Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A and B ) Immunoprecipitation followed by mass spectrometry of SIRPα-associated proteins. ( A ) Schematic representation of assay. ( B ) Plasma membrane-associated proteins found in SIRPα immunoprecipitates from WT BMDMs, but not from SIRPα KO BMDMs. c , Co-immunoprecipitation assay of SIRPα, CD18 and CD11b in WT and SIRPα KO BMDMs. IP, immunoprecipitation. Abs, antibodies. ( D to F ) FRET assays. ( D ) Schematic representation of FRET assay in HEK293T cells. ( E and F ) Representative confocal microscopy images ( E ) and compiled data ( F ) of FRET assays with donor-labeled SIRPα, acceptor-labeled CD18 and unlabeled CD11b in the presence of control (Ctrl) IgG, CD18 mAb GAME-46 or CD11b mAb 5C6. Yellow to purple spectrum denotes strong to weak FRET. DIC, differential interference contrast. Scale bars, 5 μm. ( G and H ) LUV-FRET assay. ( G ) Schematic representation of LUV-FRET assay. ( H ), Time-course of donor-labeled SIRPα fluorescence intensity after addition of acceptor-labeled CD18 or CD11b, monitored with a real-time plate reader. All data are means ± s.e.m. ns, not significant, **** p < 0.0001. Results in ( C , E and H ) are representative of 3 independent experiments. Results in ( B and F ) are pooled from a total of 3 independent experiments. Each symbol in ( F ) represents one cell.

Article Snippet: Human cDNAs for SIRPα version (V) 1 (Cat: HG11612-UT), PD-1 (Cat: HG10377-M), LILRB1 (Cat: HG16014-UT), 2B4 (Cat: HG10042-NF), CD18 (Cat: HG10970-UT) and CD11b (Cat: HG10494-UT) were obtained from Sino Biological (Beijing, China).

Techniques: Immunoprecipitation, Mass Spectrometry, Clinical Proteomics, Membrane, Co-Immunoprecipitation Assay, Confocal Microscopy, Labeling, Control, Fluorescence

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A and B ) FRET assays with SIRPα and SIRPβ1a. ( A ) A schematic representation of SIRPα and SIRPβ1a, with their 1 IgV domain and 2 IgC domains, is depicted. ( B ) Compiled data of 3 independent experiments using donor-labeled SIRPα or SIRPβ1a, acceptor-labeled CD18 and unlabeled CD11b, as done for , D to F. ( C and D ) FRET assays using SIRPα IgV domain. ( C ) A schematic representation of a SIRPα variant having only the IgV domain is shown. ( D ) Compiled data of 3 independent experiments using donor-labeled SIRPα IgV, acceptor-labeled CD18 and unlabeled CD11b, as done for , D to F. ( E - H ) FRET assays using SIRPα variants carrying non-conserved residues from SIRPβ1a. ( E and G ) Schematic representations of SIRPα variants. ( F and H ) Compiled data from 3 independent experiments using donor-labeled SIRPα variants, acceptor-labeled CD18 and unlabeled CD11b, as done for , D to F. ( I to K ) Proximity ligation assay (PLA) of SIRPα and CD18 in BMDMs expressing or not the indicated SIRPα variants. (I) Flow cytometry analyses of SIRPα expression. ( J and K ) Representative confocal microscopy images ( J ) and compiled data from 3 independent experiments ( K ) of PLA for SIRPα and CD18. Scale bar, 10 μm. All data are means ± s.e.m. ns, not significant, **** p < 0.0001. Results in ( I and J ) are representative of 3 independent experiments. Results in ( B , D , F , H and K ) are pooled from 3 independent experiments. Each symbol in ( B , D , F , H and K ) represents one cell or mouse.

Article Snippet: Human cDNAs for SIRPα version (V) 1 (Cat: HG11612-UT), PD-1 (Cat: HG10377-M), LILRB1 (Cat: HG16014-UT), 2B4 (Cat: HG10042-NF), CD18 (Cat: HG10970-UT) and CD11b (Cat: HG10494-UT) were obtained from Sino Biological (Beijing, China).

