Journal: Research

Article Title: Trabecular-Like Scaffold Dictates Osteogenesis via Fluid Shear Stress-Induced Metabolic Reprogramming through the CAV1–HIF-1α Axis

doi: 10.34133/research.1307

Figure Lengend Snippet: Integrated transcriptomic and proteomic analyses and in vitro validation of mechano-metabolic remodeling in BMSCs under HFSS. (A) Schematic workflow of the integrated analysis. DEGs and DEPs between the HFSS and Ctrl groups were identified from RNA-seq and proteomic datasets, respectively, and concordantly regulated targets showing the same direction of change at both transcriptomic and proteomic levels were selected for downstream analyses. Created with BioRender.com . (B) Venn diagram illustrating the overlap between DEGs and DEPs. (C) Nine-quadrant plot comparing log₂ fold changes at the RNA and protein levels, highlighting concordant and discordant regulation patterns. (D) GO enrichment analysis of concordantly regulated genes and proteins. (E) KEGG pathway enrichment analysis of concordant targets. (F) GSEA plots for glycolysis, HIF-1 signaling, and PI3K–AKT signaling, showing positive enrichment of all 3 pathways in the HFSS group. (G) Heatmap of selected genes and proteins involved in glycolysis and mechanosignaling. (H) Phalloidin staining of the actin cytoskeleton in BMSCs cultured on Voronoi scaffolds under dynamic FSS. (I) Seahorse extracellular flux analysis of BMSCs cultured on V50, V60, and V70 scaffolds. (J) Representative ECAR curves and quantitative analysis of glycolysis rate, glycolytic reserve, and glycolytic capacity. (K) Western blot analysis of GLUT1, HIF1A, and CAV1, and (L) PI3K, p-PI3K, AKT, and p-AKT in BMSCs cultured on scaffolds, with corresponding quantitative analyses of Western blot protein bands. (M) Quantitative analysis of multiple glycolytic flux fractions in BMSCs seeded onto scaffolds. (N) Representative immunofluorescence images of CAV1 expression in BMSCs cultured on V50, V60, and V70 scaffolds. Scale bar, 100 μm. (O) qPCR analysis of Hif1a , Ldha , Slc2a1 , and Cav1 gene expression. Data are presented as mean ± SD ( n = 3 per group). Statistical significance was assessed using unpaired 2-tailed Welch’s t test and one-way ANOVA with Tukey’s HSD post hoc test.

Article Snippet: Samples were then incubated overnight at 4 °C with primary antibodies against CAV1 or ITGA3 (ABclonal, China), followed by incubation with the corresponding fluorescent secondary antibodies.

Techniques: In Vitro, Biomarker Discovery, RNA Sequencing, Staining, Cell Culture, Western Blot, Immunofluorescence, Expressing, Gene Expression

Journal: Research

Article Title: Trabecular-Like Scaffold Dictates Osteogenesis via Fluid Shear Stress-Induced Metabolic Reprogramming through the CAV1–HIF-1α Axis

doi: 10.34133/research.1307

Figure Lengend Snippet: Cav1 knockdown attenuates HFSS-induced glycolytic activation and osteogenic differentiation in mouse BMSCs. (A) Schematic illustration of the proposed CAV1-centered mechano-metabolic axis. Caveolae-associated CAV1 at the plasma membrane is hypothesized to couple mechanical cues to PI3K–AKT and HIF-1 signaling pathways, thereby promoting glycolytic metabolism. Created with BioRender.com . (B) Experimental design for Cav1 knockdown under 3D dynamic culture conditions. BMSCs transduced with control shRNA or Cav1 shRNA were seeded onto Voronoi scaffolds and subjected to HFSS in a 3D culture system. (C) Western blot analysis of LDHA, HIF1A, CAV1, and GLUT1 in Cav1 -knockdown BMSCs under HFSS, with corresponding quantitative analyses of Western blot protein bands. (D) Representative ECAR curves and (E) quantitative analysis of glycolysis rate, glycolytic reserve, and glycolytic capacity. (F) qPCR analysis of Ldha , Hif1a , Pkm2 , and Slc2a1 messenger RNA expression in the same 3 groups. (G) Western blot analysis of osteogenic proteins COL1A1, OPN, ALP, and RUNX2 in the 3 groups. (H) qPCR analysis of Col1a1 , Ocn , and Runx2 mRNA expression. (I) Alizarin Red S staining and quantitative analysis of mineralized nodules after 21 d of 3D dynamic culture on Voronoi scaffolds. Data are presented as mean ± SD ( n = 3 per group). Statistical significance was assessed using one-way ANOVA with Tukey’s HSD post hoc test.

