Journal: International Journal of Biological Sciences

Article Title: Integrative Single-Cell and Spatial Transcriptomics Analysis Reveals ECM-remodeling Cancer-associated Fibroblast-Derived POSTN as a Key Mediator in Pancreatic Ductal Adenocarcinoma Progression

doi: 10.7150/ijbs.108618

Figure Lengend Snippet: CAF-derived POSTN induces the translation to EMT-subtype via PI3K/AKT/β-catenin signaling in PDAC Cells. (A) Differential gene expression analysis of ductal cells based on fibroblast-derived POSTN levels. Tumor samples from the CRA001160 dataset were classified into high- and low- POSTN groups (75th percentile cutoff), and ductal cell gene expression profiles were compared to identify differences linked to CAF-derived POSTN. (B) GSEA of upregulated genes in ductal cells from POSTN-high samples identified enriched KEGG pathways. (C-F) GSEA of upregulated genes revealed enrichment in the EMT pathway, focal adhesion pathway, PI3K-AKT pathway, and WNT pathway. (G-H) Western blot analysis of β-catenin expression, and phosphorylation of FAK, AKT, and GSK-3β in BxPC-3 and PANC-1 cells after 24-hour treatment with rhPOSTN at varying concentrations. (I) Schematic illustration of how CAF-derived POSTN drives the EMT phenotype in PDAC cells via integrin αvβ5/FAK/PI3K/AKT/β-catenin signaling pathway.

Article Snippet: BxPC-3 cells were pretreated for 24 h with CAF-oePOSTN CM, CAF-NC CM, recombinant human Periostin (rhPOSTN, Novoprotein, CJ39), or integrin αvβ5 receptor inhibitor (MCE, HY-16141).

Techniques: Derivative Assay, Gene Expression, Western Blot, Expressing, Phospho-proteomics

Journal: International Journal of Biological Sciences

Article Title: Integrative Single-Cell and Spatial Transcriptomics Analysis Reveals ECM-remodeling Cancer-associated Fibroblast-Derived POSTN as a Key Mediator in Pancreatic Ductal Adenocarcinoma Progression

doi: 10.7150/ijbs.108618

Figure Lengend Snippet: Integrin αvβ5 inhibitors partially reverse POSTN-induced proliferation, colony formation, migration, and invasion of PDAC cells. (A) Co-localization of POSTN and intergrin β5 in PDAC tissues. Immunofluorescence staining showing POSTN (green), integrin β5 (red), and nuclei (blue) in PDAC patient resection specimens. Scale bars, 25 μm. (B-C) Effect of integrin αvβ5 inhibition on POSTN-induced proliferation in BxPC-3 and PANC-1 cells. Cells were treated with: (1) negative control, (2) 500 ng/mL rhPOSTN, (3) integrin αvβ5 inhibitor (HY-16141) at 1/5 IC 50 concentration, or (4) integrin αvβ5 inhibitor pretreated for 24 hours, followed by 500 ng/mL rhPOSTN. Cell proliferation was assessed using CCK-8 assays. (D) Colony formation assays in BxPC-3 and PANC-1 cells following the same treatments as in (B-C). (E-F) Wound healing assays in BxPC-3 and PANC-1 cells after treatments as described in (B-C), assessing migration capacity. (G-H) Transwell assays in BxPC-3 and PANC-1 cells to assess migation (G) and invasion (H) under the same treatment as in (B-C). (I) Western blot analysis of β-catenin expression, and phosphorylation of FAK, AKT, and GSK-3β in BxPC-3 and PANC-1 cells after 24-hour treatment as described in (B-C).

Article Snippet: BxPC-3 cells were pretreated for 24 h with CAF-oePOSTN CM, CAF-NC CM, recombinant human Periostin (rhPOSTN, Novoprotein, CJ39), or integrin αvβ5 receptor inhibitor (MCE, HY-16141).

