *, p < 0.05.

Article Snippet: Heparan sulfate antibody (F58-10E4) was purchased from Amsbio, integrin α3 antibody (P1B5 from Calbiochem, integrin αV, integrin β7, EphA2, and EphA5 antibodies (MAB12191, MAB4669, AF3035, and MAB541, respectively) from R...D Systems, integrin β1 and integrin β3 antibodies (T2S/16 and PM6/13, respectively) from Novus Biologicals, integrin β5 and EphA2 antibodies (AST-3T and SHM16, respectively) from BioLegend, xct and GAPDH antibodies (ab37185 and ab181602, respectively) from Abcam, DC-SIGN antibody (DCN47.5) from Miltenyi Biotec, EphA4 antibody (4C8H5) from ThermoFisher, EphB2 antibody (2D12C6) from Santa Cruz Biotech, and Flag antibody (M2) from Sigma-Aldrich.

Techniques: Infection, Blocking Assay, Expressing, Flow Cytometry, Control, Incubation, Virus