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R&D Systems ligand
Ligand, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mouse+integrin+2/Recombinant+Mouse+Integrin+alpha+X+beta+2+Protein%2C+CF/pmc12670074-119-10-11
Average 93 stars, based on 3 article reviews

ligand - by Bioz Stars, 2026-09

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Article Title: Metalloproteinase-mediated Shedding of Integrin β2 Promotes Macrophage Efflux from Inflammatory Sites
Article Snippet: Flow data were analyzed using FlowJo software (Tree Star Inc.). .. ELISAs for Soluble Integrin 2 and Integrin M 2 Complexes—ELISAs for integrin 2 or M 2 complexes utilized monoclonal antibodies to mouse integrin 2 (MAB2618, R&D Systems) and integrin M (clone M1/70, Pharmingen) as capture antibodies, and biotin anti-mouse integrin 2 (clone C71/ 16, Pharmingen) as the detection antibody with streptavidinHRP (Jackson ImmunoResearch). .. Antibodies used for depletion of specific integrins from Pharmingen included integrin 2 (clone M18/12), integrin M (clone M1/70), integrin L (clone 2D7), and integrin X (clone HL3).

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Melanoma cell-intrinsic ITGB2 expression and activation by CD44 ( A ) Single-cell (sc) RNA-seq analysis of human ITGB2 gene ( ITGB2 ) expression by patient melanoma (MM) cells versus tumor-infiltrating T cells or endothelial cells (ECs), as depicted by violin plots (median, bold white line; top and bottom quartiles, thin white lines) overlayed with dots representing respective single cells ( B ) Percentages (mean,) of human ITGB2 surface protein expression by patient MM cells, T cells, and ECs ( n = 5 patients) as determined by flow cytometry ( C ) Mean ITGB2 + SOX10 + frequency (%) in benign nevi ( n = 7 patients), primary melanomas ( n = 24 patients), and metastatic melanomas ( n = 13 patients) as determined by multicolor immunofluorescence staining of a patient melanocytic tissue microarray (TMA). Kruskal-Wallis multiple comparisons test was used to assess statistical significance ( D ) Incidence (%) of patient sentinel lymph node (SLN) metastases versus respective primary melanoma biospecimen cohorts ( n = 105) of increasing cancer cell-ITGB2 positivity, 0–2% ( n = 40), 2–25% ( n = 36), >25% ( n = 29), as determined by immunostaining. Frequencies of ITGB2-positive (black bars) and ITGB2-negative (white bars) melanoma cells within each cohort are shown. Fisher’s exact test was performed to determine statistical significance ( E ) Representative multiplex immunofluorescence staining of a patient primary melanoma biopsy for co-expression of ITGB2 (red, all panels) and the melanocytic marker, nuclear SOX-10 (green, first panel), pan T cell marker, CD3 (green, second panel), vascular endothelial marker, CD31 (green, third panel), or macrophage marker, PU.1 (green, fourth panel). Nuclei were counterstained with DAPI (blue). Size bars, 50 μm ( F and G ), Representative immunoblots of ITGB2 protein expression by (F) human melanoma lines, A2058, A375, C8161, FEMX, LOX-IMVI, MDA-MB-435S, and control HSB-2 T lymphoblastic leukemia cells and HUVEC endothelial cells, and (G) murine melanoma lines, B16-F10, YUMM1.7, YUMM3.3, YUMM4.1, YUMM5.2, and control EL-4 T cell lymphoma cells and C166 endothelial cells ( H and I ) Effect of CD44 ab-mediated crosslinking (black bars) versus isotype control ab treatment (white bars) on ITGB2 surface protein expression level (mean fluorescence intensity, MFI, ± SEM) by (H) human and (I) murine melanoma lines and respective cell controls (gray bars) as above, based on FC analysis ( J and K ) Effect of CD44 ab crosslinking as in (H and I) on the activation state of human melanoma cell-ITGB2 as determined by FC (MFI ± SEM) using the activation-sensitive ITGB2 antibody clones (J) KIM-127 and (K) MEM-148. Results are representative of at least n = 3 independent experiments. *, p < 0.05; **, p < 0.01; NS, not significant. See also figs. S1, S2, and S3.

Journal: Molecular Cancer

Article Title: Targeting the tumor cell-intrinsic ITGB2 axis inhibits melanoma progression

doi: 10.1186/s12943-025-02527-z

Figure Lengend Snippet: Melanoma cell-intrinsic ITGB2 expression and activation by CD44 ( A ) Single-cell (sc) RNA-seq analysis of human ITGB2 gene ( ITGB2 ) expression by patient melanoma (MM) cells versus tumor-infiltrating T cells or endothelial cells (ECs), as depicted by violin plots (median, bold white line; top and bottom quartiles, thin white lines) overlayed with dots representing respective single cells ( B ) Percentages (mean,) of human ITGB2 surface protein expression by patient MM cells, T cells, and ECs ( n = 5 patients) as determined by flow cytometry ( C ) Mean ITGB2 + SOX10 + frequency (%) in benign nevi ( n = 7 patients), primary melanomas ( n = 24 patients), and metastatic melanomas ( n = 13 patients) as determined by multicolor immunofluorescence staining of a patient melanocytic tissue microarray (TMA). Kruskal-Wallis multiple comparisons test was used to assess statistical significance ( D ) Incidence (%) of patient sentinel lymph node (SLN) metastases versus respective primary melanoma biospecimen cohorts ( n = 105) of increasing cancer cell-ITGB2 positivity, 0–2% ( n = 40), 2–25% ( n = 36), >25% ( n = 29), as determined by immunostaining. Frequencies of ITGB2-positive (black bars) and ITGB2-negative (white bars) melanoma cells within each cohort are shown. Fisher’s exact test was performed to determine statistical significance ( E ) Representative multiplex immunofluorescence staining of a patient primary melanoma biopsy for co-expression of ITGB2 (red, all panels) and the melanocytic marker, nuclear SOX-10 (green, first panel), pan T cell marker, CD3 (green, second panel), vascular endothelial marker, CD31 (green, third panel), or macrophage marker, PU.1 (green, fourth panel). Nuclei were counterstained with DAPI (blue). Size bars, 50 μm ( F and G ), Representative immunoblots of ITGB2 protein expression by (F) human melanoma lines, A2058, A375, C8161, FEMX, LOX-IMVI, MDA-MB-435S, and control HSB-2 T lymphoblastic leukemia cells and HUVEC endothelial cells, and (G) murine melanoma lines, B16-F10, YUMM1.7, YUMM3.3, YUMM4.1, YUMM5.2, and control EL-4 T cell lymphoma cells and C166 endothelial cells ( H and I ) Effect of CD44 ab-mediated crosslinking (black bars) versus isotype control ab treatment (white bars) on ITGB2 surface protein expression level (mean fluorescence intensity, MFI, ± SEM) by (H) human and (I) murine melanoma lines and respective cell controls (gray bars) as above, based on FC analysis ( J and K ) Effect of CD44 ab crosslinking as in (H and I) on the activation state of human melanoma cell-ITGB2 as determined by FC (MFI ± SEM) using the activation-sensitive ITGB2 antibody clones (J) KIM-127 and (K) MEM-148. Results are representative of at least n = 3 independent experiments. *, p < 0.05; **, p < 0.01; NS, not significant. See also figs. S1, S2, and S3.