Techniques: Labeling, Variant Assay, Proximity Ligation Assay, Expressing, Flow Cytometry, Confocal Microscopy

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A to C ) The impact of SIRPα variants defective in CD18-binding, CD47-binding or phosphatase signaling, alone or in combination, expressed in BMDMs, was analyzed. ( A ) Schematic depictions of SIRPα variants, as was done for . SIRPα R91T carried an arginine (R)-to-threonine (T) mutation at position 91 (shown by blue star), which abolished CD18-binding. ( B ) Phagocytosis assays of IgG-opsonized L1210 cells by BMDMs, as was done for . ( C ) Efficiency of phagocytosis inhibition was calculated as for , using values from . ( D and E ) Representative flow cytometry profiles ( D ) and compiled data from 3 independent experiments ( E ) of ICAM-1-binding using SIRPα KO BMDMs expressing WT SIRPα or SIRPα R91T BMDMs, in the presence or absence of FcR triggering using mouse IgG2a. ( F and G ) The impact of a SIRPα variant carrying the isoleucine-to-glycine 332 (I332G) mutation, expressed in SIRPα KO BMDMs, was analyzed. (F) Flow cytometry analyses of CD11b expression. ( G ) Compiled data from 3 independent phagocytosis assays, assessed by microscopy. ( H ) FRET assays of donor-labeled SIRPα, acceptor-labeled CD18 and unlabeled CD11b in the presence of WT CD11b or CD11b I332G , as was done for , D to F. ( I ) FRET assays of donor-labeled human SIRPα version (V) 1 or V2 with acceptor-labeled human CD18 and unlabeled human CD11b, in the presence of Ctrl IgG, human CD18 mAbs CBR LFA1/2 or TS1/18, as was done for , D to F. ( J ) Phagocytosis of human lymphoma cells Raji, which were opsonized with CD20 mAbs, by human peripheral blood monocyte (PBMC)-derived macrophages, in the presence of the indicated mAbs, was assessed by microscopy. All data are means ± s.e.m. ns, not significant; * p < 0.05, ** p < 0.01 and **** p < 0.0001. Results in ( D and F ) are representative of 3 independent experiments. Results in ( B , C , E and G to J ) are pooled from 3 independent experiments. Each symbol in ( B , E and G to J ) represents one cell, mouse or healthy donor.

Article Snippet: Human cDNAs for SIRPα version (V) 1 (Cat: HG11612-UT), PD-1 (Cat: HG10377-M), LILRB1 (Cat: HG16014-UT), 2B4 (Cat: HG10042-NF), CD18 (Cat: HG10970-UT) and CD11b (Cat: HG10494-UT) were obtained from Sino Biological (Beijing, China).

Techniques: Binding Assay, Mutagenesis, Inhibition, Flow Cytometry, Expressing, Variant Assay, Microscopy, Labeling, Derivative Assay

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A ) FRET assays of donor-labeled mouse SIRPα with acceptor-labeled mouse CD18 and unlabeled mouse CD11b, in the presence of Fc-silent mouse SIRPα mAbs, as was done for , D to F. ( B ) Binding of a soluble CD47-Fc fusion protein to EL-4 cells, expressing or not expressing mouse SIRPα, was studied by flow cytometry. ( C to K ) Generation and impact of bispecific antibody (BsAb) against mouse SIRPα. ( C ) Schematic representation of Fc-silent BsAb combining one arm of mAb #17 with one arm of mAb #27, using the “knob-into-hole” technology. Phagocytosis of IgG-opsonized L1210 cells ( D ) and EL-4 cells ( E ) by WT BMDMs, in the presence of mAbs, was assessed by a microscopy assays. ( F to K ) Schematic depictions of the assays are shown in (F and I). RAG-1 KO mice injected subcutaneously with Tac + L1210 cells ( G and H ), or C57BL/6J mice injected subcutaneously with Tac + EL-4 cells ( J and K ), were treated by intraperitoneal injection of Fc-silent mAbs, alongside Tac mAb 7G7 for opsonization. Tumor volume was measured using a caliper ( G and J ) and survival was recorded ( H and K ). ( L ) FRET assays of donor-labeled human SIRPα V1 or V2 with acceptor-labeled human CD18 and unlabeled human CD11b in the presence of Fc-silent Ctrl IgG and human SIRPα mAbs KWAR23, 40A, 50A, or 18D5, as was done for , D to F. The mAbs were rendered Fc-silent by the LALAPG mutation. ( M ) Phagocytosis of IgG-opsonized Raji cells by human macrophages in the presence of Fc-silent Ctrl IgG and SIRPα mAbs KWAR23, 40A, 50A, or 18D5, was assayed as for . ( N ) FRET assays of donor-labeled human 2B4 (SLAMF4), PD-1 or LILRB1 with acceptor-labeled human CD18, in the presence of Ctrl IgG or human CD18 mAb were done as for , D to F. All data are means ± s.e.m. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Results are pooled from a total of two ( H and K ), three ( A , D , E , G , J , L and N ) or five ( B and M ) independent experiments. Each symbol in ( A , D , E and L to N ) represents one healthy donor, cell or mouse.

