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: Breast Cancer : Basic and Clinical Research

Article Title: Male Breast Cancer—Immunohistochemical Patterns and Clinical Relevance of FASN, ATF3, and Collagen IV

doi: 10.1177/11782234211002496

Figure Lengend Snippet: Antibody reagents and conditions.

Article Snippet: β6 integrin , Atlas antibodies , ITGB6 , 1:350—28 min , ULTRA CC1-48 min.

Techniques:

Journal: Cell & Bioscience

Article Title: Protein expression of eIF4E and integrin αvβ6 in colon cancer can predict clinical significance, reveal their correlation and imply possible mechanism of interaction

doi: 10.1186/2045-3701-4-23

Figure Lengend Snippet: Association between eIF4E expression, integrin αvβ6 expression and clinicopathologic variables in colon cancer cases

Article Snippet: As primary antibodies, prediluted anti-Integrin β6 polyclonal rabbit antibody (1:200; Proteintech Group, Inc., Cat.No.19695-1-AP, Chicago, USA), anti- eIF-4E rabbit monoclonal antibody (1:100; Epitomics, Inc., Cat.No.

Techniques: Expressing

Journal: Cell & Bioscience

Article Title: Protein expression of eIF4E and integrin αvβ6 in colon cancer can predict clinical significance, reveal their correlation and imply possible mechanism of interaction

doi: 10.1186/2045-3701-4-23

Figure Lengend Snippet: Integrin β6 expression in human colonic cancer. First row: (a) , (b) , (c) (Scale bar 200 μm.); second row: (d) , (e) , (f) , corresponding to each figure above (Scale bar 100 μm). (a) and (d) was obtained from paracancerous normal tissue of patient with colon cancer, with negative staining. (b) and (e) was obtained from colonic carcinoma tissue,with negative Integrin β6 expression. (c) and (f) was obtained from colonic carcinoma, with positive Integrin β6 expression.

Article Snippet: As primary antibodies, prediluted anti-Integrin β6 polyclonal rabbit antibody (1:200; Proteintech Group, Inc., Cat.No.19695-1-AP, Chicago, USA), anti- eIF-4E rabbit monoclonal antibody (1:100; Epitomics, Inc., Cat.No.

Techniques: Expressing, Negative Staining

Journal: Cell & Bioscience

Article Title: Protein expression of eIF4E and integrin αvβ6 in colon cancer can predict clinical significance, reveal their correlation and imply possible mechanism of interaction

doi: 10.1186/2045-3701-4-23

Figure Lengend Snippet: Correlation between integrin αvβ6 expression and eIF4E expression in human colonic carcinoma tissues (r = 0.299, P < 0.001)

Article Snippet: As primary antibodies, prediluted anti-Integrin β6 polyclonal rabbit antibody (1:200; Proteintech Group, Inc., Cat.No.19695-1-AP, Chicago, USA), anti- eIF-4E rabbit monoclonal antibody (1:100; Epitomics, Inc., Cat.No.

Techniques: Expressing

Journal: Cell & Bioscience

Article Title: Protein expression of eIF4E and integrin αvβ6 in colon cancer can predict clinical significance, reveal their correlation and imply possible mechanism of interaction

doi: 10.1186/2045-3701-4-23

Figure Lengend Snippet: Overall survival according to eIF4E with integrin αvβ6 expression (P = 0.028, The log-rank test).

Article Snippet: As primary antibodies, prediluted anti-Integrin β6 polyclonal rabbit antibody (1:200; Proteintech Group, Inc., Cat.No.19695-1-AP, Chicago, USA), anti- eIF-4E rabbit monoclonal antibody (1:100; Epitomics, Inc., Cat.No.

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: Immunoglobulin light chains generate proinflammatory and profibrotic kidney injury

doi: 10.1172/JCI125517

Figure Lengend Snippet: (A and B) Flow cytometric studies demonstrated that both the κ2 and λ3 FLCs increased the number of cells expressing ITGB6 (n = 4 experiments in each group). Pretreatment of HK-2 cells with ITGB6 shRNA prevented this response. (C) Along with the changes in surface localization of ITGB6, pretreatment of the cells with ITGB6 shRNA also inhibited the production of active TGF-β (n = 4 experiments in each group). Data are expressed as the mean ± SEM. *P < 0.0009 compared with the other 2 corresponding groups (ANOVA).