Article Snippet: The following abs and reagents were used for immunohistochemistry and immunofluorescence: unconjugated mouse anti-human ITGB2 ab (clone MEM-48, Novus Biologicals, Cat# NB500-379, RRID: AB_10000712), Dako REAL Detection System, Alkaline Phosphatase/RED (Agilent Dako, Santa Clara, CA, Cat# K5005), Biotin-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, Cat# 31800, RRID: AB_228305), AF546-conjugated goat anti-mouse IgG1 (Thermo Fisher Scientific, Cat# A-21123, RRID: AB_2535765), and AF488-conjugated goat anti-mouse IgG1 (Thermo Fisher Scientific, Cat# A-21121, RRID: AB_2535764), unconjugated rabbit anti-human SOX10 (clone EPR4007, Abcam, Cat# ab155279, RRID: AB_2650603), unconjugated rabbit anti-human CD3 (clone SP162, Abcam, Cat# ab135372, RRID: AB_2884903), unconjugated rabbit anti-human CD31 (clone EPR3094, Abcam, Cat# ab76533, RRID: AB_1523298), unconjugated rabbit anti-human ICAM-1 (MilliporeSigma, Cat# SAB5700809, RRID: AB_3669069) and Cy3-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Cat# A10520, RRID: AB_10563288) or AF488-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Cat# A-11008, RRID: AB_143165), unconjugated mouse anti-human SOX10 (clone 1D2C8, Proteintech, Rosemont, IL, Cat#66786-1-Ig, RRID: AB_2882131) and AF647-conjugated goat anti-mouse IgG2a (Thermo Fisher Scientific, Cat# A-21241, RRID: AB_2535810), unconjugated mouse anti-human PU.1 (clone G148-74, BD Biosciences, Cat# 554268, RRID: AB_395335) and AF488-conjugated goat anti-mouse IgG2a (Thermo Fisher Scientific, Cat# A-21131, RRID: AB_2535771).

Techniques: Expressing, Activation Assay, RNA Sequencing, Flow Cytometry, Multicolor Immunofluorescence Staining, Microarray, Immunostaining, Multiplex Assay, Immunofluorescence, Staining, Marker, Western Blot, Control, Fluorescence, Clone Assay

Antibody-based blockade of melanoma cell-intrinsic ITGB2 inhibits ICAM-1-dependent adhesion and growth ( A and B ) Relative in vitro adhesion (mean ± SEM) to immobilized ICAM-1 versus negative coating control of (A) human melanoma C8161 and MDA-MB-435S or positive control HSB-2 cells and (B) murine melanoma B16-F10 and YUMM5.2 or positive control EL-4 cells, either untreated (respective left panels) or treated with ITGB2 blocking ab or EDTA pan-integrin antagonist versus isotype control ab (respective right panels). ( C and D ) Tumor growth kinetics in vivo (mean ± SEM) of (C) human C8161 and MDA-MB-435S cells in NSG mice treated with human-specific ITGB2 blocking ab versus isotype control ab or (D) murine B16-F10 and YUMM5.2 cells in NSG mice treated with anti-murine ITGB2 blocking versus isotype control ab. Results in panels (A and B) are representative of and/or pooled from at least n = 3 independent experiments. The unpaired Student’s t test was used to statistically compare two groups and one-way ANOVA with Dunnett’s post-test for comparison of three groups. Panels (C and D) involved n = 5–20 mice per respective treatment group. Repeated-measures two-way ANOVA or mixed model followed by Šídák’s multiple comparisons correction were used to assess statistical differences in tumor growth. *, p < 0.05; **, p < 0.01; ***, p < 0.001. See also Figs. and , and , fig. S3

Journal: Molecular Cancer

Article Title: Targeting the tumor cell-intrinsic ITGB2 axis inhibits melanoma progression

doi: 10.1186/s12943-025-02527-z

Figure Lengend Snippet: Antibody-based blockade of melanoma cell-intrinsic ITGB2 inhibits ICAM-1-dependent adhesion and growth ( A and B ) Relative in vitro adhesion (mean ± SEM) to immobilized ICAM-1 versus negative coating control of (A) human melanoma C8161 and MDA-MB-435S or positive control HSB-2 cells and (B) murine melanoma B16-F10 and YUMM5.2 or positive control EL-4 cells, either untreated (respective left panels) or treated with ITGB2 blocking ab or EDTA pan-integrin antagonist versus isotype control ab (respective right panels). ( C and D ) Tumor growth kinetics in vivo (mean ± SEM) of (C) human C8161 and MDA-MB-435S cells in NSG mice treated with human-specific ITGB2 blocking ab versus isotype control ab or (D) murine B16-F10 and YUMM5.2 cells in NSG mice treated with anti-murine ITGB2 blocking versus isotype control ab. Results in panels (A and B) are representative of and/or pooled from at least n = 3 independent experiments. The unpaired Student’s t test was used to statistically compare two groups and one-way ANOVA with Dunnett’s post-test for comparison of three groups. Panels (C and D) involved n = 5–20 mice per respective treatment group. Repeated-measures two-way ANOVA or mixed model followed by Šídák’s multiple comparisons correction were used to assess statistical differences in tumor growth. *, p < 0.05; **, p < 0.01; ***, p < 0.001. See also Figs. and , and , fig. S3

Techniques: In Vitro, Control, Positive Control, Blocking Assay, In Vivo, Comparison

Antibody-based ITGB2 blockade or host Icam1 deficiency inhibit melanoma metastasis ( A to C ) Effect of anti-murine ITGB2 blocking ab versus isotype control ab on tumorigenesis of B16-F10 and YUMM5.2 cells in wildtype (WT) C57BL/6 mice. (A) Tumor growth kinetics (mean ± SEM), (B) relative intratumoral T cell levels, and (C) relative lung metastasis of GFP-expressing melanoma cells were determined by qPCR-based quantitation of genomic Cd3 or GFP in tumor and lung tissue, respectively. (B ) Primer specificity for Cd3 was validated using positive control murine T cells and negative control B16-F10 and YUMM5.2 cells. (C) Specificity of GFP primers was authenticated using positive control GFP-expressing B16-F10 and YUMM5.2 cells and negative control lungs obtained from WT mice without tumors. ( D to F ) Effect of anti-murine ITGB2 blocking ab versus isotype control ab on tumorigenesis of B16-F10 and YUMM5.2 cells in Icam1 −/− C57BL/6 mice. (D) Tumor growth kinetics (mean ± SEM), (E) intratumoral T cell levels, and (F) lung metastasis in Icam1- deficient mice were determined by qPCR analysis using positive and negative cell and sample controls, as above. Panels (A and D) involved n = 16–20 mice per respective treatment group. Results in panels (B, C, E, and F) are representative of and/or pooled from at least n = 3 independent experiments. Tumor control groups in panels B and E, C and F are identical, respectively. Repeated-measures two-way ANOVA or mixed model followed by Šídák’s multiple comparisons correction were used to assess statistical differences in tumor growth in panels (A and D). Data in (B, C, E, and F) were statistically compared using the unpaired Student’s t test. *, p < 0.05; NS, not significant; nd, not detected. See also Figs. and , fig. S3

Journal: Molecular Cancer

Article Title: Targeting the tumor cell-intrinsic ITGB2 axis inhibits melanoma progression

doi: 10.1186/s12943-025-02527-z

Figure Lengend Snippet: Antibody-based ITGB2 blockade or host Icam1 deficiency inhibit melanoma metastasis ( A to C ) Effect of anti-murine ITGB2 blocking ab versus isotype control ab on tumorigenesis of B16-F10 and YUMM5.2 cells in wildtype (WT) C57BL/6 mice. (A) Tumor growth kinetics (mean ± SEM), (B) relative intratumoral T cell levels, and (C) relative lung metastasis of GFP-expressing melanoma cells were determined by qPCR-based quantitation of genomic Cd3 or GFP in tumor and lung tissue, respectively. (B ) Primer specificity for Cd3 was validated using positive control murine T cells and negative control B16-F10 and YUMM5.2 cells. (C) Specificity of GFP primers was authenticated using positive control GFP-expressing B16-F10 and YUMM5.2 cells and negative control lungs obtained from WT mice without tumors. ( D to F ) Effect of anti-murine ITGB2 blocking ab versus isotype control ab on tumorigenesis of B16-F10 and YUMM5.2 cells in Icam1 −/− C57BL/6 mice. (D) Tumor growth kinetics (mean ± SEM), (E) intratumoral T cell levels, and (F) lung metastasis in Icam1- deficient mice were determined by qPCR analysis using positive and negative cell and sample controls, as above. Panels (A and D) involved n = 16–20 mice per respective treatment group. Results in panels (B, C, E, and F) are representative of and/or pooled from at least n = 3 independent experiments. Tumor control groups in panels B and E, C and F are identical, respectively. Repeated-measures two-way ANOVA or mixed model followed by Šídák’s multiple comparisons correction were used to assess statistical differences in tumor growth in panels (A and D). Data in (B, C, E, and F) were statistically compared using the unpaired Student’s t test. *, p < 0.05; NS, not significant; nd, not detected. See also Figs. and , fig. S3

Techniques: Blocking Assay, Control, Expressing, Quantitation Assay, Positive Control, Negative Control

