Journal: Sensors and Actuators B: Chemical

Article Title: Extracellular matrix protein microarray-based biosensor with single cell resolution: Integrin profiling and characterization of cell-biomaterial interactions

doi: 10.1016/j.snb.2019.126954

Figure Lengend Snippet: Fig. 1. Protein microarray biosensor development. (A) Scheme of the

Article Snippet: A suspension of 106 cells was dyed for 10 min at 4 oC in the dark with human CD29-PEVIO770 antibodies for β1 detection (Miltenyi Biotec, Cat. No: 130-101-281) and human CD51/CD61-APC antibodies for αvβ3 determination (Miltenyi Biotec, Cat. No: 130-103-745) following the manufacturer’s instructions.

Techniques: Microarray

Journal: Nature Communications

Article Title: Force-bearing phagocytic adhesion rings mediate the phagocytosis of surface-bound particles

doi: 10.1038/s41467-025-56404-w

Figure Lengend Snippet: a A substrate grafted with an integrative tension sensor (ITS) enables the imaging of integrin tensions generated by macrophages on the substrate. ITS is a DNA/PNA duplex conjugated with an integrin ligand and a quencher-dye pair. Densely immobilized on a substrate, ITS becomes fluorescent after integrin tension from cells ruptures the duplex, hence recording the integrin tension signal by fluorescence on the substrate. The substrate is supplemented with fibronectin to minimize the impact of ITS rupture on normal cell functions. b 3D images of F-actin (green) and integrin tensions (magenta) on substrates responding to 2 µm-sized surface-bound microbeads (blue). The beads were either casein-passivated or IgG-opsonized. c Integrin tension signals and F-actin structures of macrophages on substrates immobilized with microbeads (size: 1.1 µm) and coated with RGD-null ITS (no integrin ligand), unzip ITS (low tension tolerance, T tol ) or shear ITS (high T tol ). “BF” denotes bright field imaging. The experiment was repeated three times independently with similar results. d Force signal intensities of individual particles on surfaces coated with unzip ITS, RGD-null ITS, and shear ITS, respectively. Each point represents an individual particle, with 60 particles quantified for each condition. Data in ( d ) are collected from three independent experiments and presented as mean ± SD. p : two-tailed unpaired t -test with Welch’s correction. **** p < 0.0001. Source data are provided in the Source Data file.

Article Snippet: After cell culture for 40 h, integrin β 2 specific siRNA (Itgb2 Mouse siRNA Oligo Duplex, SR419987, Origene), integrin β 1 specific siRNA (Itgb1 Mouse siRNA Oligo Duplex, SR427527, Origene), scrambled siRNA as negative control (SR30004, Origene), or dye-labeled siRNA (SR30002, Origene) for the transfection verification was added as follows: siRNAs were dissolved in RNase-free buffer (provided by the supplier) at 10 μM as the stocking concentration.

Techniques: Imaging, Generated, Fluorescence, Shear, Two Tailed Test

Journal: Nature Communications

Article Title: Force-bearing phagocytic adhesion rings mediate the phagocytosis of surface-bound particles

doi: 10.1038/s41467-025-56404-w

Figure Lengend Snippet: a Macrophages (THP-1 cells) consistently formed circular adhesion structures that we named phagocytic adhesion rings (PARs) on the substrate, specifically surrounding surface-bound microbeads (size: 1.1 µm). The beads were passivated by casein coating. Immunostaining shows that integrin integrin β 2, but not integrin β 1 , is consistently recruited to PARs. As a control, integrin β 1 was recruited to focal adhesions in HeLa cells. The integrins in PARs and focal adhesions transmit molecular tensions (magenta) reported by ITS. b Integrin β 2 -YFP, but not integrin β 1 -YFP, clustered around microbeads and co-localized with integrin tensions in live macrophages. c Knocking down integrin β 2 with siRNA significantly reduced integrin tensions in PARs. Macrophages in null control were not transfected with siRNA. Macrophages in negative control were transfected with scrambled siRNA. Each point represents an individual PAR, with 100 PARs for each experiment condition. d Knocking down integrin β 2 significantly reduced phagocytic efficiencies of surface-bound passivated microbeads by macrophages. Each point represents an individual cell, with 10 cells for each experiment condition. e Immunostaining showed that PARs consist of talin, vinculin, F-actin, Arp3, pFAK, and Rab5, but not FCHO2. Each experiment in ( a , b , e ) was repeated three times independently with similar results. Data in ( c , d ) are collected from three independent experiments and presented as mean ± SD. p : two-tailed unpaired t-test with Welch’s correction. **** p < 0.0001. Source data are provided in the Source Data file.

