lama4  (Proteintech)

Journal: Advanced Science

Article Title: The FOXC2‐LAMA4 Axis Orchestrates Vasculogenic Mimicry and Immunosuppressive Niche Formation to Drive Metastatic Cascade in Renal Cell Carcinoma

doi: 10.1002/advs.202516382

Figure Lengend Snippet: Multi‐omics sequencing identifies LAMA4 as a key downstream effector of FOXC2. (A) Genome‐wide binding signal heatmap of FOXC2 CUT&Tag sequencing in 786‐O cells (FOXC2 vs. IgG). (B) Distribution of FOXC2 binding peaks across the genome and motif analysis of its promoter‐bound sequences in 786‐O cells. (C) Venn diagram intersecting differentially expressed genes (log2FC > 1) upon FOXC2 knockdown, FOXC2‐bound downstream genes, and Epithelial_s4 subpopulation‐enriched genes, identifying LAMA4 and VAV3 as potential FOXC2 transcriptional targets. (D) GO/KEGG functional enrichment analysis of genes with FOXC2 binding peaks from CUT&Tag sequencing. (E) GO/KEGG functional enrichment analysis of RNA‐seq differentially expressed genes in FOXC2 knockdown vs. control groups. (F) CUT&Tag sequencing reveals a specific FOXC2 binding site (highlighted in dark yellow) within the upstream promoter region of the LAMA4 gene. (G) RNA‐seq data from 786‐O cells validate LAMA4 as a FOXC2‐regulated differentially expressed gene. (H) TCGA‐KIRC dataset analysis demonstrates a significant positive correlation between FOXC2 and LAMA4 mRNA expression (R = 0.527, ** p < 0.01). (I)–(K) scRNA data show significant co‐expression of FOXC2 and LAMA4 in the Epithelial_s4 subpopulation. (L) scRNA data from 12 ccRCC patients confirm a robust expression correlation between tumor cell‐derived FOXC2 and LAMA4 (R = 0.630, * p < 0.05). (M) Western blot analysis of FOXC2 and LAMA4 protein levels in paired tumor and adjacent normal tissues from ccRCC patients. (N) Positive correlation between FOXC2 and LAMA4 protein expression. Protein levels were quantified by western blot (n=9; * p < 0.05, ** p < 0.01). A significant correlation was found by Spearman analysis (R = 0.717, p = 0.037).

Article Snippet: THP‐1 monocytes were differentiated into M0 macrophages by treatment with 100 ng/mL phorbol 12‐myristate 13‐acetate (PMA; Selleck, S7791) for 48 h. The differentiated macrophages were then stimulated for 24 h with 25 ng/mL recombinant human LAMA4 (Origene, TP761994).

Techniques: Biomarker Discovery, Sequencing, Genome Wide, Binding Assay, Knockdown, Functional Assay, RNA Sequencing, Control, Expressing, Derivative Assay, Western Blot

Journal: Advanced Science

Article Title: The FOXC2‐LAMA4 Axis Orchestrates Vasculogenic Mimicry and Immunosuppressive Niche Formation to Drive Metastatic Cascade in Renal Cell Carcinoma

doi: 10.1002/advs.202516382

Figure Lengend Snippet: FOXC2 promotes VM in ccRCC cells by transcriptionally regulating LAMA4. (A) Western blot analysis of LAMA4 protein expression upon FOXC2 knockdown. (B) qPCR detection of LAMA4 mRNA levels following FOXC2 knockdown (n=3; ** p < 0.01, *** p < 0.001). (C) Western blot analysis of FOXC2, LAMA4, VE‑cadherin, and the EMT markers E‑cadherin, N‑cadherin, and Vimentin in FOXC2‑overexpressing (OE‐ FOXC2) and LAMA4‑knockdown (sh‐LAMA4) cell models. (D) Tube formation assays demonstrate that FOXC2 overexpression significantly enhances VM capacity in renal cancer cells, while LAMA4 knockdown partially reverses this effect (n=3; *** p < 0.001). (E) Transwell invasion assays reveal that FOXC2 overexpression promotes cancer cell invasion, which is suppressed by LAMA4 knockdown (n=3; ** p < 0.01, *** p < 0.001). (F) Wound healing assays confirm that FOXC2 overexpression increases cell migration, an effect attenuated by LAMA4 knockdown (n=3; *** p < 0.001). 0.001). (G) Statistical analysis was performed on the following metrics: tube formation (number of tubes, branches, junctions, meshes, and total length), wound healing (wound healing percentage), and Transwell (number of migrated cells). ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. (H) Luciferase reporter assays using LAMA4 promoter truncations transfected into renal cancer cells. FOXC2 regulates promoter activity across distinct LAMA4 promoter regions (n=3; ns p > 0.05, *** p < 0.001). (I) ChIP with FOXC2 antibody followed by qPCR amplification of LAMA4 promoter fragments (site1–site4) (n=3; ns p > 0.05, ** p < 0.01). (J) Luciferase assays comparing wild‐type and site2‐mutated LAMA4 promoter activity in renal cancer cells. FOXC2 selectively regulates wild‐type promoter activity (n=3; ns p > 0.05, *** p < 0.001).