CRISPR/Cas9-based genetic knockout of melanoma cell-intrinsic Itgb2 suppresses adhesion to ICAM-1 and resultant tumor growth ( A ) Validation of CRISPR/Cas9-mediated stable KO of Itgb2 gene and ITGB2 protein in B16-F10 and YUMM5.2 melanoma cells as determined by RT-qPCR (left panel) and immunoblotting (right panel). ( B to F ) Itgb2 KO versus respective Cas9 control B16-F10 and YUMM5.2 tumor cell relative (B) in vitro adhesion (mean ± SEM) to immobilized ICAM-1, with or without negative control EDTA treatment, (C) in vitro growth (mean ± SEM) as determined by CellTiter-Glo-based luminescence analysis, and (D to F) in vivo tumor growth kinetics (mean ± SEM) in (D) NSG mice, (E) C57BL/6 mice, and (F) Icam1 −/− C57BL/6 mice. ( G ) Relative Icam1 gene expression in B16-F10 and YUMM5.2 tumors from C57BL/6 mice (black bars) versus Icam1 −/− C57BL/6 mice (white bars), with positive control murine T cells and C166 endothelial cells shown (gray bars). ( H ) scRNA-seq analysis of human ICAM1 gene expression in patient melanoma (MM) cells, tumor-infiltrating T cells, and endothelial cells (ECs) as depicted by violin plots (median, bold white line; top and bottom quartiles, thin white lines) overlayed with dots representing respective single cells. ( I ) Percentages (mean) of human ICAM-1 surface protein expression by patient MM cells, T cells, and ECs ( n = 5 patients) as determined by FC. ( J ) Multiplex immunofluorescence staining of a representative ( n = 4 patients) clinical melanoma biospecimen for expression of the melanocytic marker, nuclear SOX-10 (red, first panel), ITGB2 (yellow, second panel), and ICAM-1 (green, third panel). The merged image is also shown (fourth panel). Nuclei were counterstained with DAPI (blue). Size bars, 50 μm. Results in panels (A, B, C, and G) are representative of and/or pooled from at least n = 3 independent experiments. The unpaired Student’s t test was used to statistically compare two groups and one-way ANOVA with Dunnett’s post-test for comparison of three groups. Panels (D to F) involved n = 10 mice per respective melanoma cell variant. Repeated-measures two-way ANOVA was used to assess statistical differences in tumor growth. **, p < 0.01; ***, p < 0.001; NS, not significant; nd, not detected. See also Figs. and 4, figs. S3 and S4

Journal: Molecular Cancer

Article Title: Targeting the tumor cell-intrinsic ITGB2 axis inhibits melanoma progression

doi: 10.1186/s12943-025-02527-z

Figure Lengend Snippet: CRISPR/Cas9-based genetic knockout of melanoma cell-intrinsic Itgb2 suppresses adhesion to ICAM-1 and resultant tumor growth ( A ) Validation of CRISPR/Cas9-mediated stable KO of Itgb2 gene and ITGB2 protein in B16-F10 and YUMM5.2 melanoma cells as determined by RT-qPCR (left panel) and immunoblotting (right panel). ( B to F ) Itgb2 KO versus respective Cas9 control B16-F10 and YUMM5.2 tumor cell relative (B) in vitro adhesion (mean ± SEM) to immobilized ICAM-1, with or without negative control EDTA treatment, (C) in vitro growth (mean ± SEM) as determined by CellTiter-Glo-based luminescence analysis, and (D to F) in vivo tumor growth kinetics (mean ± SEM) in (D) NSG mice, (E) C57BL/6 mice, and (F) Icam1 −/− C57BL/6 mice. ( G ) Relative Icam1 gene expression in B16-F10 and YUMM5.2 tumors from C57BL/6 mice (black bars) versus Icam1 −/− C57BL/6 mice (white bars), with positive control murine T cells and C166 endothelial cells shown (gray bars). ( H ) scRNA-seq analysis of human ICAM1 gene expression in patient melanoma (MM) cells, tumor-infiltrating T cells, and endothelial cells (ECs) as depicted by violin plots (median, bold white line; top and bottom quartiles, thin white lines) overlayed with dots representing respective single cells. ( I ) Percentages (mean) of human ICAM-1 surface protein expression by patient MM cells, T cells, and ECs ( n = 5 patients) as determined by FC. ( J ) Multiplex immunofluorescence staining of a representative ( n = 4 patients) clinical melanoma biospecimen for expression of the melanocytic marker, nuclear SOX-10 (red, first panel), ITGB2 (yellow, second panel), and ICAM-1 (green, third panel). The merged image is also shown (fourth panel). Nuclei were counterstained with DAPI (blue). Size bars, 50 μm. Results in panels (A, B, C, and G) are representative of and/or pooled from at least n = 3 independent experiments. The unpaired Student’s t test was used to statistically compare two groups and one-way ANOVA with Dunnett’s post-test for comparison of three groups. Panels (D to F) involved n = 10 mice per respective melanoma cell variant. Repeated-measures two-way ANOVA was used to assess statistical differences in tumor growth. **, p < 0.01; ***, p < 0.001; NS, not significant; nd, not detected. See also Figs. and 4, figs. S3 and S4

Techniques: CRISPR, Knock-Out, Biomarker Discovery, Quantitative RT-PCR, Western Blot, Control, In Vitro, Negative Control, In Vivo, Gene Expression, Positive Control, Expressing, Multiplex Assay, Immunofluorescence, Staining, Marker, Comparison, Variant Assay

The melanoma cell-ITGB2:ICAM-1 axis stimulates downstream Wnt pathway activation, the inhibition of which suppresses cancer cell:ICAM-1 adhesion ( A ) Heatmaps of differentially expressed genes (DEGs) exhibiting pathway interconnectivity ( n = 51) in Itgb2 KO versus control YUMM5.2 tumors and which showed consistent trends in both NSG (left panel) and wildtype (WT) C57BL/6 mice (middle panel), but not in Icam1 −/− C57BL/6 hosts (right panel), as determined by RNA-seq analysis. ( B ) Protein-protein interaction and cluster map (STRING) of 22 of the 51 DEGs described in (A) exhibiting the strongest interaction scores. Respective network clusters (gray ovals) and relative strengths of direct protein-protein interactions (stronger, wider lines; weaker, thinner lines) as well as indirect associations (dashed lines) are shown. Proteins without any designated cluster associations were omitted. The paired Wilcoxon test was used to assess statistical significance. ( C ) Magnitude of difference in expression of each Wnt pathway DEG in Itgb2 KO versus Cas9 control melanomas (log fold change) as in (A) and identified in the Gene Ontology Biological Process (GOBP) database. Wnt signaling effectors were grouped into activating ( Frat2 , Kpna1 , Wnt5a , Wnt5b ) versus inhibitory ( Dkk2 , Igfbp4 , Kank1 , Notum ) cohorts. Medians are represented by horizontal bars in box and whiskers plots. ( D ) Validation by RT-qPCR (fold change) of Wnt effector DEGs as in (C) using independent Itgb2 KO versus Cas9 control YUMM5.2 tumor biospecimens from NSG, WT, or Icam1 −/− C57BL/6 mice. Medians are represented by horizontal bars in box and whiskers plots. ( E ) Representative immunoblots of canonical Wnt mediators, active (non-p) β-catenin and LEF-1, and ACTB loading control (left), and non-canonical Wnt effector, p-VANGL2, and respective total controls (right) in Itgb2 KO versus Cas9 control YUMM5.2 melanoma cells. ( F ) Representatie immunoblots of Wnt signaling mediators as in (E) of YUMM5.2 melanoma cells treated with the Wnt inhibitors, pyrvinium pamoate, LGK974, or zamaporvint, versus vehicle control. ( G and H ) Relative in vitro adhesion (mean ± SEM) to immobilized ICAM-1 as determined by CellTiter-Glo-based luminescence analysis of (G) Itgb2 KO versus Cas9 control YUMM5.2 variants and (H) anti-murine ITGB2 blocking ab versus isotype control ab treated YUMM5.2 wildtype cells, in the combined presence or absence of pyrvinium pamoate, LGK974, zamaporvint, or vehicle control. The paired Student’s t test was used to assess statistical significance. Panels (A, B, C, and D) are representative of n = 2–6 tumors per variant group in each respective animal host. Results in (E, F, G, and H) are representative of and/or pooled from at least n = 2–7 independent experiments each. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, not significant. See also Figs. and , and , figs. S5 and S6