Article Snippet: After cell culture for 40 h, integrin β 2 specific siRNA (Itgb2 Mouse siRNA Oligo Duplex, SR419987, Origene), integrin β 1 specific siRNA (Itgb1 Mouse siRNA Oligo Duplex, SR427527, Origene), scrambled siRNA as negative control (SR30004, Origene), or dye-labeled siRNA (SR30002, Origene) for the transfection verification was added as follows: siRNAs were dissolved in RNase-free buffer (provided by the supplier) at 10 μM as the stocking concentration.

Techniques: Immunostaining, Control, Transfection, Negative Control, Two Tailed Test

Journal: Nature Communications

Article Title: Force-bearing phagocytic adhesion rings mediate the phagocytosis of surface-bound particles

doi: 10.1038/s41467-025-56404-w

Figure Lengend Snippet: a Integrin tensions and F-actin structures of human monocyte-induced macrophages in response to surface-bound microbeads or E . coli. b Integrin tension and F-actin structures of human THP-1 macrophages in response to surface-bound microbeads or E . coli. c Integrin tension and F-actin structures of mouse RAW macrophages in response to surface-bound microbeads or E . coli. d Average fluorescence intensities of integrin tension signals in PARs in response to single surface-bound particles. Each point represents an individual PAR. Each experiment in ( a , b , c ) was repeated three times independently with similar results. Data in ( d ) are collected from three independent experiments. Source data are provided in the Source Data file.

Article Snippet: After cell culture for 40 h, integrin β 2 specific siRNA (Itgb2 Mouse siRNA Oligo Duplex, SR419987, Origene), integrin β 1 specific siRNA (Itgb1 Mouse siRNA Oligo Duplex, SR427527, Origene), scrambled siRNA as negative control (SR30004, Origene), or dye-labeled siRNA (SR30002, Origene) for the transfection verification was added as follows: siRNAs were dissolved in RNase-free buffer (provided by the supplier) at 10 μM as the stocking concentration.

Techniques: Fluorescence

Journal: Nature Communications

Article Title: Force-bearing phagocytic adhesion rings mediate the phagocytosis of surface-bound particles