Article Snippet: THP‐1 monocytes were differentiated into M0 macrophages by treatment with 100 ng/mL phorbol 12‐myristate 13‐acetate (PMA; Selleck, S7791) for 48 h. The differentiated macrophages were then stimulated for 24 h with 25 ng/mL recombinant human LAMA4 (Origene, TP761994).

Techniques: Western Blot, Expressing, Knockdown, Over Expression, Migration, Luciferase, Transfection, Activity Assay, Amplification

Journal: Advanced Science

Article Title: The FOXC2‐LAMA4 Axis Orchestrates Vasculogenic Mimicry and Immunosuppressive Niche Formation to Drive Metastatic Cascade in Renal Cell Carcinoma

doi: 10.1002/advs.202516382

Figure Lengend Snippet: FOXC2‐LAMA4 remodels the metastatic microenvironment by promoting TREM2 + CD206 + MAM polarization in pulmonary metastases. (A) Experimental design and grouping for orthotopic renal carcinoma xenografts. (B) In vivo imaging system dynamically monitors renal orthotopic tumor growth (days 7, 14, 21, 28) and lung metastasis formation (terminal imaging at day 28) across groups. (C) Comparison of renal tumor volumes at the experimental endpoint among groups. (D) Lung metastasis imaging and H&E staining validation in the Foxc2 overexpression group (Foxc2 OE Lama4 NC ) vs. Foxc2 overexpression + LAMA4 knockdown group (Foxc2 OE Lama4 KD ). (E) Dimensionality reduction and cell type annotation of scRNA data from lung tissues of Foxc2 OE Lama4 NC and Foxc2 OE Lama4 KD mice. (F) Heatmap of MAMs signature genes showing upregulated immunosuppressive markers (e.g., Trem2, Arg1) (log2FC >1, * p < 0.05). (G) Tissue preference analysis (R/oe score) highlights significant enrichment of MAMs (red arrow) in the Foxc2 OE Lama4 NC group. (H),(I) Flow cytometry analysis of the effect of LAMA4 on inducing immunosuppressive polarization. Shown is the MFI of CD206 (H) within the CD68 + population for human THP1‑derived macrophages and (I) within the CD11b + F4/80 + population for mouse BMDMs following treatment with LAMA4. Untreated cells and IL‑4‑treated cells served as negative and positive controls, respectively (n = 3, **** p < 0.0001). (J) scRNA data from ccRCC lung metastases show high LAMA4 expression and elevated Trem2 + macrophage markers (TREM2, C1QC, APOE, CD163) in GPNMB‐Hi and FOLR2‐Hi macrophages. (K) scRNA of murine lung metastases: Macrophages in the oeFoxc2 ncLama4 group exhibit higher M2 markers, while T cells display pronounced exhaustion markers. (L),(M) Flow cytometry analysis of immune cell phenotypes in lung metastases from the orthotopic kidney cancer model. (L) Proportion of exhausted (CD8a + PD‑1 + ) CD8 + T cells. (M) MFI of TREM2 on macrophages. Comparisons are between the FOXC2 OE LAMA4 NC and FOXC2 OE LAMA4 KD groups (n = 5). ( ** p < 0.01, *** p < 0.001).