Journal: Molecular Cancer

Article Title: Targeting the tumor cell-intrinsic ITGB2 axis inhibits melanoma progression

doi: 10.1186/s12943-025-02527-z

Figure Lengend Snippet: The melanoma cell-ITGB2:ICAM-1 axis stimulates downstream Wnt pathway activation, the inhibition of which suppresses cancer cell:ICAM-1 adhesion ( A ) Heatmaps of differentially expressed genes (DEGs) exhibiting pathway interconnectivity ( n = 51) in Itgb2 KO versus control YUMM5.2 tumors and which showed consistent trends in both NSG (left panel) and wildtype (WT) C57BL/6 mice (middle panel), but not in Icam1 −/− C57BL/6 hosts (right panel), as determined by RNA-seq analysis. ( B ) Protein-protein interaction and cluster map (STRING) of 22 of the 51 DEGs described in (A) exhibiting the strongest interaction scores. Respective network clusters (gray ovals) and relative strengths of direct protein-protein interactions (stronger, wider lines; weaker, thinner lines) as well as indirect associations (dashed lines) are shown. Proteins without any designated cluster associations were omitted. The paired Wilcoxon test was used to assess statistical significance. ( C ) Magnitude of difference in expression of each Wnt pathway DEG in Itgb2 KO versus Cas9 control melanomas (log fold change) as in (A) and identified in the Gene Ontology Biological Process (GOBP) database. Wnt signaling effectors were grouped into activating ( Frat2 , Kpna1 , Wnt5a , Wnt5b ) versus inhibitory ( Dkk2 , Igfbp4 , Kank1 , Notum ) cohorts. Medians are represented by horizontal bars in box and whiskers plots. ( D ) Validation by RT-qPCR (fold change) of Wnt effector DEGs as in (C) using independent Itgb2 KO versus Cas9 control YUMM5.2 tumor biospecimens from NSG, WT, or Icam1 −/− C57BL/6 mice. Medians are represented by horizontal bars in box and whiskers plots. ( E ) Representative immunoblots of canonical Wnt mediators, active (non-p) β-catenin and LEF-1, and ACTB loading control (left), and non-canonical Wnt effector, p-VANGL2, and respective total controls (right) in Itgb2 KO versus Cas9 control YUMM5.2 melanoma cells. ( F ) Representatie immunoblots of Wnt signaling mediators as in (E) of YUMM5.2 melanoma cells treated with the Wnt inhibitors, pyrvinium pamoate, LGK974, or zamaporvint, versus vehicle control. ( G and H ) Relative in vitro adhesion (mean ± SEM) to immobilized ICAM-1 as determined by CellTiter-Glo-based luminescence analysis of (G) Itgb2 KO versus Cas9 control YUMM5.2 variants and (H) anti-murine ITGB2 blocking ab versus isotype control ab treated YUMM5.2 wildtype cells, in the combined presence or absence of pyrvinium pamoate, LGK974, zamaporvint, or vehicle control. The paired Student’s t test was used to assess statistical significance. Panels (A, B, C, and D) are representative of n = 2–6 tumors per variant group in each respective animal host. Results in (E, F, G, and H) are representative of and/or pooled from at least n = 2–7 independent experiments each. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, not significant. See also Figs. and , and , figs. S5 and S6

Techniques: Activation Assay, Inhibition, Control, RNA Sequencing, Protein-Protein interactions, Expressing, Biomarker Discovery, Quantitative RT-PCR, Western Blot, In Vitro, Blocking Assay, Variant Assay

Wnt antagonism suppresses ITGB2:ICAM-1-dependent melanoma growth in vivo ( A and B ) Tumor growth kinetics (mean ± SEM) of (A) Itgb2 KO versus Cas9 control YUMM5.2 variant cells or (B) YUMM5.2 wildtype cells treated with anti-murine ITGB2 blocking ab versus isotype control ab, with or without concurrent administration of the Wnt inhibitors, pyrvinium pamoate, LGK974, zamaporvint, as well as vehicle control in NSG (left panel), wildtype (WT) C57BL/6 (middle panel), or Icam1 −/− C57BL/6 mice (right panel). Because tumorigenicity experiments evaluating LGK974 and zamaporvint effects were conducted concurrently, vehicle control groups for both drugs are identical. Panels (A and B) involved n = 6–10 mice per respective treatment group. Repeated-measures two-way ANOVA or mixed model followed by Šídák’s multiple comparisons correction were used to assess statistical differences in tumor growth in panels. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, not significant. See also Fig.

Journal: Molecular Cancer

Article Title: Targeting the tumor cell-intrinsic ITGB2 axis inhibits melanoma progression

doi: 10.1186/s12943-025-02527-z

Figure Lengend Snippet: Wnt antagonism suppresses ITGB2:ICAM-1-dependent melanoma growth in vivo ( A and B ) Tumor growth kinetics (mean ± SEM) of (A) Itgb2 KO versus Cas9 control YUMM5.2 variant cells or (B) YUMM5.2 wildtype cells treated with anti-murine ITGB2 blocking ab versus isotype control ab, with or without concurrent administration of the Wnt inhibitors, pyrvinium pamoate, LGK974, zamaporvint, as well as vehicle control in NSG (left panel), wildtype (WT) C57BL/6 (middle panel), or Icam1 −/− C57BL/6 mice (right panel). Because tumorigenicity experiments evaluating LGK974 and zamaporvint effects were conducted concurrently, vehicle control groups for both drugs are identical. Panels (A and B) involved n = 6–10 mice per respective treatment group. Repeated-measures two-way ANOVA or mixed model followed by Šídák’s multiple comparisons correction were used to assess statistical differences in tumor growth in panels. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, not significant. See also Fig.

Techniques: In Vivo, Control, Variant Assay, Blocking Assay

$Identification of SP65 with affinity for DCs and synthesis of SP65-conjugated lipids. ( A ) Phage-display was used to select peptides that bound to the recombinant mouse CD11c (mCD11c) and DCs, but not to CD3 + T cells. After the first five rounds of biopanning, the phage titer eluted from mCD11c increased 2,200-fold, and after the subsequent two rounds, the titer from CD3 + T cell-unbound/DC-bound fractions increased 4,000-fold compared with the first round. Pfu, plaque-forming units. (B ) Binding activity and specificity between individual phage clones and mCD11c or bovine serum albumin were tested in 5th round by ELISA. A 450 , optical density at 450 nm. ( C ) Binding activities between the phage clone 65 (PC65) or control phage and bone marrow derived dendritic cells (BMDCs) were analyzed by flow cytometry. ( D ) Hydrogen bonds between SP65 and mCD11c demonstrated in Pymol. Light pink: αX. Slate Blue: β2. Green: SP65. Yellow: hydrogen bond (red arrow highlighted). ( E ) Synthesis scheme of SP65-DOPE; The sequence of synthetic peptide 65 (SP65) is PRSMPSRHKSQRGGGC. ( F ) MALDI-TOF mass spectrum of SP65-DOPE under positive mode; upper: found MS; lower: calculated MS. ( G ) Synthesis scheme of Ctrl-DOPE; Derived from amino acid residue 561–572 of BSA, the sequence of control peptide is KATEEQLKTVMEGGGC. ( H ) MALDI-TOF mass spectrum of Ctrl-DOPE under negative mode; upper: found MS; lower: calculated MS$