doi: 10.1038/s41467-025-56404-w

Figure Lengend Snippet: a TGT, acting as integrin ligands that rupture at a critical force, was utilized to restrict the integrin tension level of whole cells under a designed level determined by the critical force. The unzip TGT has a lower critical force compared to the shear TGT, hence restricting integrin tensions under a lower level. To facilitate TGT application, a PLL-PEG polymer was coated on glass surfaces, providing non-fouling surfaces that minimize non-specific integrin-substrate interactions while still supporting cell adhesion through electrostatic attraction. b F-actin imaged on the TGT-null, the unzip TGT, and the shear TGT surfaces immobilized with casein-passivated microbeads (size: 1.1 µm). F-actin ring-like structures (F-actin rings) were formed on both TGT surfaces but were much less prevalent on the TGT-null surface. c PARs represented by F-actin rings were formed on the unzip TGT and the shear TGT surfaces, but not on the TGT-null surface. PAR formation is quantified as the percentage of microbeads surrounded by visible F-actin rings within individual cells. Each point represents an individual cell, with cell number n = 20 for each condition. d F-actin signal intensities around microbeads beneath macrophages on the TGT-null, the unzip TGT, and the shear TGT surfaces, respectively. Each point represents an individual PAR, with PAR number n = 31, 36, and 39, respectively (left to right). e The unzip TGT (fluorophore-quencher labeled) and the shear TGT were mixed at concentration ratios of 4:0, 2:2, and 1:3 and coated onto the substrates. The TGT mixture enabled both the visualization and restriction of integrin tensions. Green dots mark the presence of microbeads. Yellow arrows mark the successful phagocytosis, indicated by an empty force ring (integrin tension signal in a ring pattern marking a phagocytic site) without a microbead (already internalized). f Phagocytic efficiencies of surface-bound microbeads on TGT surfaces. Phagocytic efficiencies were assessed by quantifying the ratio of force rings without beads (successful phagocytosis) to all force rings in a macrophage. Each point represents an individual cell, with cell number n = 10 for each condition. Each experiment in ( b , e ) was repeated three times independently with similar results. Data in ( c , d , f ) are collected from three independent experiments and presented as mean ± SD. p : two-tailed unpaired t-test with Welch’s correction. *** p < 0.001, **** p < 0.0001. Source data are provided in the Source Data file.

Article Snippet: After cell culture for 40 h, integrin β 2 specific siRNA (Itgb2 Mouse siRNA Oligo Duplex, SR419987, Origene), integrin β 1 specific siRNA (Itgb1 Mouse siRNA Oligo Duplex, SR427527, Origene), scrambled siRNA as negative control (SR30004, Origene), or dye-labeled siRNA (SR30002, Origene) for the transfection verification was added as follows: siRNAs were dissolved in RNase-free buffer (provided by the supplier) at 10 μM as the stocking concentration.

Techniques: Shear, Polymer, Labeling, Concentration Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Force-bearing phagocytic adhesion rings mediate the phagocytosis of surface-bound particles

doi: 10.1038/s41467-025-56404-w

Figure Lengend Snippet: a Integrin tensions in PARs and F-actin structures of macrophages in response to surface-bound particles with the treatments of DMSO (control), Y−27632 (25 µM), blebbistatin (25 µM), CK666 (100 µM) and cytochalasin D (1 µM), respectively. b Integrin tensions in focal adhesions (FAs) and F-actin structures of HeLa cells with the same treatments as those in ( a ). c Signal intensities of integrin tensions in PARs under different inhibition conditions. Each point represents an individual PAR, with PAR number n = 121, 123, 84, and 112, respectively (left to right). d Signal intensities of integrin tensions in FAs under different inhibition conditions. Each point represents an individual FA, with FA number n = 60 for each condition. e Phagocytic efficiencies of casein-passivated microbeads (size: 1.1 µm) on fibronectin-coated (on top of the PLL coating) substrates. The microbeads were linked to the PLL with 0.4% glutaraldehyde. Each point represents an individual cell, with cell number n = 20 for each condition. Each experiment in ( a , b ) was repeated three times independently with similar results. Data in ( c , d ) are collected from three independent experiments and presented as mean ± SD. Data in ( e ) are collected from two independent experiments. p : two-tailed unpaired t -test with Welch’s correction. ns (not significant) p > 0.05, **** p < 0.0001. Source data are provided in the Source Data file.

Article Snippet: After cell culture for 40 h, integrin β 2 specific siRNA (Itgb2 Mouse siRNA Oligo Duplex, SR419987, Origene), integrin β 1 specific siRNA (Itgb1 Mouse siRNA Oligo Duplex, SR427527, Origene), scrambled siRNA as negative control (SR30004, Origene), or dye-labeled siRNA (SR30002, Origene) for the transfection verification was added as follows: siRNAs were dissolved in RNase-free buffer (provided by the supplier) at 10 μM as the stocking concentration.