Article Snippet: Samples were then incubated overnight at 4 °C with primary antibodies against CAV1 or ITGA3 (ABclonal, China), followed by incubation with the corresponding fluorescent secondary antibodies.

Techniques: Knockdown, Activation Assay, Clinical Proteomics, Membrane, Protein-Protein interactions, Transduction, Control, shRNA, Western Blot, RNA Expression, Expressing, Staining

Journal: Research

Article Title: Trabecular-Like Scaffold Dictates Osteogenesis via Fluid Shear Stress-Induced Metabolic Reprogramming through the CAV1–HIF-1α Axis

doi: 10.34133/research.1307

Figure Lengend Snippet: Pharmacological perturbation of the CAV1-centered mechano-metabolic axis required for HFSS-induced osteogenesis. (A) Schematic diagram illustrating the mechanistic intervention strategy. Created with BioRender.com . (B) Western blot analysis of PI3K–AKT signaling in Cav1 -knockdown BMSCs, with corresponding quantitative analyses of Western blot protein bands. (C) Western blot analysis of PI3K, p-PI3K, AKT, and p-AKT to assess the effects of LY294002 in inhibiting the PI3K signaling pathway, with quantitative analyses of Western blot protein bands. (D) Western blot analysis of COL1A1, HIF1A, and GLUT1 expression, with corresponding quantitative analyses. (E) qPCR analysis of Col1a1 , Runx2 , Opn , Pkm2 , Ldha , and Hif1a mRNA expression in LY294002-treated groups. (F) Representative ECAR curves and (G) quantitative analysis of glycolysis rate, glycolytic reserve, and glycolytic capacity in LY294002-treated groups. (H) Western blot analysis of LDHA, COL1A1, HIF1A, and GLUT1 to assess the effects of YC-1 in inhibiting the HIF-1 signaling pathway, and (I) quantitative analyses of Western blot protein bands. (J) qPCR analysis of Col1a1 , Runx2 , Opn , Pkm2 , Ldha , and Hif1a mRNA expression in YC-1-treated groups. (K) Representative ECAR curves and (L) quantitative analysis of glycolysis rate in YC-1-treated groups. Data are presented as mean ± SD ( n = 3 per group). Statistical significance was assessed using one-way ANOVA with Tukey’s HSD post hoc test.

Article Snippet: Samples were then incubated overnight at 4 °C with primary antibodies against CAV1 or ITGA3 (ABclonal, China), followed by incubation with the corresponding fluorescent secondary antibodies.

Techniques: Western Blot, Knockdown, Expressing

Journal: Research

Article Title: Trabecular-Like Scaffold Dictates Osteogenesis via Fluid Shear Stress-Induced Metabolic Reprogramming through the CAV1–HIF-1α Axis

doi: 10.34133/research.1307

Figure Lengend Snippet: Schematic illustration of the CAV1-centered mechano-metabolic axis by which Voronoi scaffold-regulated FSS drives glycolytic reprogramming and osteogenic differentiation of BMSCs. Created with BioRender.com .

Article Snippet: Samples were then incubated overnight at 4 °C with primary antibodies against CAV1 or ITGA3 (ABclonal, China), followed by incubation with the corresponding fluorescent secondary antibodies.

Techniques:

Journal: Cell & Bioscience

Article Title: CALR-dependent endoplasmic reticulum N-glycoprotein maturation promotes Japanese encephalitis virus entry and replication