Journal: Journal of Nanobiotechnology

Article Title: Dendritic cell-targeted liposomes for cancer immunotherapy via inhibition of aryl hydrocarbon receptor

doi: 10.1186/s12951-025-03756-6

Figure Lengend Snippet: Identification of SP65 with affinity for DCs and synthesis of SP65-conjugated lipids. ( A ) Phage-display was used to select peptides that bound to the recombinant mouse CD11c (mCD11c) and DCs, but not to CD3 + T cells. After the first five rounds of biopanning, the phage titer eluted from mCD11c increased 2,200-fold, and after the subsequent two rounds, the titer from CD3 + T cell-unbound/DC-bound fractions increased 4,000-fold compared with the first round. Pfu, plaque-forming units. (B ) Binding activity and specificity between individual phage clones and mCD11c or bovine serum albumin were tested in 5th round by ELISA. A 450 , optical density at 450 nm. ( C ) Binding activities between the phage clone 65 (PC65) or control phage and bone marrow derived dendritic cells (BMDCs) were analyzed by flow cytometry. ( D ) Hydrogen bonds between SP65 and mCD11c demonstrated in Pymol. Light pink: αX. Slate Blue: β2. Green: SP65. Yellow: hydrogen bond (red arrow highlighted). ( E ) Synthesis scheme of SP65-DOPE; The sequence of synthetic peptide 65 (SP65) is PRSMPSRHKSQRGGGC. ( F ) MALDI-TOF mass spectrum of SP65-DOPE under positive mode; upper: found MS; lower: calculated MS. ( G ) Synthesis scheme of Ctrl-DOPE; Derived from amino acid residue 561–572 of BSA, the sequence of control peptide is KATEEQLKTVMEGGGC. ( H ) MALDI-TOF mass spectrum of Ctrl-DOPE under negative mode; upper: found MS; lower: calculated MS

Article Snippet: Briefly, recombinant mouse CD11c (mCD11c) protein (R&D Systems, Cat# 7987-AX) was coated onto 96-well plates and exposed to 2 × 10 11 phages from the Ph.D.−12 library (New England Biolabs) in PBS buffer at 4 °C for 1 h. After washing, bound phages were eluted with RIPA buffer, amplified, and titrated using the ER2738 Escherichia coli strain.

Techniques: Recombinant, Binding Assay, Activity Assay, Clone Assay, Enzyme-linked Immunosorbent Assay, Control, Derivative Assay, Flow Cytometry, Sequencing, Residue

SP65 enhances liposomal Dil uptake into DCs. ( A ) Fluorescence Dil uptake of splenic DCs after 30 min–4 h of incubation with variable formulations of liposomal Dil, n = 3. ( B ) Quantitative data of mean fluorescence intensity (MFI) of Dil in splenic DCs after 4 h of incubation with lipo-Dil (non-conjugated), Ctrl-lipo-Dil (control peptide-conjugated), or SP65-lipo-Dil (SP65-conjugated) from ( A ). ( C ) Confocal microscopic images of Dil uptake in splenic DCs. Cell shape was observed using the differential interference contrast model. (Scale bar = 50 μm). ( D ) In vivo Dil uptakes in CD11c + and CD11c − cells from peripheral blood of mice receiving liposomal Dil through intravenous injection. ( n = 3 biologically independent mice). ( F ) Quantitative data of the MFI of Dil in CD11c + cells from ( E ) after 2 h of injection. ( G ) Distribution of DiR in major organs from mice receiving liposomal DiR through intravenous injection after 2 h. ( G ) Quantitative data of ( F ) ( n = 4 biologically independent mice). All data were expressed as the mean values ± SD. Statistical differences were evaluated using one-way ANOVA. *, p < 0.05, **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. All statistical analyses were performed with GraphPad Prism 10.4.0

Journal: Journal of Nanobiotechnology

Article Title: Dendritic cell-targeted liposomes for cancer immunotherapy via inhibition of aryl hydrocarbon receptor

doi: 10.1186/s12951-025-03756-6

Figure Lengend Snippet: SP65 enhances liposomal Dil uptake into DCs. ( A ) Fluorescence Dil uptake of splenic DCs after 30 min–4 h of incubation with variable formulations of liposomal Dil, n = 3. ( B ) Quantitative data of mean fluorescence intensity (MFI) of Dil in splenic DCs after 4 h of incubation with lipo-Dil (non-conjugated), Ctrl-lipo-Dil (control peptide-conjugated), or SP65-lipo-Dil (SP65-conjugated) from ( A ). ( C ) Confocal microscopic images of Dil uptake in splenic DCs. Cell shape was observed using the differential interference contrast model. (Scale bar = 50 μm). ( D ) In vivo Dil uptakes in CD11c + and CD11c − cells from peripheral blood of mice receiving liposomal Dil through intravenous injection. ( n = 3 biologically independent mice). ( F ) Quantitative data of the MFI of Dil in CD11c + cells from ( E ) after 2 h of injection. ( G ) Distribution of DiR in major organs from mice receiving liposomal DiR through intravenous injection after 2 h. ( G ) Quantitative data of ( F ) ( n = 4 biologically independent mice). All data were expressed as the mean values ± SD. Statistical differences were evaluated using one-way ANOVA. *, p < 0.05, **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. All statistical analyses were performed with GraphPad Prism 10.4.0

Techniques: Fluorescence, Incubation, Control, In Vivo, Injection

$Desialylation results in the activation of the Hippo pathway. A, MDA-MB-231 cells were pretreated with different doses of sialidase for 3 h; then, the cell membrane fractions were immunoblotted with SNA (recognizing α2,6-sialylated proteins) and ConA (an α-mannose/α-glucose-binding lectin) lectins or blotted with anti-integrin β1 antibody. B, to further determine the change of sialylation on the cell surface after sialidase treatment, the indicated cells were incubated with biotin-conjugated MAA (recognizing 2,3-sialylated proteins, dotted line ), biotin-conjugated SNA ( bold line ), or without ( gray shadow ) lectin, followed by incubation with appropriate Alexa Flour 647 conjugate and subjected to flow cytometry. C, MDA-MB-231 cells were treated as described in ( A ), and then the cell lysates were immunoblotted with anti-p-YAP S127, anti-YAP, anti-p-LATS1 T1079, anti-LATS1, and anti-GAPDH antibodies. The relative ratios (phospho-YAP and phospho-LATS1 versus YAP and LATS1, respectively) are presented as the mean ± SD ( n = 3 biological replicates, ∗∗, p < 0.01, ∗∗∗, p < 0.001 is determined by two-tail unpaired t test). SNA, Sambucus nigra; MAA, Maackia amurensis agglutinin; ConA, Concanavalin A; LATS, large tumor suppressor kinase; YAP, yes-associated protein.$

Journal: The Journal of Biological Chemistry

Article Title: Inhibitory effects of β-galactoside α2,6-sialyltransferase 1 on the Hippo pathway in breast cancer cells

doi: 10.1016/j.jbc.2025.110266

Figure Lengend Snippet: Desialylation results in the activation of the Hippo pathway. A, MDA-MB-231 cells were pretreated with different doses of sialidase for 3 h; then, the cell membrane fractions were immunoblotted with SNA (recognizing α2,6-sialylated proteins) and ConA (an α-mannose/α-glucose-binding lectin) lectins or blotted with anti-integrin β1 antibody. B, to further determine the change of sialylation on the cell surface after sialidase treatment, the indicated cells were incubated with biotin-conjugated MAA (recognizing 2,3-sialylated proteins, dotted line ), biotin-conjugated SNA ( bold line ), or without ( gray shadow ) lectin, followed by incubation with appropriate Alexa Flour 647 conjugate and subjected to flow cytometry. C, MDA-MB-231 cells were treated as described in ( A ), and then the cell lysates were immunoblotted with anti-p-YAP S127, anti-YAP, anti-p-LATS1 T1079, anti-LATS1, and anti-GAPDH antibodies. The relative ratios (phospho-YAP and phospho-LATS1 versus YAP and LATS1, respectively) are presented as the mean ± SD ( n = 3 biological replicates, ∗∗, p < 0.01, ∗∗∗, p < 0.001 is determined by two-tail unpaired t test). SNA, Sambucus nigra; MAA, Maackia amurensis agglutinin; ConA, Concanavalin A; LATS, large tumor suppressor kinase; YAP, yes-associated protein.

Article Snippet: The experiments were performed using the following antibodies: Rabbit antibodies against p-YAP(S127) (#13008S), p-LATS1(T1079) (#8654S), LATS1 (#3477S), p-Src(Y416) (#2101S), p-FAK(Y397) (#8556S), FAK (#3285S), EGFR (#4267S), p-EGFR(Y1068) (#3777S), and integrin β1 (#9699S) were from Cell Signaling Technology; mouse mAb against GAPDH (#sc-365062), and β-actin (#sc-47778) were from Santa Cruz Biotechnology; mouse mAb against integrin α5 (610633) was from BD Biosciences; rabbit pAbs against LPAR4 (22165-1-AP) and mouse mAb against YAP (66900-1-Ig) were obtained from Proteintech; rabbit pAb against ST3GAL4 (NBP1-69565) was obtained from Novus Biologicals; mouse mAbs against FLAG (clone M2, #F3165) and Src (clone GD11, #05-184) were from Sigma; goat pAb against ST6GAL1 (AF5924) was from R&D Systems; mouse mAb against integrin β1 (P5D2) was from Developmental Studies Hybridoma Bank.