Techniques: Control, Inhibition, Two Tailed Test

Journal: Nature Communications

Article Title: Force-bearing phagocytic adhesion rings mediate the phagocytosis of surface-bound particles

doi: 10.1038/s41467-025-56404-w

Figure Lengend Snippet: a Laminar actin wave pinpoints surface-bound particles. Refer to Supplementary Movie . The magenta arrows indicate the beads that are detaching. b The dynamics of an F-actin ring and integrin tensions around a detachable bead. Integrin tensions were concurrently generated with the formation of the F-actin ring and remained active during F-actin ring constriction. After the bead was detached, the F-actin ring disassembled, and the generation of integrin tensions ceased. Refer to Supplementary Movie . c A surface-bound microbead detached after the F-actin ring completed constriction. d The inner diameter of F-actin rings during the phagocytosis of surface-bound particles. The time zero is defined as the moment of a microbead detaching from the surface. e Signal intensities of integrin tensions in PARs in response to microbeads linked to substrates by glutaraldehyde at different concentrations. Each point represents an individual PAR, with PAR number n = 50 for each condition. Data are collected from three independent experiments and presented as mean ± SD. Source data are provided in the Source Data file. f The dynamics of an F-actin ring and integrin tensions in a PAR around a non-detachable microbead. The microbead was fused on the surface by heating. The F-actin ring displayed multiple rounds of polymerization, accompanied by the generation of integrin tensions. The slope of the force curve (magenta) represents the real-time activity of integrin tensions as ITS is an irreversible tension sensor. Refer to Supplementary Movie . g A simplistic model of PAR and PAR-assisted phagocytosis. (1) When actin (yellow) polymerization is obstructed by a surface-bound particle, (2) talin (pink), the adapter protein linking the actin network and integrin is tensioned, (3) recruiting vinculin (red) to reinforce the linkage and signal the formation of a PAR. (4) Integrins (green) in the PAR support local actin polymerization that squeezes beneath surface-bound particles and lifts the particles up.

Article Snippet: After cell culture for 40 h, integrin β 2 specific siRNA (Itgb2 Mouse siRNA Oligo Duplex, SR419987, Origene), integrin β 1 specific siRNA (Itgb1 Mouse siRNA Oligo Duplex, SR427527, Origene), scrambled siRNA as negative control (SR30004, Origene), or dye-labeled siRNA (SR30002, Origene) for the transfection verification was added as follows: siRNAs were dissolved in RNase-free buffer (provided by the supplier) at 10 μM as the stocking concentration.

Techniques: Generated, Activity Assay

Journal: Cancer research

Article Title: Targeting Interactions between Siglec-10 and α3β1 Integrin Enhances Macrophage-Mediated Phagocytosis of Pancreatic Cancer

doi: 10.1158/0008-5472.CAN-25-0977

Figure Lengend Snippet: (a) Experimental design for the pull-down of Siglec-10 ligands on PDAC cells. Recombinant Siglec-10 Fc (as well as a no-protein control or a Siglec-5 control) was allowed to bind its physiological ligands on the surface of PDAC cells, followed by an HRP-conjugated anti-Fc secondary antibody. In the presence of H₂O₂, HRP generated short-lived radicals that facilitated the transfer of biotin to proximal Siglec-10 ligands. Biotinylated Siglec-10 ligands were pulled down using streptavidin beads and identified by mass spectrometry. Created in BioRender. Saini, P. (2025) https://BioRender.com/lhnibjl . (b) A total of 4,044 proteins were identified, with enriched binding compared to a control using the anti-Fc antibody only without Siglec-10 protein. Of these, 110 proteins showed enrichment relative to a Siglec-5 control. Six proteins, CD47, CD59, CD73, ITGB6, ITGA3, and ITGB1, were significantly overexpressed in PAAD tissues compared to normal tissues in the TCGA dataset. (c) Response curves showing interactions between Siglec-10 and the six glycoproteins measured by surface plasmon resonance (SPR). Two concentrations (1000 nM, green; 100 nM, red) were tested for all glycoproteins, while ITGA3 was also tested at 300 nM (green) and 30 nM (red). (d) Binding of the SNA lectin (specific for sialic acid) to ITGA3 and ITGB1 recombinant glycoproteins was measured by a lectin array. Sialidase-treated glycoproteins (blue bars) showed significantly reduced binding compared to untreated glycoproteins (red bars). Unpaired t-tests. Means with SEM are shown. (e) SPR response curves comparing the binding of intact (sialylated) ITGA3 and desialylated ITGA3 to immobilized Siglec-10. (f) SPR response curves comparing the binding of intact (sialylated) ITGB1 and desialylated ITGB1 to immobilized Siglec-10.

