Journal: Journal of Integrative Agriculture

Article Title: Salmonella YrbD protein mediates invasion into the host by interacting with β2 integrin1

doi: 10.1016/j.jia.2023.12.035

Figure Lengend Snippet: Fig. 1 Overexpression of β2 integrin or siRNA silencing affect Salmonella infection. A, overexpression of β2 integrin facilitated Salmonella infection in transfected HeLa cells. B and C, flow cytometry measurement. D, CFU assays. E, transfection with siRNA resulted in the downregulation of β2 integrin mRNA in transfected HeLa cells. NT, non-targeting siRNA. F and G, silencing of β2 integrin inhibited Salmonella infection of HeLa cells. Data are mean±SD. Unpaired t-test was used for the statistical analysis, and a P-value<0.05 was considered significant. *, P<0.05; **, P<0.01; ***, P<0.001. ns, not significant. Scale bars=400 µm in A and F.

Article Snippet: For immunohistochemistry, after incubating with 1:50 diluted rabbit anti-β2 integrin antibody (NBP1-88127, Novus, St. Charles, MO, USA) overnight at 4°C, the sections were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody for 30 min at RT, then rinsed thrice with PBS for 5 min each, stained with diaminobenzidine (DAB) (Sigma-Aldrich), and counterstained with hematoxylin.

Techniques: Over Expression, Infection, Transfection, Flow Cytometry

Journal: Journal of Integrative Agriculture

Article Title: Salmonella YrbD protein mediates invasion into the host by interacting with β2 integrin1

doi: 10.1016/j.jia.2023.12.035

Figure Lengend Snippet: Fig. 2 β2 integrin is required for Salmonella binding. A, silencing of β2 integrin inhibited Salmonella binding of HeLa cells. HeLa cells were infected with Salmonella at 4˚C for 1 h. Cells were stained with anti-Salmonella antibody (green), actin-specific TRITC- phalloidin (red), and DAPI (blue). Samples were imaged using a Zeiss LSM880 laser-scanning confocal microscope and processed using Zeiss ZEN 2.3 blue edition software. Each image shown represents a set of 24 images. Presented are the combined z-stacks for each infected cell. B, number of Salmonella per cell with silencing of β2 integrin. C, overexpression of β2 integrin increased binding of Salmonella to HeLa cells. The samples were stained described as above. D, number of Salmonella per cell with overexpression of β2 integrin. Data are mean±SD. Statistical significance was determined using the Mann-Whitney test, and a P-value<0.05 was considered significant. ****, P<0.0001. ns, not significant. 63× objective lens.

Article Snippet: For immunohistochemistry, after incubating with 1:50 diluted rabbit anti-β2 integrin antibody (NBP1-88127, Novus, St. Charles, MO, USA) overnight at 4°C, the sections were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody for 30 min at RT, then rinsed thrice with PBS for 5 min each, stained with diaminobenzidine (DAB) (Sigma-Aldrich), and counterstained with hematoxylin.

Techniques: Binding Assay, Infection, Staining, Microscopy, Software, Over Expression, MANN-WHITNEY

Journal: Journal of Integrative Agriculture

Article Title: Salmonella YrbD protein mediates invasion into the host by interacting with β2 integrin1

doi: 10.1016/j.jia.2023.12.035

Figure Lengend Snippet: Fig. 3 Antibodies to β2 integrin block Salmonella infection of HeLa, Caco-2, and RAW264.7 cells in a dose-dependent manner. A and B, the mAb against β2 integrin blocked the Salmonella infection of HeLa cells. The isotype IgG2a at the highest concentration was used as control. C and D, the pAb against β2 integrin blocked the Salmonella infection of HeLa cells. The isotype IgG at the highest concentration was used as control. E and F, the mAb against β2 integrin blocked the Salmonella infection of Caco-2 cells. G and H, the mAb against β2 integrin blocked the Salmonella infection of RAW264.7 cells. Data are mean±SD. One-way ANOVA was used for the statistical analysis, and P-value<0.05 was considered significant. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. ns, not significant. Scale bars=200 µm.

Article Snippet: For immunohistochemistry, after incubating with 1:50 diluted rabbit anti-β2 integrin antibody (NBP1-88127, Novus, St. Charles, MO, USA) overnight at 4°C, the sections were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody for 30 min at RT, then rinsed thrice with PBS for 5 min each, stained with diaminobenzidine (DAB) (Sigma-Aldrich), and counterstained with hematoxylin.