Techniques: Activation Assay, Membrane, Binding Assay, Incubation, Flow Cytometry

ST6GAL1 catalyzes the α2,6-sialylation of LPAR4, EGFR, integrin α5, and integrin β1 in MDA-MB-231 cells. A, the cell lysates from Con-, ST6GAL1-KO-, ST6GAL1-Res- MDA-MB-231 cells were immunoprecipitated by SSA-agaroses and then blotted with antibodies against LPAR4, EGFR, integrin β1, and integrin α5. The whole-cell lysates were also subjected to WB with indicated antibodies. B, the cell lysates from Con- and ST3GAL4-OE- MDA-MB-231 cells were immunoprecipitated by SSA- and MAM-agaroses and then blotted with antibodies against integrin β1, EGFR, and integrin α5 separately. The whole-cell lysates were also subjected to WB with indicated antibodies. The relative ratios (the α2,6-sialylated LPAR4, EGFR, integrin α5, or integrin β1 versus total LPAR4, EGFR, integrin α5, or integrin β1, respectively) in ( A ) and (the α2,3-sialylated or α2,6-sialylated integrin β1, EGFR, or integrin α5 versus total integrin β1, EGFR, or integrin α5, respectively) in ( B ) are shown as the mean ± SD ( n = 3 biological replicates, ∗∗, p < 0.01; ∗∗∗, p < 0.001; and ∗∗∗∗, p < 0.0001 are determined by one-way ANOVA with Tukey's post hoc test and two-tail unpaired t test, respectively). WB, Western blot; ST6GAL1, β-galactoside α2,6-sialyltransferase 1; SSA, Sambucus sieboldiana agglutinin; Con, control; Res, rescue; EGFR, epidermal growth factor receptor; Maackia amurensis.

Journal: The Journal of Biological Chemistry

Article Title: Inhibitory effects of β-galactoside α2,6-sialyltransferase 1 on the Hippo pathway in breast cancer cells

doi: 10.1016/j.jbc.2025.110266

Figure Lengend Snippet: ST6GAL1 catalyzes the α2,6-sialylation of LPAR4, EGFR, integrin α5, and integrin β1 in MDA-MB-231 cells. A, the cell lysates from Con-, ST6GAL1-KO-, ST6GAL1-Res- MDA-MB-231 cells were immunoprecipitated by SSA-agaroses and then blotted with antibodies against LPAR4, EGFR, integrin β1, and integrin α5. The whole-cell lysates were also subjected to WB with indicated antibodies. B, the cell lysates from Con- and ST3GAL4-OE- MDA-MB-231 cells were immunoprecipitated by SSA- and MAM-agaroses and then blotted with antibodies against integrin β1, EGFR, and integrin α5 separately. The whole-cell lysates were also subjected to WB with indicated antibodies. The relative ratios (the α2,6-sialylated LPAR4, EGFR, integrin α5, or integrin β1 versus total LPAR4, EGFR, integrin α5, or integrin β1, respectively) in ( A ) and (the α2,3-sialylated or α2,6-sialylated integrin β1, EGFR, or integrin α5 versus total integrin β1, EGFR, or integrin α5, respectively) in ( B ) are shown as the mean ± SD ( n = 3 biological replicates, ∗∗, p < 0.01; ∗∗∗, p < 0.001; and ∗∗∗∗, p < 0.0001 are determined by one-way ANOVA with Tukey's post hoc test and two-tail unpaired t test, respectively). WB, Western blot; ST6GAL1, β-galactoside α2,6-sialyltransferase 1; SSA, Sambucus sieboldiana agglutinin; Con, control; Res, rescue; EGFR, epidermal growth factor receptor; Maackia amurensis.

Techniques: Immunoprecipitation, Western Blot, Control

ST6GAL1 mediates integrin β1–LPAR4/EGFR complex formation. A and B, the cell lysates from Con-, ST6GAL1-KO-, ST6GAL1-Res- MDA-MB-231 ( A ) and BT549 ( B ) cells were immunoprecipitated by anti-integrin β1 antibody and then blotted with antibodies against LPAR4, EGFR, and integrin β1. The whole-cell lysates were also subjected to WB with indicated antibodies. The relative ratios (the association of EGFR or LPAR4 with integrin β1, respectively) in ( A ) and ( B ) are presented as the mean ± SD ( n = 3 biological replicates, ∗∗∗∗, p < 0.0001 is determined by one-way ANOVA with Tukey's post hoc test). ST6GAL1, β-galactoside α2,6-sialyltransferase 1; Con, control; Res, rescue; EGFR, epidermal growth factor receptor; WB, Western blot.

Journal: The Journal of Biological Chemistry

Article Title: Inhibitory effects of β-galactoside α2,6-sialyltransferase 1 on the Hippo pathway in breast cancer cells

doi: 10.1016/j.jbc.2025.110266

Figure Lengend Snippet: ST6GAL1 mediates integrin β1–LPAR4/EGFR complex formation. A and B, the cell lysates from Con-, ST6GAL1-KO-, ST6GAL1-Res- MDA-MB-231 ( A ) and BT549 ( B ) cells were immunoprecipitated by anti-integrin β1 antibody and then blotted with antibodies against LPAR4, EGFR, and integrin β1. The whole-cell lysates were also subjected to WB with indicated antibodies. The relative ratios (the association of EGFR or LPAR4 with integrin β1, respectively) in ( A ) and ( B ) are presented as the mean ± SD ( n = 3 biological replicates, ∗∗∗∗, p < 0.0001 is determined by one-way ANOVA with Tukey's post hoc test). ST6GAL1, β-galactoside α2,6-sialyltransferase 1; Con, control; Res, rescue; EGFR, epidermal growth factor receptor; WB, Western blot.

Techniques: Immunoprecipitation, Control, Western Blot

Schematic diagram of the proposed molecular mechanism for negative regulation of Hippo signaling via ST6GAL1. Various upstream cell membrane receptors of the Hippo pathway have been identified, including the RTKs ( e.g. , EGFR), GPCRs ( e.g. , LPAR4), and integrins ( e.g. , integrin α5β1). The RTK, GPCR, and integrin signals transduced by growth factors (GFs, e.g. , EGF), extracellular factors ( e.g. , LPA), and the extracellular matrix (ECM, e.g. , FN) can facilitate Hippo pathway effectors ( e.g. , PI3K and FAK) association, which promote LATS1/2-mediated regulation of YAP. In the cells with ST6GAL1 expression ( left ), the cell membrane receptors, such as EGFR, LPAR4, and integrin α5β1, are modified by α2,6-sialylation, which mediate the integrin β1–EGFR/LPAR4 complex formation and in turn facilitate their responses to EGF, LPA, and FN, respectively. These signalings inactivate LATS1/2 kinases or induce the dephosphorylation of YAP, finally leading to hypophosphorylated YAP (p-YAP S127). Hypophosphorylated YAP accumulates in the nucleus, where it can bind to various transcription factors (TFs, e.g. , TEAD family) to enhance the expression of target genes ( e.g. , ANKRD1 , CTGF , and CYR61 ) expression that promote cell adhesion, spreading, proliferation, migration, and metastasis. The Hippo signaling can be inhibited by the verteporfin (VP) inhibitor, which targets YAP-TEAD activity. In the ST6GAL1 deficiency cells ( right ), the N -glycans on cell membrane receptors are without α2,6-sialylation, which exhibit weak integrin β1–EGFR/LPAR4 complex formation and delayed responses to EGF, LPA, and FN stimulation and activate the LATS1/2 kinases and phosphorylate YAP on S127. The phosphorylated YAP (p-YAP S127) is retained in the cytoplasm, inhibiting YAP/TEAD-dependent transcription. The p of the red background represents the activation of related proteins, while gray background represents the inactivation. LATS, large tumor suppressor kinase; YAP, yes-associated protein; ST6GAL1, β-galactoside α2,6-sialyltransferase 1; RTK, receptor tyrosine kinase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FN, fibronectin; GPCR, G protein–coupled receptor; GT, glycosyltransferase; LPA, lysophosphatidic acid; FAK, focal adhesion kinase.