doi: 10.1186/s13578-026-01607-x

Figure Lengend Snippet: CALR deficiency remodels the cell-surface adhesion landscape and reduces cellular permissiveness to JEV infection. A Co-immunoprecipitation analysis of interactions between CALR and full-length or domain-truncated JEV E constructs. HEK293T cells were co-transfected with HA-tagged CALR and Flag-tagged full-length or truncated E variants (E-Flag, DI + DII-Flag, DIII + TMD-Flag, E-ΔDII-Flag, and E-ΔDIII-Flag). B Antibody-blocking assay using anti-CALR antibody to evaluate effects on JEV infection, assessed by NS3 protein abundance. Scale bar, 100 μm. C Quantitative proteomic analysis of differentially expressed proteins in CALR-KO versus WT PK-15 cells. DOWN, downregulated; NS, not significant change; UP, upregulated proteins. D Immunoblot validation of representative adhesion- and glycan-associated proteins identified by proteomic analysis. E Generation and validation of ITGA3-KO and ITGB1-KO PK-15 cell lines using CRISPR/Cas9. F Indirect immunofluorescence analysis of NS3 protein expression in WT, ITGA3-KO, and ITGB1-KO PK-15 cells at 24 hpi following JEV infection at an MOI of 0.1. Scale bar, 100 μm. G Quantification of NS3 protein fluorescence intensity in WT, ITGA3-KO, and ITGB1-KO PK-15 cells at 24 hpi following JEV infection at an MOI of 0.1. H Absolute RT-qPCR quantification of extracellular viral genome copy numbers in culture supernatants from WT, ITGA3-KO, and ITGB1-KO PK-15 cells at 24 hpi following JEV infection at an MOI of 0.1. I Confocal microscopy showing co-localization of ITGA3 or ITGB1 with JEV E protein in PK-15 cells. Scale bar, 20 μm. J Co-immunoprecipitation analysis of interactions between ITGA3 or ITGB1 and JEV E protein. Data are presented as mean ± SD from at least three independent experiments. Statistical significance was determined using a two-sided Student’s t-test. KO, knockout; WT, wild-type. ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: Membranes were blocked in 5% milk and incubated with primary antibodies against Flag (Abbkine, ABT2011), HA (Abbkine, ABT2041), JEV-NS3 (GeneTex, GTX125868), JEV-E (GeneTex, GTX638878), ITGA2 (Abcam, ab133557), ITGA3 (Abclonal, A17502), ITGB1 (Abcam, EPR16895 ), LAMB1 (Abclonal, A19970), LAMC1 (Abclonal, A16020), CALR (Abclonal, A1066), CANX (Proteintech, 10427-2-AP), BiP (Proteintech, 11587-1-AP), PERK (Abcam, EPR19876-294), EIF2α (Proteintech, 11170-1-AP), p-EIF2α (Affinity, #AF3087), ATF6 (Abcam, EPR22690-84), LC3 (Abcam, ab192890), p62 (Abclonal, A19700), EDEM1 (Proteintech, 26226-1-AP), EDEM3 (Proteintech, 27310-1-AP), HSPG2 (Abcam, ab2501), β-actin (Abbkine, A01010-SK) and GAPDH (Abclonal, AC033).

Techniques: Infection, Immunoprecipitation, Construct, Transfection, Antibody Blocking Assay, Quantitative Proteomics, Western Blot, Biomarker Discovery, Glycoproteomics, CRISPR, Immunofluorescence, Expressing, Fluorescence, Quantitative RT-PCR, Confocal Microscopy, Knock-Out

Journal: Cell & Bioscience

Article Title: CALR-dependent endoplasmic reticulum N-glycoprotein maturation promotes Japanese encephalitis virus entry and replication

doi: 10.1186/s13578-026-01607-x

Figure Lengend Snippet: CALR selectively sustains ITGA3 abundance through N-linked glycoprotein maturation. A Schematic illustration of ER-associated quality control and degradation pathways involved in nascent N-glycoprotein processing, including a potential defect in N-glycan maturation upstream of the CALR/CANX cycle. B Glycomics profiling of N-glycan compositions in CALR-KO and WT PK-15 cells. C Lectin immunofluorescence staining of CALR-KO and WT PK-15 cells using ConA, AAL, and PHA-E, with corresponding quantification of fluorescence intensity. Scale bar, 50 μm. D Immunoblot analysis of ITGA3 protein abundance in CALR-KO and WT PK-15 cells treated with the α-glucosidase inhibitor 1-deoxynojirimycin (1-DNJ). E Immunoblot analysis of ITGB1 protein abundance in CALR-KO and WT PK-15 cells treated with 1-DNJ. F Dose-dependent effects of 1-DNJ (0, 0.5, 1, and 2 mM) on ITGA3 protein abundance in WT PK-15 cells. G Dose-dependent effects of 1-DNJ (0, 0.5, 1, and 2 mM) on ITGB1 protein abundance in WT PK-15 cells. H Immunoblot analysis showing the dose-dependent effects of CALR overexpression on ITGA3 protein abundance in HEK293T cells co-transfected with a constant amount of Flag-tagged ITGA3 expression plasmid (500 ng) and increasing amounts of HA-tagged CALR expression plasmid (0, 500, 1500, and 3000 ng). Total transfected DNA was normalized to 3500 ng using the empty vector. I Immunoblot analysis showing the dose-dependent effect of CALR overexpression on ITGB1 protein abundance in HEK293T cells co-transfected with a constant amount of Flag-tagged ITGB1 expression plasmid (500 ng) and increasing amounts of HA-tagged CALR expression plasmid (0, 500, 1500, and 3000 ng). Total transfected DNA was normalized to 3500 ng using the empty vector. J Confocal microscopy analysis showing co-localization of CALR and ITGA3 in PK-15 cells. Scale bar, 20 μm. K Co-immunoprecipitation analysis demonstrating the interaction between CALR and ITGA3. Data are presented as mean ± SD from at least three independent experiments. Statistical significance was determined using a two-sided Student’s t-test. KO, knockout; WT, wild-type. ns, not significant. *** P < 0.001