Article Snippet: Human cDNAs for SIRPα version (V) 1 (Cat: HG11612-UT), PD-1 (Cat: HG10377-M), LILRB1 (Cat: HG16014-UT), 2B4 (Cat: HG10042-NF), CD18 (Cat: HG10970-UT) and CD11b (Cat: HG10494-UT) were obtained from Sino Biological (Beijing, China).

Techniques: Labeling, Binding Assay, Expressing, Flow Cytometry, Microscopy, Injection, Mutagenesis

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

doi: 10.3390/cells11091532

Figure Lengend Snippet: Figure 1. Integrin β2 can be substituted by its human homolog in mouse Hoxb8 FL cells. (A) Scheme of workflow. Hoxb8 FL cells were generated from bone marrow of integrin β2-deficient mice and retrovirally transduced with mouse or human integrin β2. Human/mouse integrin β2-positive cells were FACS-sorted. (B) Surface expression of different integrin subunits and cell markers on neutrophil-like cells differentiated from control (β2+/+), integrin β2 ko (β2−/−), and human or mouse integrin β2-rescued integrin β2−/−(β2−/−/hβ2 and β2−/−/mβ2) Hoxb8 FL cells assessed by FACS analysis. (C) Integrin β2 surface expression levels on neutrophil-like cells differentiated from integrin β2−/−Hoxb8 FL cells retrovirally transduced with human integrin β2 (β2−/−/hβ2) compared to PMNs isolated from human blood. (D) Static adhesion of untreated, TNFα-treated, or PMA-treated Hoxb8 FL-derived β2+/+, β2−/−, β2−/−/hβ2, and β2−/−/mβ2 neutrophils on ICAM1. N = 4. Individual data points of the 4 independent experiments are shown. (E,F) Adhesion (E) and rolling velocities (F) of neutrophil-like cells differentiated from β2+/+, β2−/−, β2−/−/hβ2, and β2−/−/mβ2 Hoxb8 FL cells in flow chambers coated with ICAM1 and P-selectin with or without CXCL1 under constant shear rate of 1 dyn/cm2. N = 10/11 chambers with CXCL1 (rolling and adhesion); N = 3/4 without CXCL1 (adhesion). (G) Neutrophil-like β2+/+, β2−/−, β2−/−/hβ2, and β2−/−/mβ2 cells plated on a P-selectin-, ICAM1-, and CXCL1-coated surface for 10 min. Scale bar: 100 µm. All values are given as mean ± SD. * p < 0.05, *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Generated, Transduction, Expressing, Control, Isolation, Derivative Assay, Shear

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

doi: 10.3390/cells11091532

Figure Lengend Snippet: Figure 2. Expression of human integrin β2 rescues spreading and adhesion defects in mouse integrin β2 knockout macrophages. (A) Adhesion of integrin β2−/−and human or mouse integrin β2 ex- pressing integrin β2−/−macrophages to ICAM1 and fibronectin in relation to control macrophages. N = 5. Individual data points represent the 5 independent experiments. (B) Spreading of control, integrin β2−/−, and human or mouse integrin β2 expressing integrin β2−/−macrophages to ICAM1 and fibronectin assessed 2 h after plating. N = 3 independent experiments, shown as individual data points. (C) Hoxb8-derived β2+/+, β2−/−, β2−/−/hβ2, and β2−/−/mβ2 macrophages plated on an ICAM1- or fibronectin-coated surface for 2 h. Scale bar: 100 µm. (D) Alignment of the amino acid sequence of the mouse and human β2 integrin cytoplasmic tails. Talin-binding NPLF and kindlin- binding NPKF motives are highlighted in red. MP, membrane proximal; MD, membrane distal. (E) Control, integrin β2−/−, and human or mouse integrin β2 expressing integrin β2−/−

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Expressing, Knock-Out, Control, Derivative Assay, Sequencing, Binding Assay, Membrane