Journal: The Journal of Biological Chemistry

Article Title: Inhibitory effects of β-galactoside α2,6-sialyltransferase 1 on the Hippo pathway in breast cancer cells

doi: 10.1016/j.jbc.2025.110266

Figure Lengend Snippet: Schematic diagram of the proposed molecular mechanism for negative regulation of Hippo signaling via ST6GAL1. Various upstream cell membrane receptors of the Hippo pathway have been identified, including the RTKs ( e.g. , EGFR), GPCRs ( e.g. , LPAR4), and integrins ( e.g. , integrin α5β1). The RTK, GPCR, and integrin signals transduced by growth factors (GFs, e.g. , EGF), extracellular factors ( e.g. , LPA), and the extracellular matrix (ECM, e.g. , FN) can facilitate Hippo pathway effectors ( e.g. , PI3K and FAK) association, which promote LATS1/2-mediated regulation of YAP. In the cells with ST6GAL1 expression ( left ), the cell membrane receptors, such as EGFR, LPAR4, and integrin α5β1, are modified by α2,6-sialylation, which mediate the integrin β1–EGFR/LPAR4 complex formation and in turn facilitate their responses to EGF, LPA, and FN, respectively. These signalings inactivate LATS1/2 kinases or induce the dephosphorylation of YAP, finally leading to hypophosphorylated YAP (p-YAP S127). Hypophosphorylated YAP accumulates in the nucleus, where it can bind to various transcription factors (TFs, e.g. , TEAD family) to enhance the expression of target genes ( e.g. , ANKRD1 , CTGF , and CYR61 ) expression that promote cell adhesion, spreading, proliferation, migration, and metastasis. The Hippo signaling can be inhibited by the verteporfin (VP) inhibitor, which targets YAP-TEAD activity. In the ST6GAL1 deficiency cells ( right ), the N -glycans on cell membrane receptors are without α2,6-sialylation, which exhibit weak integrin β1–EGFR/LPAR4 complex formation and delayed responses to EGF, LPA, and FN stimulation and activate the LATS1/2 kinases and phosphorylate YAP on S127. The phosphorylated YAP (p-YAP S127) is retained in the cytoplasm, inhibiting YAP/TEAD-dependent transcription. The p of the red background represents the activation of related proteins, while gray background represents the inactivation. LATS, large tumor suppressor kinase; YAP, yes-associated protein; ST6GAL1, β-galactoside α2,6-sialyltransferase 1; RTK, receptor tyrosine kinase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FN, fibronectin; GPCR, G protein–coupled receptor; GT, glycosyltransferase; LPA, lysophosphatidic acid; FAK, focal adhesion kinase.

Techniques: Membrane, Expressing, Modification, De-Phosphorylation Assay, Migration, Activity Assay, Activation Assay

(A) β1 integrin is diminished in the liver of conditional knockout (cKO) animals carrying the albumin promoter to drive cre expression in mice homozygous for floxed β1 integrin (Alb-cKO) compared to control mice (CT) by western blotting. N=4 CT and 4 Alb-cKO replicates. **p<0.005. (B) Western blot analysis shows successful depletion of β1 integrin in hepatocytes isolated from Alb-cKO animals. Flow cytometry confirms a decrease in the percentage of cells expressing β1 integrin in Alb-cKO hepatocytes. The number of replicates for the groups is shown in the order presented in the graphs: N=12/10 for western blot and 6/6 for flow cytometry. *p<0.05, ****p<0.0001. (C) Sirius-red and trichrome staining suggest an increase in extracellular matrix in Alb-cKO liver sections. Bars represent 100μm. (D) mRNA expression of collagen I, III and IV as well as fibronectin was increased in Alb-cKO livers compared to CT. N=13/21 for collagen I, 10/10 for collagen III, 10/10 for collagen IV, 8/9 for fibronectin. *p<0.05. (E) Collagen is increased in the liver of Alb-cKO animals. Collagen content was evaluated using a biochemical method to quantify hydroxyproline followed by adjustment to collagen amount. N=18/21, ***p<0.001. (F) Collagen I is increased in Alb-cKO livers by western blotting. N=8/10. (G) An increase in collagen I is suggested by immunofluorescence staining. Bars represent 100μm. (H) Despite the increase in matrix, no evidence for liver-related blood laboratory abnormalities. N=31/40. Livers and blood was examined in 14-16-week-old animals, while hepatocytes were isolated by liver perfusion from 8-10 week-old animals and examined immediately. Data were analyzed using unpaired t-tests for all graphs presented in this figure. In the case of collagen IV mRNA expression Welch’s correction was applied because of the significant difference in the variances between CT and Alb-cKO. All graphs show CT to the left and Alb-cKO to the right.

Journal: bioRxiv

Article Title: MODULATION OF COLLAGEN-BINDING INTEGRINS AFFECTS FIBROBLAST ACTIVATION AND INHIBITS FIBROSIS

doi: 10.1101/2025.05.14.653428

Figure Lengend Snippet: (A) β1 integrin is diminished in the liver of conditional knockout (cKO) animals carrying the albumin promoter to drive cre expression in mice homozygous for floxed β1 integrin (Alb-cKO) compared to control mice (CT) by western blotting. N=4 CT and 4 Alb-cKO replicates. **p<0.005. (B) Western blot analysis shows successful depletion of β1 integrin in hepatocytes isolated from Alb-cKO animals. Flow cytometry confirms a decrease in the percentage of cells expressing β1 integrin in Alb-cKO hepatocytes. The number of replicates for the groups is shown in the order presented in the graphs: N=12/10 for western blot and 6/6 for flow cytometry. *p<0.05, ****p<0.0001. (C) Sirius-red and trichrome staining suggest an increase in extracellular matrix in Alb-cKO liver sections. Bars represent 100μm. (D) mRNA expression of collagen I, III and IV as well as fibronectin was increased in Alb-cKO livers compared to CT. N=13/21 for collagen I, 10/10 for collagen III, 10/10 for collagen IV, 8/9 for fibronectin. *p<0.05. (E) Collagen is increased in the liver of Alb-cKO animals. Collagen content was evaluated using a biochemical method to quantify hydroxyproline followed by adjustment to collagen amount. N=18/21, ***p<0.001. (F) Collagen I is increased in Alb-cKO livers by western blotting. N=8/10. (G) An increase in collagen I is suggested by immunofluorescence staining. Bars represent 100μm. (H) Despite the increase in matrix, no evidence for liver-related blood laboratory abnormalities. N=31/40. Livers and blood was examined in 14-16-week-old animals, while hepatocytes were isolated by liver perfusion from 8-10 week-old animals and examined immediately. Data were analyzed using unpaired t-tests for all graphs presented in this figure. In the case of collagen IV mRNA expression Welch’s correction was applied because of the significant difference in the variances between CT and Alb-cKO. All graphs show CT to the left and Alb-cKO to the right.

Article Snippet: The following murine integrin pairs were purchased from R&D systems: α1β1 #8188-AB, α2β1 #7828-A2, α10β1 #7827-AB, α11β1 #7808-AB, α5β1 #7728-A5, αvβ1 # 7705-AV, αvβ3 #7889-AV, as well as human α11β1 #6357-AB.

