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Santa Cruz Biotechnology sc 35674 itga3 sirna human santa cruz biotechnology
Sc 35674 Itga3 Sirna Human Santa Cruz Biotechnology, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 62 article reviews

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( A ) Flow cytometry analysis of surface expression of indicated integrins 2 days after reseeding of NGN3-GFP-derived pancreatic progenitors on FN or LN411. Data were analyzed by two-tailed paired t-tests and are shown as boxplots with box boundaries extending to the minimum and maximum with a line at the mean ( n = 6 for ITGα6, n = 3 for ITGα3, ITGα5, and ITGαV, biological replicates). ( B ) qPCR analysis of cell-ECM adhesion genes ( ITGA6 , ITGA3 , ITGA5 and ITGAV ) and the endocrine marker gene NEUROG3 at indicated time points after reseeding on LN411 or FN. The data were analyzed by paired t -tests with Holm–Šídák adjustment for multiple comparisons and are shown as mean expression ± SEM ( n = 3, biological replicates). ( C ) qPCR analysis of ITGA3 and NEUROG3 2 days after transfection of pancreatic progenitors with siRNA targeting ITGA3 . The data were analyzed using two-tailed paired t -tests and are shown as mean expression ± SEM ( n = 7, biological replicates). ( D ) qPCR analysis of ITGA6 and NEUROG3 2 days after transfection of pancreatic progenitors with siRNA targeting ITGA6 . The data were analyzed using two-tailed paired t -tests and are shown as mean fold change in expression ± SEM ( n = 5, biological replicates). .

Journal: EMBO Reports

Article Title: Extracellular matrix-driven metabolic control of pancreatic endocrine lineage allocation

doi: 10.1038/s44319-025-00610-6

Figure Lengend Snippet: ( A ) Flow cytometry analysis of surface expression of indicated integrins 2 days after reseeding of NGN3-GFP-derived pancreatic progenitors on FN or LN411. Data were analyzed by two-tailed paired t-tests and are shown as boxplots with box boundaries extending to the minimum and maximum with a line at the mean ( n = 6 for ITGα6, n = 3 for ITGα3, ITGα5, and ITGαV, biological replicates). ( B ) qPCR analysis of cell-ECM adhesion genes ( ITGA6 , ITGA3 , ITGA5 and ITGAV ) and the endocrine marker gene NEUROG3 at indicated time points after reseeding on LN411 or FN. The data were analyzed by paired t -tests with Holm–Šídák adjustment for multiple comparisons and are shown as mean expression ± SEM ( n = 3, biological replicates). ( C ) qPCR analysis of ITGA3 and NEUROG3 2 days after transfection of pancreatic progenitors with siRNA targeting ITGA3 . The data were analyzed using two-tailed paired t -tests and are shown as mean expression ± SEM ( n = 7, biological replicates). ( D ) qPCR analysis of ITGA6 and NEUROG3 2 days after transfection of pancreatic progenitors with siRNA targeting ITGA6 . The data were analyzed using two-tailed paired t -tests and are shown as mean fold change in expression ± SEM ( n = 5, biological replicates). .

Article Snippet: ITGA3 TaqMan probe , Thermo Fisher , Hs01076879_m1.

Techniques: Flow Cytometry, Expressing, Derivative Assay, Two Tailed Test, Marker, Transfection

( A ) Schematic of the explant experimental setup. Due to embryonic lethality of Itga3 −/− ; Itga6 −/− mouse embryos, pancreata were explanted at E11.5. Explants were then cultured for 3 days, after which hypoplasia was apparent in double knockout explants. ( B ) Images showing part of the dissected gastrointestinal tract from E14.5 WT and Itga3 −/− ; Itga6 −/− mouse embryos. The double knockout exhibits hypoplasia of the pancreas (outlined). ( C ) Representative whole-mount staining of Itga3 +/− / Itga6 +/− , and Itga3 −/− / Itga6 −/− pancreata explanted at E11.5 and cultured for 3 days ex vivo. Immunostaining with DAPI (blue), insulin (red), glucagon (green), and E-cadherin (E-cad, White). Scale bar = 200 μm. ( D ) Quantification of the staining for Itga3 +/− / Itga6 −/− and Itga3 −/− / Itga6 +/− pancreatic explants. The data were analyzed by Tukey’s test and are shown as mean expression ± SEM ( n = 14 for wild type, 4 for double heterozygotes, 7 for Itga6 single knockout, and 5 for Itga3 single knockout and Itga3 / Itga6 double knockout, biological replicates). .