Article Snippet: Additional antibodies obtained commercially included those directed against SMAD2/3 (C4T) (MilliporeSigma); κ FLC (no. A0100, Dako, Agilent Technologies); ITGB6 (catalog TA321227, OriGene Technologies); GAPDH (catalog Ab8245, Abcam); HO-1 (ADI-SPA-894-F; Enzo Life Sciences); rat F4/80 (catalog 14480182, Invitrogen, Thermo Fisher Scientific); IL-1β (catalog Ab9722, Abcam); NIMP-R14 (sc-59338, Santa Cruz Biotechnology); and KIM-1 (AF1817, R&D Systems).

Techniques: Expressing, shRNA

Journal: The Journal of Clinical Investigation

Article Title: Immunoglobulin light chains generate proinflammatory and profibrotic kidney injury

doi: 10.1172/JCI125517

Figure Lengend Snippet: (A) Neutrophils, detected using anti–NIMP-R14 antibodies (red fluorescence), were present in greater numbers (white arrowheads) only in the Stat1+/+ mice treated with the higher dose of κ2 FLC. Proximal tubule expression of ITGB6 (green fluorescence) was readily detected. The IgG controls (left panel) showed minimal background staining of the samples. (B) M2 macrophage numbers, detected by colocalization of F4/80 (red fluorescence) with HO-1 (green fluorescence), were increased (white arrowheads) only in the Stat1+/+ mice treated with κ2 FLC. The inset shows a magnified image of a F4/80+HO-1+ cell (colocalized stain is shown in yellow). The IgG controls (left panel) showed minimal background staining of the samples. T, tubule. n = 8–10 mice/group. Data are expressed as the mean ± SEM of cells per HPF. *P < 0.0001 compared with the other 3 groups (ANOVA). Scale bars: 50 μm. Original magnification ×1200.

Article Snippet: Additional antibodies obtained commercially included those directed against SMAD2/3 (C4T) (MilliporeSigma); κ FLC (no. A0100, Dako, Agilent Technologies); ITGB6 (catalog TA321227, OriGene Technologies); GAPDH (catalog Ab8245, Abcam); HO-1 (ADI-SPA-894-F; Enzo Life Sciences); rat F4/80 (catalog 14480182, Invitrogen, Thermo Fisher Scientific); IL-1β (catalog Ab9722, Abcam); NIMP-R14 (sc-59338, Santa Cruz Biotechnology); and KIM-1 (AF1817, R&D Systems).

Techniques: Fluorescence, Expressing, Staining

Journal: The Journal of Clinical Investigation

Article Title: Immunoglobulin light chains generate proinflammatory and profibrotic kidney injury

doi: 10.1172/JCI125517

Figure Lengend Snippet: (A) Both doses of the κ2 FLCs prompted increases in ITGB6 levels of cortical lysates from Stat1+/+ mice (n = 6 mice/group). Data are expressed as the mean ± SEM. **P ≤ 0.006 compared with each of the other 4 groups (ANOVA). Relative ITGB6 levels were greater (P = 0.0096, ANOVA) in the PBS-treated Stat1+/+ mice compared with levels in the PBS-treated Stat1–/– mice. (B) A dose-dependent effect of κ2 FLC on p-SMAD2/3 was observed in Stat1+/+ mice but produced no changes in p-SMAD2/3 in Stat1–/– mice. Because SMAD2/3 levels were remarkably low in the kidney cortex of Stat1–/– mice, the data were factored by the density of GAPDH in the samples. n = 6 mice/group. Data are expressed as the mean ± SEM. *P < 0.005 compared with each of the other 5 groups in the experiment (ANOVA).

Article Snippet: Additional antibodies obtained commercially included those directed against SMAD2/3 (C4T) (MilliporeSigma); κ FLC (no. A0100, Dako, Agilent Technologies); ITGB6 (catalog TA321227, OriGene Technologies); GAPDH (catalog Ab8245, Abcam); HO-1 (ADI-SPA-894-F; Enzo Life Sciences); rat F4/80 (catalog 14480182, Invitrogen, Thermo Fisher Scientific); IL-1β (catalog Ab9722, Abcam); NIMP-R14 (sc-59338, Santa Cruz Biotechnology); and KIM-1 (AF1817, R&D Systems).