Article Snippet: THP‐1 monocytes were differentiated into M0 macrophages by treatment with 100 ng/mL phorbol 12‐myristate 13‐acetate (PMA; Selleck, S7791) for 48 h. The differentiated macrophages were then stimulated for 24 h with 25 ng/mL recombinant human LAMA4 (Origene, TP761994).

Techniques: In Vivo Imaging, Imaging, Comparison, Staining, Biomarker Discovery, Over Expression, Knockdown, Flow Cytometry, Expressing

Journal: Advanced Science

Article Title: The FOXC2‐LAMA4 Axis Orchestrates Vasculogenic Mimicry and Immunosuppressive Niche Formation to Drive Metastatic Cascade in Renal Cell Carcinoma

doi: 10.1002/advs.202516382

Figure Lengend Snippet: LAMA4‐ITGA6 binding activates STAT6 phosphorylation to drive GATA3‐dependent TREM2 + CD206 + MAM polarization, promoting metastatic outgrowth. (A) Schematic representation of the experimental setup to investigate the role of LAMA4 in macrophage polarization. THP‐1 cells were treated with PMA to differentiate into macrophages, followed by stimulation with LAMA4 (5 ng/mL). (B) Volcano plot comparing gene expression profiles between control and LAMA4‐treated THP‐1 macrophages. Red dots indicate upregulated genes; blue dots indicate downregulated genes. GATA3 was highly expressed in the LAMA4 treatment group. (C) Heatmap showing DEGs between control and LAMA4‐treated THP‐1 macrophages. Upregulated and downregulated genes are represented in red and blue, respectively. CXCL2, CXCL1, and TNF (inflammatory factors) exhibited elevated expression in controls, whereas ARG1, GATA3, and HES3 (immunosuppressive markers) showed significant upregulation in LAMA4‐treated macrophages. (D) GSEA demonstrating LAMA4‐mediated upregulation of the Fatty Acid Metabolic signaling pathway and downregulation of the TNF/NF‐κB signaling signaling pathway. (E) Pseudotime trajectory constructed by Monocle revealed that MAMs are derived from monocytes/macrophages (Mono/Mac) in mouse lung metastatic niches. (F) SCENIC analysis revealed heightened Gata3 regulon activity score (Gata3 RAS) enriched in MAMs, with concurrent elevation of Gata3 activity in Mono/Mac from oeFOXC2_ncLama4 mice group. (G) Cell‐cell communication analysis indicated that tumor cells in the oeFOXC2_ncLama4 group release enhanced LAMININ signals, which significantly activate downstream pathways in monocytes/macrophages (Mono/Mac), suggesting LAMA4‐mediated LAMININ signaling drives Mono/Mac differentiation into MAMs via specific receptor engagement. (H) Western blot analysis confirmed significant downregulation of GATA3 protein following siRNA‐mediated silencing, with concomitant reduction in TREM2 and CD206 expression. (I) Western blot analysis demonstrated that escalating LAMA4 concentrations (0, 1, 5, 25 ng/mL) induced progressive upregulation of immunosuppressive markers CD206 and TREM2 in macrophages. (J) Molecular docking of human and murine LAMA4 with ITGA6 reveals conserved binding capacity. Structures depict ITGA6 (cyan cartoon; extracellular domain in yellow) and LAMA4 (blue cartoon). (K) Comparative analysis of Itga6 expression in Mono/Mac and MAM populations within lung metastases revealed significantly elevated levels in oeFoxc2_ncLama4 vs. oeFoxc2_kdLama4 cohorts, suggesting enhanced responsiveness to LAMA4‐mediated signaling. (L) Validation of the interaction between LAMA4 and ITGA6. Lysates from co‐cultures of 786‐O renal carcinoma cells and THP‐1 macrophages were subjected to Co‐IP using an anti‐LAMA4 antibody, followed by immunoblotting with an anti‐ITGA6 antibody to confirm their direct binding.(M) FOXC2 knockdown attenuates the LAMA4‐ITGA6 interaction. Co‐IP was performed on lysates from co‐cultures of THP‐1 macrophages with 786‐O cells stably expressing either sh‐Ctrl or sh‐FOXC2, using an anti‐LAMA4 antibody. Western blot analysis for ITGA6 shows reduced complex formation upon FOXC2 knockdown. (N) STAT6‐neutralizing antibody (anti‐ ITGA6 Ab, 5 µg/mL) treatment significantly blocked LAMA4 (25 ng/mL)‐induced upregulation of p‐STAT6, GATA3, TREM2, and CD206 proteins in Western blot analysis. (O) In vivo imaging of orthotopic Renca‐luciferase Foxc2‐overexpressing (Renca‐luc Foxc2 oe ) renal tumors in BALB/c mice treated with ITGA6‐neutralizing antibody (10 mg/kg, i.v., weekly) vs. Rat‐IgG control, showing differential tumor progression and metastatic burden at days 7, 14, and 28 post‐implantations.