Techniques: Blocking Assay, Infection, Concentration Assay, Control

Journal: Journal of Integrative Agriculture

Article Title: Salmonella YrbD protein mediates invasion into the host by interacting with β2 integrin1

doi: 10.1016/j.jia.2023.12.035

Figure Lengend Snippet: Fig. 4 The β2 integrin ectodomain soluble protein (β2 integrin-GST) neutralizes the Salmonella infection in HeLa, Caco-2, and RAW264.7 cells. A and B, β2 integrin-GST neutralizes the Salmonella infection in HeLa cells in a dose-dependent manner. C and D, β2 integrin-GST neutralizes the Salmonella infection in Caco-2 cells. E and F, β2 integrin-GST neutralizes the Salmonella infection in RAW264.7 cells. Data are mean±SD. One-way ANOVA was used for the statistical analysis, and P-value<0.05 was considered significant. **, P<0.01; ****, P<0.0001. ns, not significant. Scale bars=200 µm.

Article Snippet: For immunohistochemistry, after incubating with 1:50 diluted rabbit anti-β2 integrin antibody (NBP1-88127, Novus, St. Charles, MO, USA) overnight at 4°C, the sections were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody for 30 min at RT, then rinsed thrice with PBS for 5 min each, stained with diaminobenzidine (DAB) (Sigma-Aldrich), and counterstained with hematoxylin.

Techniques: Infection

Journal: Journal of Integrative Agriculture

Article Title: Salmonella YrbD protein mediates invasion into the host by interacting with β2 integrin1

doi: 10.1016/j.jia.2023.12.035

Figure Lengend Snippet: Fig. 5 The β2 integrin ectodomain soluble protein neutralizes Salmonella infection in mice. A, β2 integrin-GST neutralizes the Salmonella infection in mice. The log-rank (Mantel-Cox) test was used to analyze the statistical difference between the survival rates of the challenged mice. B and C, representative images of H&E-stained sections of caecum. D and E, immunohistochemical staining for β2 integrin in caecum of infected mice and uninfected mice (negative control). F, SDS-PAGE analysis of GST pull- down assay. G, some differentially expressed proteins based on mass spectrometry analysis. H, immunoelectron microscopy analysis for YrbD localization at both OM and IM of Salmonella. SL1344 was immunolabelled for YrbD with 5-nm gold particles. Black squares represent the labeling of YrbD on the OM of Salmonella. Red squares represent the labeling of YrbD on the IM of Salmonella. The zoom-in windows of the positive labeling were provided (right). Black arrows and red arrows represent the location of the OM and IM, respectively. Scale bars=200 nm.

Article Snippet: For immunohistochemistry, after incubating with 1:50 diluted rabbit anti-β2 integrin antibody (NBP1-88127, Novus, St. Charles, MO, USA) overnight at 4°C, the sections were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody for 30 min at RT, then rinsed thrice with PBS for 5 min each, stained with diaminobenzidine (DAB) (Sigma-Aldrich), and counterstained with hematoxylin.

Techniques: Infection, Staining, Immunohistochemical staining, Negative Control, SDS Page, Pull Down Assay, Mass Spectrometry, Immuno-Electron Microscopy, Labeling

Journal: Journal of Integrative Agriculture

Article Title: Salmonella YrbD protein mediates invasion into the host by interacting with β2 integrin1

doi: 10.1016/j.jia.2023.12.035

Figure Lengend Snippet: Fig. 6 Interactions between β2 integrin and Salmonella YrbD. A, the β2 integrin-Myc interacted with YrbD-Flag in Co-IP assays with plasmid-transfected HEK293 cell lysates. B, purified β2 integrin-GST pulled down YrbD-Flag. C, purified β2 integrin-GST pulled down purified YrbD-His. D, the extracellular domain of β2 integrin interacted with YrbD-Flag. E and F, deletion of β2 integrin I-like domain abolished the interaction between YrbD and β2 integrin.

Article Snippet: For immunohistochemistry, after incubating with 1:50 diluted rabbit anti-β2 integrin antibody (NBP1-88127, Novus, St. Charles, MO, USA) overnight at 4°C, the sections were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody for 30 min at RT, then rinsed thrice with PBS for 5 min each, stained with diaminobenzidine (DAB) (Sigma-Aldrich), and counterstained with hematoxylin.

Techniques: Co-Immunoprecipitation Assay, Plasmid Preparation, Transfection, Purification

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

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

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

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

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

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

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: 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

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

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

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.

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: In Vivo, Control, Variant Assay, Blocking Assay