Techniques: Migration, Immunofluorescence, Staining, Inhibition, Negative Control, Concentration Assay, CCK-8 Assay, Western Blot, Expressing, Phospho-proteomics

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: The Journal of Clinical Investigation

Article Title: Nonimmune cell–derived ICOS ligand functions as a renoprotective αvβ3 integrin–selective antagonist

doi: 10.1172/JCI123386

Figure Lengend Snippet: (A) Schematic of a gold surface with ICOSL protein on a sensor chip CM5 and associated protein (αvβ3 integrin) over which buffer is flown in SPR assay. (B–I) SPR sensorgrams depicting interaction of immobilized human ICOSL (hICOSL, B–D) or mouse ICOSL (mICOSL, E–I) with αvβ3 integrin. These bindings were tested in the presence (B and E, active form of αvβ3) or absence (C and F, inactive form of one with EDTA in the binding buffer) of Mn2+. (D and G) SPR used in an inhibition experiment with cRGDfv. Injection of αvβ3 integrin only (D, 120 nM αvβ3 or G, 150 nM αvβ3) resulted in a binding signal for immobilized hICOSL or mICOSL alone (pink line). Preincubation with cRGDfv (3 μM or 15 μM) significantly reduced the binding for ICOSL, indicating that the RGD peptide competes with ICOSL for binding to αvβ3 (orange line). cRGDfv alone was used as a control (green line). (H and I) SPR sensorgrams showing the binding between WT (H) or mutant (I) mICOSL protein and αvβ3 integrin in the presence of physiologically relevant divalent ions, Ca2+ (0.2 mM) and Mg2+ (0.1 mM). The average KD values were determined from at least 3 independent experiments. Rate constants (ka and kd) were determined by kinetic fitting (black dotted line) of the sensorgrams using 1-to-1 Langmuir binding equation, and KD values for B, E, and H were calculated by kd/ka (B, KD = 16.2 ± 4.0 nM for hICOSL/αvβ3 with Mn2+; E, KD = 24.2 ± 6.5 nM for mICOSL/αvβ3 with Mn2+; H, KD = 21.3 ± 1.2 nM for WT mICOSL/αvβ3 with Ca2+/Mg2+). KD values for C, F, and I were calculated from steady-state affinity fittings (C, KD = 411.8 ± 164.1 nM for hICOSL/αvβ3; F, KD ≥ 2 mM for mICOSL/αvβ3; I, KD = 0.83 ± 0.8 mM for mutant mICOSL/αvβ3 with Ca2+/Mg2+).

Article Snippet: Recombinant proteins used in this study were as follows: human integrin αvβ3 (R&D Systems, 3050-AV), human integrin α3β1 (R&D Systems, 2840-A3), human integrin αIIbβ3 (R&D Systems, 7148-A2), human integrin αvβ5 (R&D Systems, 2528-AV), human fibronectin (R&D Systems, 4305-FNB-200), human ICOSL-His Tag (Thermo Fisher Scientific, 11559H08H50), mouse integrin αvβ3 (R&D Systems, 7889-AV-050), mouse ICOSL (Sino Biological, 50-190-M08H25), human B7.1 (R&D Systems, 140-B1), and human ICOS (R&D Systems, 169-CS).

Techniques: SPR Assay, Binding Assay, Inhibition, Injection, Control, Mutagenesis

Journal: The Journal of Clinical Investigation

Article Title: Nonimmune cell–derived ICOS ligand functions as a renoprotective αvβ3 integrin–selective antagonist

doi: 10.1172/JCI123386

Figure Lengend Snippet: (A) Schematic representation of the protocol to measure relative cell adhesion levels in cultured human podocytes. Image analysis and quantification by high-content screening technology were described in Methods. (B) Phase-contrast microscopy images show that cultured human podocytes confer enhanced adhesion to ICOSL mediated by β3 integrin treated with Mn2+, but do not adhere on albumin (protein control). Increased adhesion levels were completely prevented by incubation with the integrin inhibitors, including cRGD peptide and anti-β3 integrin antibody. Scale bar 100 μm. (C) Quantification of the cell adhesion using the images in B. ICOSL induced cell adhesion to RGD-dependent β3 integrin on cultured podocytes. (D) Cell adhesion analysis of cultured podocytes plated on vitronectin. Data are shown as mean ± SD; ***P < 0.001; 1-way ANOVA with Tukey’s multiple comparison test (C and D).