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

doi: 10.3390/cells11091532

Figure Lengend Snippet: Figure 3. Expression of human integrin β2 in mouse integrin β2−/−Hoxb8 cells allows assessment of integrin activity using conformation-specific anti-human integrin β2 antibodies. (A,B) FACS analyses of integrin β2 activation of Hoxb8-derived neutrophils expressing human or mouse integrin β2 by measuring staining intensities of conformation-specific antibodies mAb24 (A) and KIM127 (B) either untreated, EDTA-treated, or in response to TNFα, fMLP, or PMA. (C,D) Relative mAb24 (C) and KIM127 (D) binding to resting or EDTA-, TNFα-, fMLP-, or PMA-stimulated Hoxb8-derived neutrophil-like cells upon treatment with DMSO, the phosphatidylinositol-3-kinase (PI3K) inhibitor Wortmannin, the phospholipase C (PLC) inhibitor U-73122, or the protein kinase C (PKC) inhibitor Gö6983. N = 7 (mAb24) or 5 (KIM127) independent experiments, shown as individual data points. All values are given as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Expressing, Activity Assay, Activation Assay, Derivative Assay, Staining, Binding Assay

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

doi: 10.3390/cells11091532

Figure Lengend Snippet: Figure 4. Both talin1 and kindlin3 are required for β2 integrin activation. (A) Scheme of work- flow. Talin1, kindlin3, and talin1/kindlin3 double-deficient Hoxb8 FL cells were generated via the CRISPR/Cas9 system. Single cell clones were screened for talin1 and kindlin3 expression. Four clones per genotype were subjected to CRISPR/Cas9-mediated integrin β2 ablation and retrovirally trans- duced to express human integrin β2. Mouse integrin β2-negative and human integrin β2-positive cells were FACS-sorted. (B) Western blot analysis of neutrophil-like cells differentiated from different human integrin β2 expressing Hoxb8 single cell clones, in which talin1 and/or kindlin3 were ablated with the CRISPR/Cas9 system. GAPDH served as loading control. (C,D) Relative mAb24 (C) and KIM127 (D) binding to neutrophil-like cells derived from Hoxb8 single cell clones, expressing human integrin β2 and lacking talin1 and/or kindlin3 expression. N = 4 clones. Result of each clone is plotted as individual data point. All values are given as mean ± SD. ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Activation Assay, Generated, CRISPR, Clone Assay, Expressing, Western Blot, Control, Binding Assay, Derivative Assay

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

doi: 10.3390/cells11091532

Figure Lengend Snippet: Figure 5. Kindlin3 is dispensable for P-selectin-induced integrin αLβ2-mediated slow rolling but required for chemokine-induced slower rolling. (A–C) Adhesion (A), rolling velocities (B), and rolling velocities after LFA-1 blocking (C) of Hoxb8 cell-derived PMN-LCs in flow chambers coated with ICAM1, P-selectin, and CXCL1 under constant shear rate of 1 dyn/cm2. N = 12–16 (A,B) and 6–9 (C) flow chambers. (D) Rolling velocities of PMN-LCs on ICAM1- and P-selectin-coated surfaces (without CXCL1) under constant shear rate of 1 dyn/cm2. N = 8–9 flow chambers. Cells were differentiated from different single cell clones, in which talin1 and/or kindlin3 were ablated with the CRISPR/Cas9 system. Rolling velocities are shown as cumulative distribution of the velocities of approximately 500 (B), 300 (C), and 400 (D) cells. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Blocking Assay, Derivative Assay, Shear, Clone Assay, CRISPR

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

doi: 10.3390/cells11091532

Figure Lengend Snippet: Figure 6. Talin and/or kindlin3 binding-deficient human integrin β2 exhibit impaired integrin ac- tivation. (A) Scheme of the integrin β2 cytoplasmic domain, which binds talin1 via a membrane proximal NPLF motif and kindlin3 via a membrane-distal NPKF and a TTT motif (left). Muta- tion of these motifs to NPLA and AAA prevent talin1 and kindlin3 binding, respectively (right). (B) Relative mAb24 binding to neutrophil-like cells derived from Hoxb8 cells expressing human wild-type integrin β2, mutant integrin β2 F/A (F754A), mutant integrin β2 TTT/AAA (T758A, T759A, T760A), or double-mutant integrin F/A TTT/AAA in response to TNFα, CXCL1, fMLP, and PMA or left untreated. N = 5 experiments indicated as individual data points. All values are given as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Binding Assay, Membrane, Derivative Assay, Expressing, Mutagenesis