Article Snippet: The following recombinant proteins were tested: CD47 (His tag, Acrobiosystems, Catalog# CD7-H5227), CD59 (His, Avitag, Acrobiosystems, Catalog# CD9-H82E3), NT5E/CD73 (His, Avitag, Acrobiosystems, Catalog# CD3-H82E3), ITGB6 (C-Myc/DDK, Origene, Catalog# TP317387), ITGA3 (C-Myc/DDK, Origene, Catalog# TP320975), and ITGB1 (C-Myc/DDK, Origene, Catalog# TP303818).

Techniques: Recombinant, Control, Generated, Mass Spectrometry, Binding Assay, SPR Assay

Journal: Cancer research

Article Title: Targeting Interactions between Siglec-10 and α3β1 Integrin Enhances Macrophage-Mediated Phagocytosis of Pancreatic Cancer

doi: 10.1158/0008-5472.CAN-25-0977

Figure Lengend Snippet: ( a–c ) Comparison of CD24 (a), ITGA3 (b), and ITGB1 (c) expression between normal tissues and PAAD tissues in the TCGA dataset. Unpaired t tests. ( d–f ) Survival analyses of pancreatic tumor patients in the TCGA dataset showing the correlation between CD24 (d), ITGA3 (e), and ITGB1 (f) expression and overall survival. ( g–i ) Expression of CD24 (g), ITGA3 (h), and ITGB1 (i) in the PDAC TME across different disease states. Kruskal–Wallis test with Dunn’s multiple comparisons correction. Means with SEM are shown.

Article Snippet: The following recombinant proteins were tested: CD47 (His tag, Acrobiosystems, Catalog# CD7-H5227), CD59 (His, Avitag, Acrobiosystems, Catalog# CD9-H82E3), NT5E/CD73 (His, Avitag, Acrobiosystems, Catalog# CD3-H82E3), ITGB6 (C-Myc/DDK, Origene, Catalog# TP317387), ITGA3 (C-Myc/DDK, Origene, Catalog# TP320975), and ITGB1 (C-Myc/DDK, Origene, Catalog# TP303818).

Techniques: Comparison, Expressing

Journal: Cancer research

Article Title: Targeting Interactions between Siglec-10 and α3β1 Integrin Enhances Macrophage-Mediated Phagocytosis of Pancreatic Cancer