Journal: EMBO Reports

Article Title: Extracellular matrix-driven metabolic control of pancreatic endocrine lineage allocation

doi: 10.1038/s44319-025-00610-6

Figure Lengend Snippet: ( A ) Schematic of the explant experimental setup. Due to embryonic lethality of Itga3 −/− ; Itga6 −/− mouse embryos, pancreata were explanted at E11.5. Explants were then cultured for 3 days, after which hypoplasia was apparent in double knockout explants. ( B ) Images showing part of the dissected gastrointestinal tract from E14.5 WT and Itga3 −/− ; Itga6 −/− mouse embryos. The double knockout exhibits hypoplasia of the pancreas (outlined). ( C ) Representative whole-mount staining of Itga3 +/− / Itga6 +/− , and Itga3 −/− / Itga6 −/− pancreata explanted at E11.5 and cultured for 3 days ex vivo. Immunostaining with DAPI (blue), insulin (red), glucagon (green), and E-cadherin (E-cad, White). Scale bar = 200 μm. ( D ) Quantification of the staining for Itga3 +/− / Itga6 −/− and Itga3 −/− / Itga6 +/− pancreatic explants. The data were analyzed by Tukey’s test and are shown as mean expression ± SEM ( n = 14 for wild type, 4 for double heterozygotes, 7 for Itga6 single knockout, and 5 for Itga3 single knockout and Itga3 / Itga6 double knockout, biological replicates). .

Article Snippet: ITGA3 TaqMan probe , Thermo Fisher , Hs01076879_m1.

Techniques: Cell Culture, Double Knockout, Staining, Ex Vivo, Immunostaining, Expressing, Knock-Out

OTUD1 directly interacts with β-catenin. (A) Schematic illustration of the quantitative proteomic screening used to identify OTUD1-binding proteins. Eleven potential binding partners were found, including CD44, BASP1, VARS1, LMAN1, GNG12, ITGA3, IARS1, DHX15, TMED10, JUP, and β-catenin. (B) HUVEC lysates were immunoprecipitated using OTUD1 or IgG antibodies, followed by immunoblotting with OTUD1 and β-catenin antibodies (n = 3). (C) Hek 293T cells were transfected for 24 h with Flag-vector or Flag-OTUD1 plasmids. Cell lysates underwent immunoprecipitation with anti-Flag or IgG antibodies, followed by immunoblotting using anti-Flag and β-catenin antibodies (n = 3). (D) BLI assay assessing β-catenin binding to in vitro -biotinylated OTUD1. Sensorgrams show the binding at different β-catenin concentrations (color lines). (E) Immunofluorescence staining for OTUD1 (red), β-catenin (green), and nuclei (DAPI, blue) in HUVECs. Scale bar = 50 μm. (F) Quantification of fluorescence intensity showing OTUD1 and β-catenin co-localization and (G) nuclear translocation of β-catenin (n = 7). White arrows indicate OTUD1 and β-catenin co-localization. (H) Diagram of the β-catenin domain deletion construct used in (I, J). (I, J) Full-length β-catenin or truncated forms were co-expressed with OTUD1 in Hek 293T cells. Immunoprecipitation was performed with anti-His antibody, followed by an immunoblot assay with specific antibodies (n = 3). Protein levels were normalized to GAPDH. Data are presented as mean ± SEM. Statistical analysis used a two-tailed unpaired Student's t -test (F, G). Abbreviations: IP, immunoprecipitation; IgG, immunoglobulin G; WCL, whole cell lysates; BLI, bio-layer interferometry; NTD, N-terminal domain; CTD, C-terminal domain; ARM, armadillo repeats 1–12.

