Journal: Nature Structural & Molecular Biology

Article Title: A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens

doi: 10.1038/s41594-024-01211-y

Figure Lengend Snippet: ( a ) RFP growth competition assay (used in Figs. , , ): The ipUSEPR vector expresses a sgRNA together with a puromycin-resistant gene (PuroR) and a TagRFP fluorescent protein. The RFP fluorescent signal of live (DAPI – ) singlet cells was detected by an Attune NxT flow cytometer with an HTS autosampler. The sgRNA targeting a functionally important gene will result in a reduced RFP + population in the culture. ( b ) Gating strategy for detecting the cell surface αVβ5 expression (used in Fig. ).

Article Snippet: Cell surface integrin αVβ5 was recognized by a mouse monoclonal anti-human αVβ5 antibody (clone P1F76; sc-13588, Santa Cruz Biotech; 1:200) and stained with AF488-conjugated donkey anti-mouse IgG (ab150105, Abcam) secondary antibody.

Techniques: Competitive Binding Assay, Plasmid Preparation, Flow Cytometry, Expressing

Journal: Nature Structural & Molecular Biology

Article Title: A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens

doi: 10.1038/s41594-024-01211-y

Figure Lengend Snippet: a , Model of integrin α (red) and β (blue) subunits and domain structures. The binding site of the extracellular ligand (yellow) is assembled upon heterodimerization of the α/β subunits. b , Schematic outline of integrin family CRISPR screens (712 sgRNAs) in Cas9-expressing MDA231 and PANC1 cells. c , Fold change of each sgRNA from day 0 to day 24 in MDA231-Cas9 + ( x axis) and PANC1-Cas9 + ( y axis) cells. The sgRNAs targeting ITGAV (red dots), ITGB5 (blue dots), positive controls (yellow triangles), negative controls (green triangles) and the total library (gray dots) are indicated. d , Heatmap showing CRISPR impact scores (median log 10 fold change of 25 sgRNAs) of each integrin subunit in the integrin network consisting of 24 distinct integrin α/β heterodimers. The solid lines indicate the integrin α/β pairs forming the RGD receptors (yellow), collagen receptors (pink), laminin receptors (brown) and leukocyte-specific receptors (green). The red dotted circle highlights αVβ5 as the top essential integrin heterodimer in cancer cells. e , Growth competition assay of MDA231-Cas9 + cells transduced with RFP-labeled sgCtrl (gray lines; two independent sgRNA sequences) and sgITGB1/3/5/6/8 (blue lines; three independent sgRNA sequences for each gene). Asterisk indicates that all three sgRNAs for each ITGB gene group were significantly different ( P < 0.01) from the two sgCtrl groups ( n = 3 for each group). f , Western blot of ITGB5 and β-actin in MDA231-Cas9 + cells transduced with sgCtrl ( n = 2 independent sgRNA sequences) and sgITGB5 ( n = 3 independent sgRNA sequences) for 3 days. g , h , Cellular apoptosis detected by Annexin V + /DAPI − ( g ) and cell cycle monitored by EdU incorporation ( h ) in MDA231-Cas9 + cells transduced with sgCtrl and sgITGB5 for 3 days ( n = 3 for each group). i , Gene ranking based on the Pearson coefficient ( r ) of CERES scores between ITGAV and ITGB5 (blue) compared with other ITGAV partner β subunit genes ITGB1 / 3 / 6 / 8 (yellow) in the 769 tested cell models (Extended Data Fig. ). Data are presented as the mean ± s.e.m. P values were calculated by two-sided Student’s t -test.

Article Snippet: Cell surface integrin αVβ5 was recognized by a mouse monoclonal anti-human αVβ5 antibody (clone P1F76; sc-13588, Santa Cruz Biotech; 1:200) and stained with AF488-conjugated donkey anti-mouse IgG (ab150105, Abcam) secondary antibody.

Techniques: Binding Assay, CRISPR, Expressing, Competitive Binding Assay, Transduction, Labeling, Western Blot