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

doi: 10.3390/cells11091532

Figure Lengend Snippet: Figure 7. Rapid CRISPR-based screening approach to study putative β2 integrin regulators. (A) Scheme

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: CRISPR

Journal: mBio

Article Title: The Extracellular Domain of the β 2 Integrin β Subunit (CD18) Is Sufficient for Escherichia coli Hemolysin and Aggregatibacter actinomycetemcomitans Leukotoxin Cytotoxic Activity

doi: 10.1128/mBio.01459-19

Figure Lengend Snippet: β 2 integrin β subunit is necessary for U-937 cell sensitivity to HlyA cytotoxic activity. (A) PEG-precipitated HlyA was incubated at various concentrations with the U-937 wild-type and individual-integrin-subunit-knockout cell lines at 2 × 10 6 cells/ml for 1 h. Cells were washed, and cell viability was measured by XTT assay. The percentage of cytotoxicity was normalized to Triton X-100-treated cells at 100% and RPMI-only-treated cells at 0%. The CD 50 was calculated in GraphPad Prism, and bars represent the average and SEM from 3 biological replicates. One-way ANOVA with Bonferroni’s multiple-comparison test was performed in GraphPad Prism, with significance of each cell type compared to U-937 cells represented. (B) As in panel A, a standard XTT cytotoxicity assay was performed with multiple-integrin-subunit-knockout cell lines as indicated. Results were normalized and statistics determined as described above. (C) β 2 expression was assessed by flow cytometry on intact cells from the cell lines indicated. Bars represent the average mean fluorescent intensity (MFI) and SEM from 3 biological replicates with at least 50,000 events recorded per replicate. One-way ANOVA with Bonferroni’s multiple-comparison test was performed in GraphPad Prism, with the significance of each cell type compared to U-937 Δβ 2 cells represented. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) Recombinant integrin pairs and human ICAM-1 were separated by a 4 to 20% gradient SDS-PAGE gel, transferred to nitrocellulose, and probed with HlyA at 1 μg/ml. (Left) Bound HlyA was detected with polyclonal anti-HlyA, and integrin α subunits were detected with monoclonal antibodies. (Center) Bound HlyA was detected with a pool of monoclonal anti-HlyA antibodies, and the integrin β 2 subunit was detected with a polyclonal antibody. (Right) Single-channel images of the center blot. Multiplexed near-infrared fluorescence was used to detect multiple proteins on the same blot using a Licor Odyssey imager. Blots are representative of three biological replicates.

Article Snippet: Recombinant human integrin α L β 2 (3868-AV), human integrin α M β 2 (4047-AM), human integrin α X β 2 (5755-AX), and human ICAM-1/CD54 (720-IC) were obtained from R&D Systems.

Techniques: Activity Assay, Incubation, Knock-Out, XTT Assay, Comparison, Cytotoxicity Assay, Expressing, Flow Cytometry, Recombinant, SDS Page, Bioprocessing, Fluorescence

Journal: mBio

Article Title: The Extracellular Domain of the β 2 Integrin β Subunit (CD18) Is Sufficient for Escherichia coli Hemolysin and Aggregatibacter actinomycetemcomitans Leukotoxin Cytotoxic Activity

doi: 10.1128/mBio.01459-19

Figure Lengend Snippet: β 2 integrins specifically enhance the activity of LtxA. (A, B, and D) PEG-precipitated LtxA was incubated at various concentrations with U-937 wild-type, individual- or multiple-subunit-knockout cell lines, or complemented Δβ 2 cells as indicated for each panel at 2 × 10 6 cells/ml for 3 h. Following toxin incubations, cells were washed, and cell viability was measured by XTT assay. The percentage of cytotoxicity was normalized to Triton X-100-treated cells at 100% and RPMI-only-treated cells at 0%. The CD 50 was calculated in GraphPad Prism, and bars represent the average and SEM from 3 biological replicates. The dashed line indicates the limit of detection. One-way ANOVA with Bonferroni’s multiple-comparison test was performed in GraphPad Prism, with significance of each cell type compared to U-937 cells represented. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) β 2 expression was assessed by flow cytometry on intact cells from the cell lines indicated. Bars represent the average MFI and SEM from 3 biological replicates with at least 50,000 events recorded per replicate. One-way ANOVA with Bonferroni’s multiple-comparison test was performed in GraphPad Prism, with significance of each cell type compared to U-937 cells represented. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (E) Recombinant integrin pairs and human ICAM-1 were separated by a 4 to 20% gradient SDS-PAGE gel, transferred to nitrocellulose, and probed with LtxA at 1 μg/ml. Bound LtxA was detected with cross-reactive monoclonal anti-HlyA antibodies. The integrin β 2 subunit was detected with a polyclonal antibody. Multiplexed near-infrared fluorescence was used to detect multiple proteins on the same blot using a Licor Odyssey imager. Blot is representative of three biological replicates.