Article Snippet: Membranes were blocked in 5% milk and incubated with primary antibodies against Flag (Abbkine, ABT2011), HA (Abbkine, ABT2041), JEV-NS3 (GeneTex, GTX125868), JEV-E (GeneTex, GTX638878), ITGA2 (Abcam, ab133557), ITGA3 (Abclonal, A17502), ITGB1 (Abcam, EPR16895 ), LAMB1 (Abclonal, A19970), LAMC1 (Abclonal, A16020), CALR (Abclonal, A1066), CANX (Proteintech, 10427-2-AP), BiP (Proteintech, 11587-1-AP), PERK (Abcam, EPR19876-294), EIF2α (Proteintech, 11170-1-AP), p-EIF2α (Affinity, #AF3087), ATF6 (Abcam, EPR22690-84), LC3 (Abcam, ab192890), p62 (Abclonal, A19700), EDEM1 (Proteintech, 26226-1-AP), EDEM3 (Proteintech, 27310-1-AP), HSPG2 (Abcam, ab2501), β-actin (Abbkine, A01010-SK) and GAPDH (Abclonal, AC033).

Techniques: Control, Glycoproteomics, Immunofluorescence, Staining, Fluorescence, Western Blot, Quantitative Proteomics, Over Expression, Transfection, Expressing, Plasmid Preparation, Confocal Microscopy, Immunoprecipitation, Knock-Out

Journal: Cell & Bioscience

Article Title: CALR-dependent endoplasmic reticulum N-glycoprotein maturation promotes Japanese encephalitis virus entry and replication

doi: 10.1186/s13578-026-01607-x

Figure Lengend Snippet: Proposed model illustrating how CALR regulates cellular permissiveness to JEV infection through dual glycoprotein maturation-associated mechanisms. In WT cells (left), the CANX/CALR lectin chaperone cycle maintains an ER folding environment that supports efficient maturation and abundance of the viral E glycoprotein. Simultaneously, CALR-dependent N-glycosylation selectively supports ITGA3 maturation and steady-state abundance, establishing a key CALR-dependent node within a broader adhesion- and glycan-associated cell-surface network. Together, these processes facilitate early viral entry and productive replication. In CALR-KO cells (right), disruption of the CANX/CALR cycle impairs ER glycoprotein maturation, leading to reduced ITGA3 and viral E protein abundance and consequently restricting both early infection and downstream viral replication. Broader glycosylation remodeling may additionally alter other adhesion- and glycan-associated surface components (e.g., CAV1, HSPG2, and HS chains)

Article Snippet: Membranes were blocked in 5% milk and incubated with primary antibodies against Flag (Abbkine, ABT2011), HA (Abbkine, ABT2041), JEV-NS3 (GeneTex, GTX125868), JEV-E (GeneTex, GTX638878), ITGA2 (Abcam, ab133557), ITGA3 (Abclonal, A17502), ITGB1 (Abcam, EPR16895 ), LAMB1 (Abclonal, A19970), LAMC1 (Abclonal, A16020), CALR (Abclonal, A1066), CANX (Proteintech, 10427-2-AP), BiP (Proteintech, 11587-1-AP), PERK (Abcam, EPR19876-294), EIF2α (Proteintech, 11170-1-AP), p-EIF2α (Affinity, #AF3087), ATF6 (Abcam, EPR22690-84), LC3 (Abcam, ab192890), p62 (Abclonal, A19700), EDEM1 (Proteintech, 26226-1-AP), EDEM3 (Proteintech, 27310-1-AP), HSPG2 (Abcam, ab2501), β-actin (Abbkine, A01010-SK) and GAPDH (Abclonal, AC033).

Techniques: Infection, Glycoproteomics, Disruption, Quantitative Proteomics

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

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

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: 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

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.

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: Expressing, Multiplex Assay, Immunofluorescence, Staining, Fluorescence

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 ) 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 .

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: 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

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