Journal: Head & face medicine

Article Title: CRISPR/Cas9-mediated knock out of ITGB6 in human OSCC cells reduced migration and proliferation ability.

doi: 10.1186/s13005-024-00437-x

Figure Lengend Snippet: Fig. 1 Impact of ITGB6 mRNA expression on patient outcome. A Kaplan-Meier plot on the overall survival of patients afflicted with OSCC. Comparison of “high expression” (n = 83) mRNA ITGB6 levels and “low expression” (n = 82) mRNA ITGB6 levels. Significant longer overall survival of the “low expression” group, * p = 0.0355. B Plot of the mRNA expression levels of the group “low expression” and “high expression”. Significant difference in the mRNA expres sion levels of ITGB6, * p < 0.000001. C Comparison of perineural invasion in both groups. Significant increased perineural invasion in the “high expression” group, * p = 0.001214

Article Snippet: Subsequently the cells were stained with an APC-labeled anti-ITGB6 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany; Catalog#: 130-111-454) for 30 min in phosphate-buffered saline (PBS) supplemented with 2% (v/v) FCS and 2 mM EDTA.

Techniques: Expressing, Comparison

Journal: Head & face medicine

Article Title: CRISPR/Cas9-mediated knock out of ITGB6 in human OSCC cells reduced migration and proliferation ability.

doi: 10.1186/s13005-024-00437-x

Figure Lengend Snippet: Fig. 2 CRISPR/Cas9-mediated knockout of ITGB6 in HN cells. A Sanger sequencing of HN WT (top) and HN ITGB6KO (bottom). Traces and sequences are presented. PAM sequence, gRNA binding and Cas9 cut site is marked. B Sequencing data of HN ITGB6KO analyzed by ICE online tool (Synthego). Decomposition of the found sequences with the individual contributions are presented (Sequence 1 1: 48%; Sequence 2: 41%). Sequences with contribu tions ≤ 2% are considered as background signal and are not shown. Sequence 1 shows an insertion of one base (marked in red color) (+ 1). Sequence 2 indicates a deletion of 1 base (-1) (deleted base shown in red color in the WT sequence). Overall, a strong indication of a heterozygous KO of ITGB6 with frameshift mutations on both alleles is presented. C Verification of ITGB6 gene KO of HN ITGB6KO on the protein level by FACS analysis. HN WT cells pres ent with a homogenous ITGB6 expression, whereas HN ITGB6KO indicates a loss of ITGB6 expression

Article Snippet: Subsequently the cells were stained with an APC-labeled anti-ITGB6 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany; Catalog#: 130-111-454) for 30 min in phosphate-buffered saline (PBS) supplemented with 2% (v/v) FCS and 2 mM EDTA.

Techniques: CRISPR, Knock-Out, Sequencing, Binding Assay, Expressing

Journal: PLoS Pathogens

Article Title: EphrinA2 Regulates Clathrin Mediated KSHV Endocytosis in Fibroblast Cells by Coordinating Integrin-Associated Signaling and c-Cbl Directed Polyubiquitination

doi: 10.1371/journal.ppat.1003510

Figure Lengend Snippet: ( A ) Serum-starved (8 h) HFF cells were either mock infected (UI) or infected with KSHV (30 DNA copies/cell) for the indicated time periods and subjected to Western blot analysis for phospho-EphA2 (Y594) (pEphA2). The blot was stripped and reprobed for total EphA2 and tubulin was used as loading control. ( B ) Serum starved HFF cells were either mock infected or infected with KSHV (30 DNA copies/cell) for 5 and 10 min and processed for immunofluorescence analysis using rabbit anti-EphA2 and mouse monoclonal anti-KSHV gpK8.1A antibodies for overnight at 4°C followed by staining with anti-rabbit Alexa 488 and anti-mouse Alexa 594 secondary antibodies. Representative 2D convoluted images are shown. The white boxes within the merged panels are shown as enlarged pictures and the white arrows represent colocalization of the indicated molecules. ( C ) Serum-starved HFF cells were left uninfected or infected with KSHV for the indicated periods of time and immunoprecipitated with anti-α3β1, αVβ3 or αVβ5 antibodies and analyzed for EphA2 by Western blot (first, third and fifth panels). These blots were stripped and reprobed for total β1, β3 and β5 integrin subunits, respectively (second, fourth and sixth panels). For negative control, cell lysates from uninfected or KSHV infected HFF cells were immunoprecipitated with anti-β6 antibody and analyzed for EphA2 by western blotting (seventh panel). The blot was stripped and reprobed with total β6 antibodies (eighth panel). Whole cell lysates were subjected to western blot analysis for input expression of EphA2 (ninth panel) and β6 (tenth panel) and β-actin was used as loading control (eleventh panel). ( D ) Serum starved HFF cells were either left uninfected or infected with KSHV (30 DNA copies/cell) for 5, 10 and 30 min, washed and processed for immunofluorescence analysis. Cells were permeabilized, blocked with blocking reagents and incubated with ( D1 ) rabbit anti-EphA2 and mouse anti-αVβ3 antibodies or ( D2 ) rabbit anti-EphA2 and mouse anti-αVβ5 antibodies for 2 h at room temperature followed by staining with anti-rabbit Alexa fluor 488 or anti-mouse Alexa fluor 594. Representative 2D deconvoluted images are shown. The enlarged pictures represent boxed regions within the merged panels and the white arrows represent colocalization of the indicated molecules.