Journal: Journal of Advanced Research

Article Title: OTUD1 delays wound healing by regulating endothelial function and angiogenesis in diabetic mice

doi: 10.1016/j.jare.2025.04.038

Figure Lengend Snippet: OTUD1 directly interacts with β-catenin. (A) Schematic illustration of the quantitative proteomic screening used to identify OTUD1-binding proteins. Eleven potential binding partners were found, including CD44, BASP1, VARS1, LMAN1, GNG12, ITGA3, IARS1, DHX15, TMED10, JUP, and β-catenin. (B) HUVEC lysates were immunoprecipitated using OTUD1 or IgG antibodies, followed by immunoblotting with OTUD1 and β-catenin antibodies (n = 3). (C) Hek 293T cells were transfected for 24 h with Flag-vector or Flag-OTUD1 plasmids. Cell lysates underwent immunoprecipitation with anti-Flag or IgG antibodies, followed by immunoblotting using anti-Flag and β-catenin antibodies (n = 3). (D) BLI assay assessing β-catenin binding to in vitro -biotinylated OTUD1. Sensorgrams show the binding at different β-catenin concentrations (color lines). (E) Immunofluorescence staining for OTUD1 (red), β-catenin (green), and nuclei (DAPI, blue) in HUVECs. Scale bar = 50 μm. (F) Quantification of fluorescence intensity showing OTUD1 and β-catenin co-localization and (G) nuclear translocation of β-catenin (n = 7). White arrows indicate OTUD1 and β-catenin co-localization. (H) Diagram of the β-catenin domain deletion construct used in (I, J). (I, J) Full-length β-catenin or truncated forms were co-expressed with OTUD1 in Hek 293T cells. Immunoprecipitation was performed with anti-His antibody, followed by an immunoblot assay with specific antibodies (n = 3). Protein levels were normalized to GAPDH. Data are presented as mean ± SEM. Statistical analysis used a two-tailed unpaired Student's t -test (F, G). Abbreviations: IP, immunoprecipitation; IgG, immunoglobulin G; WCL, whole cell lysates; BLI, bio-layer interferometry; NTD, N-terminal domain; CTD, C-terminal domain; ARM, armadillo repeats 1–12.

Article Snippet: Phospho-eNOS (28939-1-AP), eNOS (27120-1-AP), β-catenin (51067–2-AP), JUP (11146-1-AP), CD44 (30854-1-AP), ITGA3 (21992-1-AP), LaminB (66095-1-Ig), and GAPDH (60004-1-Ig) were obtained from Proteintech.

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, In Vitro, Immunofluorescence, Staining, Fluorescence, Translocation Assay, Construct, Two Tailed Test

( A ) ELISA assessing the binding specificity of 2E7 scFv-Fc binding to the ITGA3B1 heterodimer and its individual subunits ( ITGA3 and ITGB1 ), as well as to the structurally related integrin complex ITGA6B4 and its subunits ( ITGA6 and ITGB4 ). HuIgG and BSA were included as negative controls. Data represent the mean ± SD from three technical replicates. A 450 , absorbance at 450 nm. ( B ) Dose-dependent binding of 2E7 scFv-Fc to immobilized ITGA3 , ITGB1 , and ITGA3B1 , as determined by ELISA. ( C ) Flow cytometry analysis of HEK293 cells transiently transfected with ITGA3 , ITGB1 , or both. ( D ) Flow cytometry of MDA-MB-231 cells following siRNA knockdown of ITGA3 , ITGB1 , or both ITGA3 and ITGB1 . ( E ) SPR sensorgrams showing 2E7 scFv-Fc binding to recombinant ITGA3B1 in the presence of either divalent cations or EDTA. Data shown are representative of three independent experiments.

Journal: Science Advances

Article Title: Phenotypic discovery and therapeutic evaluation of an ITGA3B1 -targeting antibody-drug conjugate for bladder cancer