Techniques: Produced

Journal: Developmental cell

Article Title: A Cdh3-β-catenin-laminin signaling axis in a subset of breast tumor leader cells control leader cell polarization and directional collective migration.

doi: 10.1016/j.devcel.2022.12.005

Figure Lengend Snippet: Figure 7. Cdh3 controls b-catenin activity to stimulate local laminin 332 production that is required for collective migration (A) Subcellular fractionation and quantitative western blots of confluent mouse 4T1 cells, ±Cdh3. Nuclear/cytoplasmic ratio of b-catenin levels was determined by comparing subcellular extract levels normalized to equivalent number of cells. (B) Quantitative western blot analyses of confluent mouse 4T1 and human BT549 cells, ±Cdh3, with indicated antibodies. b-cat-pS552 in 4T1 cells not determined (nd) as total b-cat in Cdh3-depleted cells was too low. Cartoon on left highlights signaling pathways interrogated. (C) Chromatin IP of confluent WT mouse 4T1 cells with indicated antibodies and PCR of promoter regions of Lam332 genes. (D) IF images of PyMT breast tumor organoids, ±Lama3, exposed to a SDF1 gradient (arrow) and stained for K14-Actin.GFP (green) and active b-integrin (9EG7) (red). Arrowheads identify co-stained cells at leading edge. 9EG7 fluorescence in K14+ cells quantified (right). (E) Western blot of confluent mouse 4T1 cells, ±Cdh3, with indicated antibodies. (F) Migration tracking maps and migration efficiency of PyMT tumor organoids, ±Cdh3, in microfluidic devices containing collagen I: laminin 332 (1:1) and exposed to a SDF1 gradient.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies P-cadherin Invitrogen Cat# MA1-2003; RRID: AB_2077774 P-cadherin Thermofisher Cat# 132000z Collagen 17 (COL17) Invitrogen Cat# PA5-1033778 Keratin 14 (K14) Biolegend Cat# 905301; RRID: AB_2565048 Laminin-a3 Dr. Takako Sasaki 1110 Laminin-g2 (Alexafluor 647) Millipore Sigma Cat# MAB19562- AF647 0FFD-actin Sigma-Aldrich Cat#A1978 Anti-rabbit IgG, HRP-linked antibody Cell Signaling Technology Cat# 7074S Anti-mouse IgG, HRP-linked antibody Cell Signaling Technology Cat# 7076S Alexa Fluor 488 goat anti-rabbit IgG (H+L) Invitrogen Cat# A11008 Alexa Fluor 594 goat anti-rabbit IgG (H+L) Invitrogen Cat# A11012 Alexa Fluor 594 goat anti-rat IgG (H+L) Invitrogen Cat# A-11007 DAPI (4’,6-Diamidino-2-2Phenylindole, Dihydrochloride) Thermofisher Cat#: D1306 b-Catenin Santa Cruz Cat# sc-7963; RRID: AB_626807 Phospho-FAK.Y397 Cell Signaling Cat# 8556; RRID: AB_10891442 FAK Cell Signaling Cat# 3285; RRID: AB_2269034 Phospho-Src.Y416 Cell Signaling Cat# 6943; RRID: AB_10013641 Src R&D System Cat# AF3389; RRID: AB_2196155 TCF4 Millipore Cat# CS204338 Mouse IgG Millipore Cat# 12-371B; RRID: AB_2617156 Goat anti-rabbit Fab fragment Jackson ImmunoResearch Cat# 111-007-003; RRID: AB_2337925 Rabbit anti-rat antibody Vector AI Cat# 4001 3-amino-9-ethylcarbaole (AEC) Abcam Cat$ Ab64252 ITG 0FFD1 BD Pharmingen Cat# 553715 Fxycle Violet Thermofisher Cat# F10347 Cdh3-PE R&D systems Cat# FAB761P; RRID: AB_2291533 PDGFRb-PE-Cy7 Thermofisher Cat# 25140282 Thy-1.1-PE-Cy7 Thermofisher Cat# 25090082 Bacterial and virus strains pGFP-C-shLenti-Cdh3 (human) Origene Cat# TL314033 pRFP-C-shLenti-Cdh3 (mouse) Origene Cat# TL500332 pGFP-C-shLenti-scramble (human) Origene Cat# TR30021 pRFP-C-shLenti-scramble (mouse) Origene Cat# TR30032 pLKO.1-puro-laminina3 (mouse) Sigma Aldrich Cat# XM_140451 pLKO.1-puro-scramble (mouse) Sigma Aldrich Cat# SHC016 pGFP-C-shLenti-scramble (mouse) Origene Cat# TR30021 pGFP-C-shLenti-laminina3 (human) Origene Cat# TL311804 Biological samples Human tissue microarray Washington University in St Louis N/A Chemicals, peptides, and recombinant proteins DMEM high glucose Gibco Cat# 11995073 DMEM/F12 Gibco Cat# 11330057 FBS Corning Cat# MT35011CV (Continued on next page) Developmental Cell 58, 34–50.e1–e9, January 9, 2023 e1

Techniques: Activity Assay, Migration, Fractionation, Western Blot, Protein-Protein interactions, Chromatin Immunoprecipitation, Staining