Article Snippet: Goat anti-β6 integrin antibody was purchased from Santa Cruz Biotechnology, CA.

Techniques: Infection, Western Blot, Control, Immunofluorescence, Staining, Immunoprecipitation, Negative Control, Expressing, Blocking Assay, Incubation

Journal: Cancer Cell International

Article Title: Targeting of 3D oral cancer spheroids by αVβ6 integrin using near-infrared peptide-conjugated IRDye 680

doi: 10.1186/s12935-024-03417-y

Figure Lengend Snippet: Analysis of αVβ6 expression. ( A ) ITGB6 proteomic expression profile in normal tissue and primary tumor (data from UALCAN) ( n = 71–108; *** p < 0.001; Two sample t test). ( B ) RT-qPCR analysis of ITGB6 mRNA expression; β-actin was used as housekeeping gene. The (αVβ6+) cell line HT-29 was used as reference ( n = 3–8; *** p < 0.001; One-way ANOVA followed by Tukey’s assay). ( C - D ) β6 integrin subunit protein expression assessed by Western-Blot; tubulin was used as a loading control ( n = 3–7; *** p < 0.001; One way ANOVA followed by Tukey’s assay). Data are presented as mean ± SEM

Article Snippet: 40 µg of protein lysate was loaded to 7.5% non-reducing SDS PAGE and after 150 min of migration and 90 min of blotting, PVDF membrane was saturated with 5% (w/v) solution of non-fat powered milk in TBST (Tris buffer solution with 0.1% Tween-20) for 1 h. Membrane was incubated overnight at 4 °C with either 1:100 mouse anti-human β6 integrin subunit antibody (Merck Millipore corp, USA, 407,317) or 1:1000 mouse anti-α-tubulin antibody (Santa Cruz Biotechnology, 23,948), followed by incubation with 1:2000 anti-mouse IgG HRP-conjugated secondary antibody (Cell signaling technology, 7076 S) for 1 h at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Cancer Cell International

Article Title: Targeting of 3D oral cancer spheroids by αVβ6 integrin using near-infrared peptide-conjugated IRDye 680

doi: 10.1186/s12935-024-03417-y

Figure Lengend Snippet: Multicellular tumor spheroid characterization. ( A ) Typical cryosection images of H10 and H5M5 spheroids after KI-67 immunohistochemistry staining (scale bar = 150 μm). ( B ) Typical immunofluorescence images of H10 and H5M5 spheroids cryosections stained with antibodies against αVβ6 integrin, cytokeratin 19, E-cadherin, vimentin, and fibronectin at day 4 post-seeding. Proteins of interest are in red and nuclei are in bleu after counterstaining with Vectashield-DAPI (scale bar = 100 μm at x40 and 40 μm at x100). ( C ) Relative quantification of immunofluorescence markers expression using ImageJ software ( n = 3–4; * p < 0.05, ** p < 0.01; Two sample t test). Data are presented as mean ± SEM

Article Snippet: 40 µg of protein lysate was loaded to 7.5% non-reducing SDS PAGE and after 150 min of migration and 90 min of blotting, PVDF membrane was saturated with 5% (w/v) solution of non-fat powered milk in TBST (Tris buffer solution with 0.1% Tween-20) for 1 h. Membrane was incubated overnight at 4 °C with either 1:100 mouse anti-human β6 integrin subunit antibody (Merck Millipore corp, USA, 407,317) or 1:1000 mouse anti-α-tubulin antibody (Santa Cruz Biotechnology, 23,948), followed by incubation with 1:2000 anti-mouse IgG HRP-conjugated secondary antibody (Cell signaling technology, 7076 S) for 1 h at room temperature.

Techniques: Immunohistochemistry, Staining, Immunofluorescence, Expressing, Software