doi: 10.1158/0008-5472.CAN-25-0977

Figure Lengend Snippet: ( a ) Schematic model illustrating Siglec-10-mediated suppression of macrophage phagocytosis. In the left panel, Siglec-10 on macrophages binds to glycan ligands on PDAC cells, including ITGA3, ITGB1, and CD24, triggering inhibitory signaling and suppressing phagocytosis. In the right panel, blocking Siglec-10 with an antibody prevents inhibitory signaling and enhances macrophage phagocytic capacity. ( b ) ELISA screening of recombinant antibodies from the top hybridoma clones for Siglec-10 binding. Binding to immobilized Siglec-10 (blue) and Siglec-5 (gray) proteins is shown. ( c ) Flow cytometric analysis of antibody selectivity, showing binding to CHO-K1 cells expressing Siglec-10 (blue) but not Siglec-5 (gray). ( d ) AUC analysis of in vitro phagocytosis assays screening various Siglec-10 antibody clones, along with commercially available anti-CD24 and anti-Siglec-10 antibodies, for their ability to enhance macrophage-mediated phagocytosis of AsPC-1 PDAC cells. Means with SEM are shown. ( e ) AUC analysis of the in vitro phagocytosis assay using the top-performing Siglec-10 antibody clone with macrophages differentiated from monocytes of four healthy donors. Statistical significance was determined using Friedman’s ANOVA test. Means with SEM are shown. ( f ) Time-course analysis of the in vitro phagocytosis assay comparing the top Siglec-10 blocking antibody clone (68A11A1, blue) with the isotype control (gray). Data represent n = 4 independent experiments. ( g ) ELISA-based binding analysis of the recombinant 68A11A1 antibody to immobilized recombinant Siglec-10 and Siglec-5 proteins across different dilutions. ( h ) Evaluation of the recombinant Siglec-10 antibody (clone 68A11A1) and anti-CD24 antibody in enhancing macrophage-mediated phagocytosis of multiple PDAC cell lines (AsPC-1, MIA PaCa-2, and PANC-1). Phagocytosis was normalized to the isotype control for each cell line and conducted using macrophages derived from monocytes of 5–8 healthy donors. Each symbol represents an individual donor; statistical significance was assessed using ratio paired t-tests compared to isotype control. Means with SEM are shown. ( i ) Triple co-culture assay involving cancer-associated fibroblasts (CAFs), PANC-1 PDAC cells, and monocyte-derived macrophages, showing phagocytosis kinetics, AUC quantification, and representative images. Statistical significance assessed using paired t-tests. ( j-k ) Flow cytometry analysis of CellTrace Violet (CTV)-labeled human CD8 + T cells co-cultured with human monocytes ± anti-Siglec-10 antibody in the presence of anti-CD3/CD28 beads for 5 days. ( j ) T cell proliferation; ( k ) granzyme B expression. ANOVA with post hoc comparisons.

Article Snippet: The following recombinant proteins were tested: CD47 (His tag, Acrobiosystems, Catalog# CD7-H5227), CD59 (His, Avitag, Acrobiosystems, Catalog# CD9-H82E3), NT5E/CD73 (His, Avitag, Acrobiosystems, Catalog# CD3-H82E3), ITGB6 (C-Myc/DDK, Origene, Catalog# TP317387), ITGA3 (C-Myc/DDK, Origene, Catalog# TP320975), and ITGB1 (C-Myc/DDK, Origene, Catalog# TP303818).

Techniques: Blocking Assay, Inhibition, Activation Assay, In Vitro, Glycoproteomics, Enzyme-linked Immunosorbent Assay, Recombinant, Clone Assay, Binding Assay, Expressing, Phagocytosis Assay, Control, Derivative Assay, Co-culture Assay, Flow Cytometry, Labeling, Cell Culture

Journal: bioRxiv

Article Title: Thrombospondin-1 Promotes Circuit-Specific Synapse Formation via β1-Integrin