Article Snippet: Recombinant human integrin α L β 2 (3868-AV), human integrin α M β 2 (4047-AM), human integrin α X β 2 (5755-AX), and human ICAM-1/CD54 (720-IC) were obtained from R&D Systems.

Techniques: Activity Assay, Incubation, Knock-Out, XTT Assay, Comparison, Expressing, Flow Cytometry, Recombinant, SDS Page, Fluorescence

Journal: Journal of Biological Chemistry

Article Title: The cell surface hyaluronidase TMEM2 regulates cell adhesion and migration via degradation of hyaluronan at focal adhesion sites

doi: 10.1016/j.jbc.2021.100481

Figure Lengend Snippet: Figure 5. Association of transmembrane protein 2 (TMEM2) with integrins via interactions between the extracellular domains. A and B, targeting of TMEM2 to focal adhesions (FAs) does not require the cyto- plasmic domain of TMEM2. In this experiment, mCherry-mTMEM2 (full length) and mCherry-mTMEM2/Δcyto (Δcyto) cells were analyzed for their in situ hyaluronan (HA) degradation activities. To allow specific analysis of the activity of the full-length mouse TMEM2 and its Δcyto deletion mutant, expression of endogenous human TMEM2 was silenced by siRNA treatment prior to the assay. A, in situ HA degradation assays were performed on substrate immobilized with FA-HA, as described in Experimental procedures section. Note that the pattern of in situ HA degradation is indistinguishable between mCherry-mTMEM2/Δcyto and mCherry-mTMEM2 cells. The scale bar represents 10 μm. B, immunostaining for vinculin in mCherry-mTMEM2 and mCherry-mTMEM2/Δcyto cells on the FA-HA substrate. Note that the sites of HA degradation colocalize with vinculin-immunoreactive puncta in both mCherry-mTMEM2/Δcyto and mCherry-mTMEM2 cells. The scale bar represents 2 μm. C–E, TMEM2 associates with integrins via extracellular interactions. C, cell surface–expressed TMEM2 is coimmunoprecipitated with integrin α5β1. mCherry-mTMEM2 cells were treated with the membrane-impermeable crosslinker 3’,3’-dithiobis(sulfosuccinimidyl

Article Snippet: Two micrograms of recombinant heterodimer of integrin ECD (α5β1; R&D Systems: 3230-A5-050; αLβ2; R&D Systems: 3868-AV-050) were applied to the TMEM2–ECD–bound and control unbound resin and incubated in HBSS++ overnight at 4 C. After extensive washing, bound materials were eluted by boiling in SDS-PAGE sample buffer, and eluents were analyzed by SDSPAGE and immunoblotting with rabbit polyclonal antiintegrin α5 (Proteintech; 10569-1-AP), rabbit monoclonal anti-integrin β1 (Abcam; ab52971), rabbit polyclonal antiintegrin β2 (Proteintech; 10544-1-AP), or mouse monoclonal anti-polyhistidine (Sigma; A7058; clone: HIS-1, peroxidase conjugated).

Techniques: In Situ, Activity Assay, Mutagenesis, Expressing, Immunostaining, Membrane

Journal: Journal of Biological Chemistry

Article Title: The cell surface hyaluronidase TMEM2 regulates cell adhesion and migration via degradation of hyaluronan at focal adhesion sites

doi: 10.1016/j.jbc.2021.100481

Figure Lengend Snippet: Figure 6. A model for the role of transmembrane protein 2 (TMEM2) in integrin-mediated cell adhesion and migration. Our results suggest that TMEM2-dependent degradation of hyaluronan (HA) is critical for cells to form strong cell–matrix adhesion on HA-rich extracellular matrix (ECM). A, high levels of HA in the ECM are inhibitory to the direct engagement of integrins to their ECM ligands. B, in the presence of TMEM2, HA in the ECM is locally removed, which generates a microenvironment that is permissible to the direct integrin–ECM engagement. C, the association between TMEM2 and integrins promotes the FA formation and maturation via further removal of HA in the vicinity of the integrin–ECM engagement. D, this in turn facilitates integrin clustering, integrin-mediated downstream signaling, and cellular responses. See the text for further discussion.