doi: 10.1126/sciadv.ady0041

Figure Lengend Snippet: ( A ) ELISA assessing the binding specificity of 2E7 scFv-Fc binding to the ITGA3B1 heterodimer and its individual subunits ( ITGA3 and ITGB1 ), as well as to the structurally related integrin complex ITGA6B4 and its subunits ( ITGA6 and ITGB4 ). HuIgG and BSA were included as negative controls. Data represent the mean ± SD from three technical replicates. A 450 , absorbance at 450 nm. ( B ) Dose-dependent binding of 2E7 scFv-Fc to immobilized ITGA3 , ITGB1 , and ITGA3B1 , as determined by ELISA. ( C ) Flow cytometry analysis of HEK293 cells transiently transfected with ITGA3 , ITGB1 , or both. ( D ) Flow cytometry of MDA-MB-231 cells following siRNA knockdown of ITGA3 , ITGB1 , or both ITGA3 and ITGB1 . ( E ) SPR sensorgrams showing 2E7 scFv-Fc binding to recombinant ITGA3B1 in the presence of either divalent cations or EDTA. Data shown are representative of three independent experiments.

Article Snippet: Recombinant human integrin subunits ( ITGA3 , ITGA6 , ITGB1 , and ITGB4 ; OriGene Technologies, Rockville, MD) and heterodimers ( ITGA3B1 and ITGA6B4 ; Acrobiosystems, Newark, DE) were each coated onto 96-well Maxisorp plates (Thermo Fisher Scientific) at a concentration of 2 μg/ml in PBS and incubated overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Flow Cytometry, Transfection, Knockdown, Recombinant

Journal: Science Advances

Article Title: Phenotypic discovery and therapeutic evaluation of an ITGA3B1 -targeting antibody-drug conjugate for bladder cancer

doi: 10.1126/sciadv.ady0041

Article Snippet: Target-specific siRNAs against ITGA3 (sc-35684) and ITGB1 (sc-35674), along with a nontargeting control siRNA (sc-37007), were obtained from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Flow Cytometry, Transfection, Knockdown, Recombinant

( A ) ITGA3 mRNA expression in normal bladder tissues ( n = 18) and primary bladder tumors ( n = 408) based on TCGA data. ( B ) ITGA3 expression across consensus molecular subtypes of bladder cancer from five aggregated clinical cohorts. ( C ) Representative multiplex immunofluorescence images of TMA cores stained for ITGA3 (green), ITGB1 (red), and nuclei [DAPI (4′,6-diamidino-2-phenylindole), blue] of primary bladder carcinomas and normal tissues. Scale bars, 100 μm. ( D to F ) Quantification of ITGA3 expression in tumor versus normal tissues: fluorescence intensity (D), percentage of ITGA3 -positive cells (E), and H -scores (F). **** P < 0.0001.

Journal: Science Advances

Article Title: Phenotypic discovery and therapeutic evaluation of an ITGA3B1 -targeting antibody-drug conjugate for bladder cancer

doi: 10.1126/sciadv.ady0041

Figure Lengend Snippet: ( A ) ITGA3 mRNA expression in normal bladder tissues ( n = 18) and primary bladder tumors ( n = 408) based on TCGA data. ( B ) ITGA3 expression across consensus molecular subtypes of bladder cancer from five aggregated clinical cohorts. ( C ) Representative multiplex immunofluorescence images of TMA cores stained for ITGA3 (green), ITGB1 (red), and nuclei [DAPI (4′,6-diamidino-2-phenylindole), blue] of primary bladder carcinomas and normal tissues. Scale bars, 100 μm. ( D to F ) Quantification of ITGA3 expression in tumor versus normal tissues: fluorescence intensity (D), percentage of ITGA3 -positive cells (E), and H -scores (F). **** P < 0.0001.

Techniques: Expressing, Multiplex Assay, Immunofluorescence, Staining, Fluorescence

(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.

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

( 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.

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.