Journal: Nature Structural & Molecular Biology

Article Title: A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens

doi: 10.1038/s41594-024-01211-y

Figure Lengend Snippet: a , 3D ‘docking box’ (cube) defined by the CRISPR-hypersensitive regions (numbers 1–7) within the ITGAV β-propeller domain. b , Compound (Cpd) ranking based on free binding energy (ΔG°) to the ‘docking box’ within the β-propeller domain predicted by AutoDock Vina. c , d , Heatmap showing relative CellTiter Glo (left) and CCK8 (right) signals (percentage of the signal for dimethyl sulfoxide; DMSO) in MDA231 cells incubated with 10 µM of 500 selected compounds ( c ) and the top nine effective compounds ( d ) for 3 days. Effective cell killing was defined as less than 10% relative signals for both CellTiter Glo and CCK8 assays. e , Schematic outline of flow cytometric measurement of cell surface integrin αVβ5 using a monoclonal antibody against integrin αVβ5 heterodimers. f , Effects of the top nine candidate compounds on cell surface integrin αVβ5 levels upon 1 h compound treatments ( n = 4 for each condition). g , h , Cellular apoptosis detected by Annexin V + /DAPI − ( g ) and cell cycle monitored by EdU incorporation ( h ) in MDA231 cells treated with Cpd_AV2 (40 µM) for 0 to 3 h ( n = 3 for each time point). i , Representative fluorescence images of F-actin (FITC, green) and nucleus (DAPI, blue) staining in MDA231 cells treated with control (DMSO) and Cpd_AV2 (40 µM) for 10 min. Scale bars, 20 µm. j , Violin plot showing the distribution of cell size (µm 2 ) in MDA231 cells treated with control (DMSO) and Cpd_AV2 (40 µM) for 10 min. Data are presented as the mean ± s.e.m. P values were calculated by two-sided Student’s t -test.

Article Snippet: Cell surface integrin αVβ5 was recognized by a mouse monoclonal anti-human αVβ5 antibody (clone P1F76; sc-13588, Santa Cruz Biotech; 1:200) and stained with AF488-conjugated donkey anti-mouse IgG (ab150105, Abcam) secondary antibody.

Techniques: CRISPR, Binding Assay, Incubation, Fluorescence, Staining, Control

Journal: Nature Structural & Molecular Biology

Article Title: A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens

doi: 10.1038/s41594-024-01211-y

Figure Lengend Snippet: a , Purification of bacterial-expressed recombinant ITGAV β-propeller domain (peptide region 31–492 aa; N-terminal His 6 -tagged) using immobilized metal affinity chromatography (IMAC) and anion exchange chromatography (IEX). The input and purified ITGAV β-propeller domain samples were visualized by gel electrophoresis and silver staining (right; gel representative of two independent protein purification experiments). b , Protein thermal stability as estimated by fluorescent dye incorporation of the purified ITGAV β-propeller domain under control (DMSO) and Cpd_AV2 (40 µM) conditions. c , Protein surface model (left) showing a docking simulation of the ITGAV β-propeller domain (colored by NCS) interacting with Cpd_AV2 (yellow). Protein ribbon model (right) illustrates an overlap of ITGB5 lysine 287 (within βA loop; cyan) and Cpd_AV2 (yellow) binding on the β-propeller HIP of ITGAV. d , e , Effects of cilengitide and Cpd_AV2 on cell surface integrin αVβ5 levels after 1 h treatment ( d ) and cell expansion after 72 h treatment in MDA231 cells ( e ) ( n = 3 for each group). f , Effects of 72 h Cpd_AV2 treatment on expansion of six cancer cell models ( n = 3 for each group). g , Chemical structure of Cpd_AV2 (source: NCI/DTP Open Chemicals Repository). h , Model showing distinct mechanisms of action between Cpd_AV2 (left) and cilengitide (right) for suppressing ECM-to-integrin αVβ5 signaling (middle). Data are presented as the mean ± s.e.m. P values were calculated by two-sided Student’s t -test. NSCLC, nonsmall-cell lung cancer.

Article Snippet: Cell surface integrin αVβ5 was recognized by a mouse monoclonal anti-human αVβ5 antibody (clone P1F76; sc-13588, Santa Cruz Biotech; 1:200) and stained with AF488-conjugated donkey anti-mouse IgG (ab150105, Abcam) secondary antibody.

Techniques: Purification, Recombinant, Affinity Chromatography, Chromatography, Nucleic Acid Electrophoresis, Silver Staining, Protein Purification, Control, Binding Assay