Article Snippet: Two micrograms of recombinant heterodimer of integrin ECD (α5β1; R&D Systems: 3230-A5-050; αLβ2; R&D Systems: 3868-AV-050) were applied to the TMEM2–ECD–bound and control unbound resin and incubated in HBSS++ overnight at 4 C. After extensive washing, bound materials were eluted by boiling in SDS-PAGE sample buffer, and eluents were analyzed by SDSPAGE and immunoblotting with rabbit polyclonal antiintegrin α5 (Proteintech; 10569-1-AP), rabbit monoclonal anti-integrin β1 (Abcam; ab52971), rabbit polyclonal antiintegrin β2 (Proteintech; 10544-1-AP), or mouse monoclonal anti-polyhistidine (Sigma; A7058; clone: HIS-1, peroxidase conjugated).

Techniques: Migration

Journal: BMC Cancer

Article Title: LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

doi: 10.1186/1471-2407-12-455

Figure Lengend Snippet: Primer sequences and PCR settings

Article Snippet: Specific direct primary antibodies CD11a antibody (FAB35951A) and CD18 (FAB1730P) from R&D system (Minneapolis, MN, USA) or isotypic control antibody (BD Pharmingen, San Diego, CA, USA) were used at 1 μg ml.

Techniques: Hybridization

Journal: BMC Cancer

Article Title: LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

doi: 10.1186/1471-2407-12-455

Figure Lengend Snippet: Expression of CD11a and CD18 in melanoma A375, 1205LU and SLM8 cell lines with conditioned medium. Cell-surface expression of CD11a and CD18 on indicated melanoma cell lines treated for 24 hrs with HUVEC conditioned medium was analyzed by flow cytometry. Isotypic controls are represented as empty histograms and specific antibody-labelling is displayed as shaded histograms. Histograms obtained with cells incubated with FCS-complete medium and labeled with specific antibodies, which overlap with the isotypic control are not shown. Data from obtained with 3 independent experiments.

Article Snippet: Specific direct primary antibodies CD11a antibody (FAB35951A) and CD18 (FAB1730P) from R&D system (Minneapolis, MN, USA) or isotypic control antibody (BD Pharmingen, San Diego, CA, USA) were used at 1 μg ml.

Techniques: Expressing, Flow Cytometry, Incubation, Labeling, Control

Journal: BMC Cancer

Article Title: LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

doi: 10.1186/1471-2407-12-455

Figure Lengend Snippet: Effect of CD11a and CD18-blocking antibodies on the transendothelial migration of A375, 1205LU and SLM8 cell lines. The experiments were performed as detailed in Figure , except that 2μg/ml of CD11a or CD18-blocking antibodies were introduced in the upper chamber of the Transwells when indicated. Histograms represent 3 independent experiments. In each experiment each condition was analyzed in duplicate.

Article Snippet: Specific direct primary antibodies CD11a antibody (FAB35951A) and CD18 (FAB1730P) from R&D system (Minneapolis, MN, USA) or isotypic control antibody (BD Pharmingen, San Diego, CA, USA) were used at 1 μg ml.

Techniques: Blocking Assay, Migration

Journal: BMC Cancer

Article Title: LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

doi: 10.1186/1471-2407-12-455

Figure Lengend Snippet: Effect of CD11 and CD18-blocking antibodies on the formation of clumps. A Semi-quantitative PCRs were performed to detect the expression of the ICAM-1 transcript. GAPDH is used as a DNA amount control. B A375, 1205LU and SLM8 cell lines were treated with 2 μg/ml of CD11a or CD18-blocking antibodies as indicated. Melanoma cells were labeled with DiO then fixed and labeled with DAPI prior to their observation under an epifluorescence microscope using a magnification of x10. Data were obtained from 3 independent experiments.

Article Snippet: Specific direct primary antibodies CD11a antibody (FAB35951A) and CD18 (FAB1730P) from R&D system (Minneapolis, MN, USA) or isotypic control antibody (BD Pharmingen, San Diego, CA, USA) were used at 1 μg ml.

Techniques: Blocking Assay, Expressing, Control, Labeling, Microscopy