Techniques: Knock-Out, Expressing, Control, Western Blot, Isolation, Flow Cytometry, Staining, Immunofluorescence

(A) Total TGF-β protein decreased when hepatocytes were cultured on collagen type I, compared to cells cultured on fibronectin or vitronectin. Freshly isolated hepatocytes were cultured in wells precoated with poly-L-lysine 0.01% or the matrix proteins at a concentration of 10μg/mL for 24 hours. Total TGF-β protein was evaluated in the medium and cells and corrected to protein content. N=11/6/11/13 replicates. *p<0.05. (B) Integrin subunits expression on the surface of freshly isolated hepatocytes was evaluated by flow cytometry. N= 3 experiments. (C-D) Knockdown of β1 or α11 integrin using siRNA in hepatocytes increased total TGF-β protein (C). Depletion was confirmed by western blotting (D). Isolated cells were cultured and treated with siRNA targeting integrin subunits that bind to collagen, fibronectin and vitronectin. TGF-β: N=36/36/21/25/33/25/25/25/25 replicates in the order of the bars, β1 depletion: N=2/2, α11 depletion N=4/2. (E) Structure of cyclic GLQGE. (F) GLQGE did not affect hepatocyte proliferation (ki67 staining, left graph) or apoptosis (annexinv-propidium-iodide staining, right graph). N=7/8/7/7 for proliferation and 7/8/8/8 for apoptosis. Cells were cultured 24 hours in the presence of 50μg/mL of the molecules, stained and evaluated by flow cytometry. (G) In hepatocytes, only GLQGE diminishes TGF-β mRNA and protein compared to control (CT). Hepatocytes cultured on vitronectin were treated with 50μg/mL of the peptides and evaluated 24 hours later. N=10/9/10/10. *p<0.05. (H) TGF-β protein in the Huh-7 hepatoma cell line was reduced after GLQGE or GLNGE treatment. Huh7 cells were cultured on vitronectin, and treated for 24 hours with 50μg/mL of the molecules. N= 5 experiments with 2-9 replicates per experiment. *p<0.05. (I) pFAK is increased after treatment of hepatocytes with GLQGE. Cells were cultured in suspension without FCS for 2 hours and treated with 50μg/mL of the peptides for 30 minutes. N=35/29/30/30. *p<0.05, ***p<0.0005. (J) Increased pFAK in hepatocytes does not require β3 integrin expression. β3 hepatocytes were obtained from global β3 knockout mice (deletion confirmed using flow cytometry in peripheral blood and shown) and compared to wildtype littermate controls. Cells isolated were treated as in I. N=4/5/5/5. **p<0.01. Comparisons were performed using unpaired t-tests.

Journal: bioRxiv

Article Title: MODULATION OF COLLAGEN-BINDING INTEGRINS AFFECTS FIBROBLAST ACTIVATION AND INHIBITS FIBROSIS

doi: 10.1101/2025.05.14.653428

Figure Lengend Snippet: (A) Total TGF-β protein decreased when hepatocytes were cultured on collagen type I, compared to cells cultured on fibronectin or vitronectin. Freshly isolated hepatocytes were cultured in wells precoated with poly-L-lysine 0.01% or the matrix proteins at a concentration of 10μg/mL for 24 hours. Total TGF-β protein was evaluated in the medium and cells and corrected to protein content. N=11/6/11/13 replicates. *p<0.05. (B) Integrin subunits expression on the surface of freshly isolated hepatocytes was evaluated by flow cytometry. N= 3 experiments. (C-D) Knockdown of β1 or α11 integrin using siRNA in hepatocytes increased total TGF-β protein (C). Depletion was confirmed by western blotting (D). Isolated cells were cultured and treated with siRNA targeting integrin subunits that bind to collagen, fibronectin and vitronectin. TGF-β: N=36/36/21/25/33/25/25/25/25 replicates in the order of the bars, β1 depletion: N=2/2, α11 depletion N=4/2. (E) Structure of cyclic GLQGE. (F) GLQGE did not affect hepatocyte proliferation (ki67 staining, left graph) or apoptosis (annexinv-propidium-iodide staining, right graph). N=7/8/7/7 for proliferation and 7/8/8/8 for apoptosis. Cells were cultured 24 hours in the presence of 50μg/mL of the molecules, stained and evaluated by flow cytometry. (G) In hepatocytes, only GLQGE diminishes TGF-β mRNA and protein compared to control (CT). Hepatocytes cultured on vitronectin were treated with 50μg/mL of the peptides and evaluated 24 hours later. N=10/9/10/10. *p<0.05. (H) TGF-β protein in the Huh-7 hepatoma cell line was reduced after GLQGE or GLNGE treatment. Huh7 cells were cultured on vitronectin, and treated for 24 hours with 50μg/mL of the molecules. N= 5 experiments with 2-9 replicates per experiment. *p<0.05. (I) pFAK is increased after treatment of hepatocytes with GLQGE. Cells were cultured in suspension without FCS for 2 hours and treated with 50μg/mL of the peptides for 30 minutes. N=35/29/30/30. *p<0.05, ***p<0.0005. (J) Increased pFAK in hepatocytes does not require β3 integrin expression. β3 hepatocytes were obtained from global β3 knockout mice (deletion confirmed using flow cytometry in peripheral blood and shown) and compared to wildtype littermate controls. Cells isolated were treated as in I. N=4/5/5/5. **p<0.01. Comparisons were performed using unpaired t-tests.

Techniques: Cell Culture, Isolation, Concentration Assay, Expressing, Flow Cytometry, Knockdown, Western Blot, Staining, Control, Suspension, Knock-Out

(A) Fibrosis was induced in animals by a single intratracheal bleomycin instillation. Starting on day 11, subcutaneous injections were administered of either NaCl 0.9% (CT) or GLQGE, GLNGE or GLOGE (in NaCl 0.9%) at a dose of 1 mg/mouse/day. On day 21, the animals were euthanized. (B) GLQGE treatment diminished collagen accumulation in the lung compared to fibrotic mice receiving 0.9% NaCl, GLNGE or GLOGE. Lung lysates were evaluated biochemically. N=18/18/17/17/18 replicates in the order of the bars. *p<0.05, ***p<0.0005, ****p<0.0001. (C) Western blot analysis shows that GLQGE diminishes collagen type I in lungs of fibrotic GLQGE-treated animals compared to fibrotic NaCl-treated animals. N=4/3/3/4/4. *p<0.05, **p<0.01, ****p<0.0001. (D) GLQGE did not alter the levels of TGF-β compared to fibrotic controls. N=18/17/16/17/17. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (E) αSMA protein was diminished by western blot analysis of the lungs in GLQGE-treated fibrotic animals compared to NaCl-injected fibrotic CT animals. N=4/4/4/4/4 replicates. *p<0.05, **p<0.005, ***p<0.001. (F) Circulating levels of LDH, reflecting cell turnover induced by injury, were elevated in all fibrotic groups, but GLQGE treatment did not diminish LDH. N=18/18/17/17/18 replicates. *p<0.05, **p<0.01, ***p<0.0005. All numerical data were evaluated by analysis of variance (ANOVA) and when significant, t-tests were performed.

Journal: bioRxiv

Article Title: MODULATION OF COLLAGEN-BINDING INTEGRINS AFFECTS FIBROBLAST ACTIVATION AND INHIBITS FIBROSIS

doi: 10.1101/2025.05.14.653428

Figure Lengend Snippet: (A) Fibrosis was induced in animals by a single intratracheal bleomycin instillation. Starting on day 11, subcutaneous injections were administered of either NaCl 0.9% (CT) or GLQGE, GLNGE or GLOGE (in NaCl 0.9%) at a dose of 1 mg/mouse/day. On day 21, the animals were euthanized. (B) GLQGE treatment diminished collagen accumulation in the lung compared to fibrotic mice receiving 0.9% NaCl, GLNGE or GLOGE. Lung lysates were evaluated biochemically. N=18/18/17/17/18 replicates in the order of the bars. *p<0.05, ***p<0.0005, ****p<0.0001. (C) Western blot analysis shows that GLQGE diminishes collagen type I in lungs of fibrotic GLQGE-treated animals compared to fibrotic NaCl-treated animals. N=4/3/3/4/4. *p<0.05, **p<0.01, ****p<0.0001. (D) GLQGE did not alter the levels of TGF-β compared to fibrotic controls. N=18/17/16/17/17. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (E) αSMA protein was diminished by western blot analysis of the lungs in GLQGE-treated fibrotic animals compared to NaCl-injected fibrotic CT animals. N=4/4/4/4/4 replicates. *p<0.05, **p<0.005, ***p<0.001. (F) Circulating levels of LDH, reflecting cell turnover induced by injury, were elevated in all fibrotic groups, but GLQGE treatment did not diminish LDH. N=18/18/17/17/18 replicates. *p<0.05, **p<0.01, ***p<0.0005. All numerical data were evaluated by analysis of variance (ANOVA) and when significant, t-tests were performed.

Techniques: Western Blot, Injection

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