doi: 10.1101/866590

Figure Lengend Snippet: (A) Schematic representation of the experimental design. Purified RGCs isolated from P7 rat retinas are treated with recombinant human TSP1 or TSP2 (150 ng/mL) in the presence of the function blocking antibody against Integrin β1 or control IgG. RGCs were labeled with a ooDSGC marker, CART (blue), and synaptic markers, Bassoon (pre-, green) and PSD95 (post-, red). (B) Representative images of the synapses formed on the RGCs treated with either full length TSP1 or TSP2 in the presence of integrin β1 function blocking antibody or control IgG. Synapses, co-localized synaptic puncta (yellow) of pre- (Bassoon, green) and post-synaptic (PSD95, red) markers, are marked with white arrows. CART (blue) visualizes ooDSGCs. Full images of RGCs are available in the supplementary figure. Quantification of fold increase in the number of co-localized synaptic puncta from the RGCs treated with (C) function blocking antibody or control IgG demonstrates blocking Integrin β1 specifically inhibits TSP1-mediated synaptogenic activity but not TSP2. Fold increase is calculated by normalizing the number of synapses per cell with the number of synapses per cell in CART+ RGCs in GM Only-Control condition (n=30 cells/condition, One-way ANOVA, *** p<0.0001). (D) Schematic representation of the experimental design. Purified RGCs isolated from P7 rat retinas are transfected with the DNA plasmids transcribing ITGB1 and BFP, then treated with synaptogenic fragments, SD1 or SD2 (150 ng/mL). Then the synapses formed onto the RGCs expressing BFP but negative for CART were imaged for synapse quantification. (E) Representative images of the synapses formed on the RGCs expressing ITGB1 and BFP. The RGCs are treated with either SD1 or SD2, then synapses (white arrows) are labeled as co-localized puncta (yellow) of pre- (Bassoon, green) and post-synaptic (PSD95, red) markers. (F) Quantification of fold increase in the number of co-localized synaptic puncta demonstrates that expression of ITGB1 sufficient for TSP1-mediated synapse formation in CART-negative RGCs. (n=20-30 cells/condition, One-way ANOVA, *** p<0.0001).

Article Snippet: Briefly, 125 ng of each DNA construct expressing BFP and ITGB1 (Addgene 51920) and 1.25μl of Lipofectamine LTX was used in each well (in a 24 well plate).

Techniques: Purification, Isolation, Recombinant, Blocking Assay, Labeling, Marker, Activity Assay, Transfection, Expressing

Journal: bioRxiv

Article Title: Thrombospondin-1 Promotes Circuit-Specific Synapse Formation via β1-Integrin

doi: 10.1101/866590

Figure Lengend Snippet: (A) Schematic representation of the transgenic mice breeding strategy. Retinas from the littermate wild-type (WT, Itgb1 +/+) and Integrin β1 cKO (Itgb1 f/f) were collected at P30. (B) Tamoxifen (20 mg/mL) was subcutaneously delivered twice at P9 and P10 (0.6 mg each day) then retinas were collected at P30. (C) Schematic of IPL and cell types that express Cadherin 6 (blue). (D) Representative images of the excitatory synapses in WT and Itgb1 cKO retinas at P30. Cadherin6-positive DS circuit IPL sublayers are visualized by tdTomato (blue, left panels) and excitatory synapses are labeled by VGluT1 (pre-, green) and PSD95 (post-, red). Only the excitatory synapses within tdTomato positive dendrites are shown by masking with tdTomato staining (right panels). The inlets (white boxes) are shown in higher magnification and the co-localized synaptic puncta (merge, yellow) are marked with white arrows. (E) There is no significant change in the number of excitatory synapses within the IPL between control (+/+) and cKO (f/f). (F) Quantification of excitatory synapses within tdTomato-positive dendrites demonstrates significant reduction in synapse number in Itgb1 cKO retinas (n=3 animals per genotype, t-test, *** p<0.0001). (G) Representative images of the inhibitory synapses in WT and Itgb1 cKO retinas at P30. The inhibitory synapses are labeled by Bassoon (pre-, green) and Gephyrin (post-, red)). Only the synapses within tdTomato positive dendrites are shown by masking with tdTomato staining. The inlets (white boxes) are shown in higher magnification and the co-localized synaptic puncta (merge, yellow) are marked with white arrows. (H) There is a significant reduction in the number of inhibitory synapses within the IPL between control (+/+) and cKO (f/f) (n=3 animals per genotype, t-test, *** p<0.0001). (I) Quantification of inhibitory synapses within tdTomato-positive dendrites demonstrates that there is no change in inhibitory synapse number between Itgb1 WT and cKO retinas (n=3 animals per genotype, t-test, n.s., not significant)

Article Snippet: Briefly, 125 ng of each DNA construct expressing BFP and ITGB1 (Addgene 51920) and 1.25μl of Lipofectamine LTX was used in each well (in a 24 well plate).

Techniques: Transgenic Assay, Labeling, Staining