Techniques: Comparison, Expressing

( a ) ITGA3 knock-down in PANC-1 PDAC cells was achieved using CRISPR-Cas9. Flow cytometry confirmed the reduction of ITGA3 expression in knockout cells (ITGA3-KO) compared to non-targeting gRNA-treated cells and untreated controls. Data from three independent replicates show the percentage of ITGA3-expressing cells and the mean fluorescence intensity (MFI). Means and SEM are shown. Unpaired t-tests. ( b ) Experimental schematic illustrating co-culture of red-labeled PDAC cells (ITGA3-KO or non-targeting gRNA-treated) with monocyte-derived macrophages. Phagocytosis was monitored over time using live-cell imaging on the Incucyte Live-Cell Analysis System. ( c ) (left) Phagocytosis over time and area under the curve (AUC). Data show macrophage-mediated phagocytosis of PDAC cells, normalized to the 0-hour time point. Paired t-tests. N = 3 donors, with experiments performed in triplicate for each donor. (right) Representative images showing increased red fluorescence as an indicator of enhanced phagocytosis. ( d ) Schematic of ITGA3 low PDAC cell enrichment. ITGA3 high PDAC cells were separated using anti-ITGA3 antibody-coated columns, and the flow-through, containing ITGA3 low cells, was collected. ( e ) Flow cytometry analysis of ITGA3 expression in MIA PaCa-2 cells after enrichment for ITGA3 low cells and comparison with non-enriched controls (Ctrl cells). ( f ) Experimental schematic illustrating the phagocytic assay setup, where monocyte-derived macrophages were co-cultured with either ITGA3 high (control) or ITGA3 low PDAC cells. ( g-h ) Phagocytosis analysis of ITGA3 high (control) versus ITGA3 low MIA PaCa-2 (g) or PANC1 (h) cells by macrophages derived from different donors. N = 5–6, with experiments performed in triplicate for each donor. Top: Representative images showing increased red fluorescence as an indicator of enhanced phagocytosis. Bottom left: Live imaging data from individual donors (each symbol represents one donor’s data done in triplicate per donor). Bottom right: AUC data from multiple donors, showing significantly higher phagocytic activity with ITGA3 low cells. Paired t-tests. Some images in panels B, D, and F were created in BioRender. Saini, P. (2025) https://BioRender.com/lbo67eu .

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 ) ITGA3 knock-down in PANC-1 PDAC cells was achieved using CRISPR-Cas9. Flow cytometry confirmed the reduction of ITGA3 expression in knockout cells (ITGA3-KO) compared to non-targeting gRNA-treated cells and untreated controls. Data from three independent replicates show the percentage of ITGA3-expressing cells and the mean fluorescence intensity (MFI). Means and SEM are shown. Unpaired t-tests. ( b ) Experimental schematic illustrating co-culture of red-labeled PDAC cells (ITGA3-KO or non-targeting gRNA-treated) with monocyte-derived macrophages. Phagocytosis was monitored over time using live-cell imaging on the Incucyte Live-Cell Analysis System. ( c ) (left) Phagocytosis over time and area under the curve (AUC). Data show macrophage-mediated phagocytosis of PDAC cells, normalized to the 0-hour time point. Paired t-tests. N = 3 donors, with experiments performed in triplicate for each donor. (right) Representative images showing increased red fluorescence as an indicator of enhanced phagocytosis. ( d ) Schematic of ITGA3 low PDAC cell enrichment. ITGA3 high PDAC cells were separated using anti-ITGA3 antibody-coated columns, and the flow-through, containing ITGA3 low cells, was collected. ( e ) Flow cytometry analysis of ITGA3 expression in MIA PaCa-2 cells after enrichment for ITGA3 low cells and comparison with non-enriched controls (Ctrl cells). ( f ) Experimental schematic illustrating the phagocytic assay setup, where monocyte-derived macrophages were co-cultured with either ITGA3 high (control) or ITGA3 low PDAC cells. ( g-h ) Phagocytosis analysis of ITGA3 high (control) versus ITGA3 low MIA PaCa-2 (g) or PANC1 (h) cells by macrophages derived from different donors. N = 5–6, with experiments performed in triplicate for each donor. Top: Representative images showing increased red fluorescence as an indicator of enhanced phagocytosis. Bottom left: Live imaging data from individual donors (each symbol represents one donor’s data done in triplicate per donor). Bottom right: AUC data from multiple donors, showing significantly higher phagocytic activity with ITGA3 low cells. Paired t-tests. Some images in panels B, D, and F were created in BioRender. Saini, P. (2025) https://BioRender.com/lbo67eu .

Techniques: Expressing, Knockdown, CRISPR, Flow Cytometry, Knock-Out, Fluorescence, Co-Culture Assay, Labeling, Derivative Assay, Live Cell Imaging, Cell Analysis, Comparison, Cell Culture, Control, Imaging, Activity Assay

( 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.

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.

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

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