Journal: Nature Structural & Molecular Biology

Article Title: A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens

doi: 10.1038/s41594-024-01211-y

Figure Lengend Snippet: ( a ) 3D structure of the extracellular domain of ITGB5 was modeled by AlphaFold2 (cyan) and overlaid with the ITGB3 portion of integrin αVβ3 structure resolved by Xiong et al. (PDB ID: 3IJE, chain B; blue). Overall, we observed high concordance of the 3D structures between ITGB3 and ITGB5, including the highly conserved basic amino acid (ITGB3’s R287 or ITGB5’s K287) in the loop motif of the βA domain highlighted in ( b ). ( c ) Modeling of ITGAV/ITGB5 interaction using the AlphaFold2 predicted ITGB5 structure (cyan) and the ITGAV portion of integrin αVβ3 structure (PDB ID: 3IJE, chain A; red). ( d and e ) Molecular dynamics simulation using GROMACS 2022 with CHARMM36m force field indicates ( d ) a close contact between ITGB5’s K287 and ITGAV’s β-propeller HIP pocket (purple box), and ( e ) the occupancy of Cpd_AV2 (yellow) into ITGAV’s HIP pocket disengaged the side chain of ITGB5’s K287 from stably interacting with ITGAV. ( f ) Substitution of ITGB5’s K287 with an alanine (K287A) significantly attenuated the ITGAV/ITGB5 NanoBRET signal, highlighting an essential role of this basic residue in integrin αVβ5 assembly (n = 3 for each group). Data are represented as mean ± s.e.m. P value was calculated by two-sided Student’s t-test.

Article Snippet: Cell surface integrin αVβ5 was recognized by a mouse monoclonal anti-human αVβ5 antibody (clone P1F76; sc-13588, Santa Cruz Biotech; 1:200) and stained with AF488-conjugated donkey anti-mouse IgG (ab150105, Abcam) secondary antibody.

Techniques: Stable Transfection, Residue

Journal: Molecular Vision

Article Title: Establishment of a continuous untransfected human corneal endothelial cell line and its biocompatibility to denuded amniotic membrane

doi:

Figure Lengend Snippet: Immunofluorescence staining patterns of cell-junction proteins of HCE cells at passage 101. A : human zonula occludens protein 1 (ZO-1). B : human N-cadherin. C : human connecxin-43. D : human integrin αv/β5. Positive expression of the cell-junction proteins of HCE cells was shown. Scale bar: 50 μm.

Article Snippet: Immunofluorescence staining of cell-junction protein expression of HCE cells at passage 101 was performed with goat anti-human zonula occludens protein 1 (ZO-1) polyclonal antibody (1:50; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human N-cadherin monoclonal antibody (1:50; Santa Cruz), mouse anti-human connexin 43 monoclonal antibody (1:250; Chemicon, Temecula, CA), mouse anti-human integrin αv/β5 monoclonal antibody (1:50; Santa Cruz), and fluorescent isothiocyanate (FITC)-conjugated rabbit anti-goat IgG antibody or rhodamine B isothiocyanate (RBITC)-conjugated goat anti-mouse IgG antibody or FITC-conjugated goat anti-mouse IgG antibody (1:100; Biosynthesis Biotechnology) as previously described [ ], and visualized with a Nikon Eclipse TE2000-U inverted fluorescent microscope (Nikon, Tokyo, Japan).

Techniques: Immunofluorescence, Staining, Expressing

Journal: Molecular Vision

Article Title: Establishment of a continuous untransfected human corneal endothelial cell line and its biocompatibility to denuded amniotic membrane

doi:

Figure Lengend Snippet: Immunofluorescence staining patterns of cell-junction proteins of the HCE cell sheet formed on dAM. A : human zonula occludens protein 1 (ZO-1). B : human N-cadherin. C : human connexin-43. D : human integrin αv/β5. Positive expression of the cell-junction proteins of passage 101 HCE cells on dAM was shown. Scale bar: 50 μm.

Article Snippet: Immunofluorescence staining of cell-junction protein expression of HCE cells at passage 101 was performed with goat anti-human zonula occludens protein 1 (ZO-1) polyclonal antibody (1:50; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human N-cadherin monoclonal antibody (1:50; Santa Cruz), mouse anti-human connexin 43 monoclonal antibody (1:250; Chemicon, Temecula, CA), mouse anti-human integrin αv/β5 monoclonal antibody (1:50; Santa Cruz), and fluorescent isothiocyanate (FITC)-conjugated rabbit anti-goat IgG antibody or rhodamine B isothiocyanate (RBITC)-conjugated goat anti-mouse IgG antibody or FITC-conjugated goat anti-mouse IgG antibody (1:100; Biosynthesis Biotechnology) as previously described [ ], and visualized with a Nikon Eclipse TE2000-U inverted fluorescent microscope (Nikon, Tokyo, Japan).

Techniques: Immunofluorescence, Staining, Expressing

Journal: Cancer cell

Article Title: Binary pan-cancer classes with distinct vulnerabilities defined by pro- or anti-cancer YAP/TEAD activity

doi: 10.1016/j.ccell.2021.06.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-ITGB5 (clone F-5) , Santa Cruz Biotechnology , Cat# sc-398214.

Techniques: Virus, Plasmid Preparation, Derivative Assay, Recombinant, Electron Microscopy, Staining, SYBR Green Assay, Reverse Transcription, Sample Prep, Microarray, Knockdown, Gene Expression, CRISPR, Software, Flow Cytometry