Article Snippet: Recombinant proteins used in this study were as follows: human integrin αvβ3 (R&D Systems, 3050-AV), human integrin α3β1 (R&D Systems, 2840-A3), human integrin αIIbβ3 (R&D Systems, 7148-A2), human integrin αvβ5 (R&D Systems, 2528-AV), human fibronectin (R&D Systems, 4305-FNB-200), human ICOSL-His Tag (Thermo Fisher Scientific, 11559H08H50), mouse integrin αvβ3 (R&D Systems, 7889-AV-050), mouse ICOSL (Sino Biological, 50-190-M08H25), human B7.1 (R&D Systems, 140-B1), and human ICOS (R&D Systems, 169-CS).

Techniques: Cell Culture, High Content Screening, Microscopy, Control, Incubation, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Nonimmune cell–derived ICOS ligand functions as a renoprotective αvβ3 integrin–selective antagonist

doi: 10.1172/JCI123386

Figure Lengend Snippet: In this study, it is shown that ICOSL binds podocyte αvβ3 integrin through its RGD motif. Kidney injury results in a rapid increase of ICOSL expression, leading to podocyte protection by blocking active αvβ3 integrin. ICOSL acts as a regulatory brake to modulate active αvβ3 integrin–mediated signaling. Fp, foot process.

Article Snippet: Recombinant proteins used in this study were as follows: human integrin αvβ3 (R&D Systems, 3050-AV), human integrin α3β1 (R&D Systems, 2840-A3), human integrin αIIbβ3 (R&D Systems, 7148-A2), human integrin αvβ5 (R&D Systems, 2528-AV), human fibronectin (R&D Systems, 4305-FNB-200), human ICOSL-His Tag (Thermo Fisher Scientific, 11559H08H50), mouse integrin αvβ3 (R&D Systems, 7889-AV-050), mouse ICOSL (Sino Biological, 50-190-M08H25), human B7.1 (R&D Systems, 140-B1), and human ICOS (R&D Systems, 169-CS).

Techniques: Expressing, Blocking Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: WISP1 induces the expression of macrophage migration inhibitory factor in human lung fibroblasts through Src kinases and EGFR-activated signaling pathways

doi: 10.1152/ajpcell.00410.2023

Figure Lengend Snippet: αvβ5-integrin and chondroitin sulfate proteoglycans are implicated in Wnt1-inducible signaling protein 1 (WISP1)-induced production of migration inhibitory factor (MIF) from human lung fibroblasts (HLFs). HLFs were pretreated for 30 minutes with anti-αvβ5-integrin antibody (6 µg/mL) ( A ) or for 2 h with chondroitinase ABC or chondroitinase AC II (3 units/mL) ( B ), followed by stimulation with WISP1 (100 ng/mL) for 48 h, and the levels of MIF in conditioned media were determined by ELISA. Data represent the means ± SD ( n = 3). *Statistically significant increase ( P < 0.05) compared to control. #Statistically significant decrease ( P < 0.05) compared to WISP1-treated cells. IgG represents immunoglobulins from nonimmunized mouse.

Article Snippet: Recombinant human EGF and anti-integrin αvβ5 mouse mAb (no. MAB2528-SP) were purchased from R&D Systems (Bio-Techne Ltd., Abingdon, UK).

Techniques: Migration, Enzyme-linked Immunosorbent Assay, Control

Journal: American Journal of Physiology - Cell Physiology

Article Title: WISP1 induces the expression of macrophage migration inhibitory factor in human lung fibroblasts through Src kinases and EGFR-activated signaling pathways

doi: 10.1152/ajpcell.00410.2023

Figure Lengend Snippet: Proposed model of the effects of Wnt1-inducible signaling protein 1 (WISP1) on the expression of migration inhibitory factor (MIF) and its receptors, as well as on the expression of cyclooxygenase (COX)-2, IL-6, and matrix metalloproteinase (MMP)-2, and production of prostaglandin E 2 (PGE 2 ) from human lung fibroblasts (HLFs). The effect of WISP-1 on the expression of MIF in HLFs with the complex interactions between WISP-1, αvβ5, Src kinases, EGF receptor (EGFR), and CD74/CD44 receptors, reveal intricate regulatory networks that could contribute to the expression of proinflamamtory molecules and remodeling enzymes secretion.

Article Snippet: Recombinant human EGF and anti-integrin αvβ5 mouse mAb (no. MAB2528-SP) were purchased from R&D Systems (Bio-Techne Ltd., Abingdon, UK).

Techniques: Expressing, Migration