Article Snippet: THP‐1 monocytes were differentiated into M0 macrophages by treatment with 100 ng/mL phorbol 12‐myristate 13‐acetate (PMA; Selleck, S7791) for 48 h. The differentiated macrophages were then stimulated for 24 h with 25 ng/mL recombinant human LAMA4 (Origene, TP761994).

Techniques: Binding Assay, Phospho-proteomics, Gene Expression, Control, Expressing, Construct, Derivative Assay, Activity Assay, Western Blot, Biomarker Discovery, Co-Immunoprecipitation Assay, Knockdown, Stable Transfection, In Vivo Imaging, Luciferase

Journal: Advanced Science

Article Title: The FOXC2‐LAMA4 Axis Orchestrates Vasculogenic Mimicry and Immunosuppressive Niche Formation to Drive Metastatic Cascade in Renal Cell Carcinoma

doi: 10.1002/advs.202516382

Figure Lengend Snippet: Multi‐omics sequencing identifies LAMA4 as a key downstream effector of FOXC2. (A) Genome‐wide binding signal heatmap of FOXC2 CUT&Tag sequencing in 786‐O cells (FOXC2 vs. IgG). (B) Distribution of FOXC2 binding peaks across the genome and motif analysis of its promoter‐bound sequences in 786‐O cells. (C) Venn diagram intersecting differentially expressed genes (log2FC > 1) upon FOXC2 knockdown, FOXC2‐bound downstream genes, and Epithelial_s4 subpopulation‐enriched genes, identifying LAMA4 and VAV3 as potential FOXC2 transcriptional targets. (D) GO/KEGG functional enrichment analysis of genes with FOXC2 binding peaks from CUT&Tag sequencing. (E) GO/KEGG functional enrichment analysis of RNA‐seq differentially expressed genes in FOXC2 knockdown vs. control groups. (F) CUT&Tag sequencing reveals a specific FOXC2 binding site (highlighted in dark yellow) within the upstream promoter region of the LAMA4 gene. (G) RNA‐seq data from 786‐O cells validate LAMA4 as a FOXC2‐regulated differentially expressed gene. (H) TCGA‐KIRC dataset analysis demonstrates a significant positive correlation between FOXC2 and LAMA4 mRNA expression (R = 0.527, ** p < 0.01). (I)–(K) scRNA data show significant co‐expression of FOXC2 and LAMA4 in the Epithelial_s4 subpopulation. (L) scRNA data from 12 ccRCC patients confirm a robust expression correlation between tumor cell‐derived FOXC2 and LAMA4 (R = 0.630, * p < 0.05). (M) Western blot analysis of FOXC2 and LAMA4 protein levels in paired tumor and adjacent normal tissues from ccRCC patients. (N) Positive correlation between FOXC2 and LAMA4 protein expression. Protein levels were quantified by western blot (n=9; * p < 0.05, ** p < 0.01). A significant correlation was found by Spearman analysis (R = 0.717, p = 0.037).

Article Snippet: In parallel, immature BMDMs were divided into three groups and treated for 24 h: an untreated control, a positive control stimulated with 25 ng/mL IL‐4 (SinoBiological, 51084‐MNAE), and an experimental group treated with 25 ng/mL LAMA4 (Origene, TP724250).

Techniques: Biomarker Discovery, Sequencing, Genome Wide, Binding Assay, Knockdown, Functional Assay, RNA Sequencing, Control, Expressing, Derivative Assay, Western Blot

Journal: Advanced Science

Article Title: The FOXC2‐LAMA4 Axis Orchestrates Vasculogenic Mimicry and Immunosuppressive Niche Formation to Drive Metastatic Cascade in Renal Cell Carcinoma

doi: 10.1002/advs.202516382

Figure Lengend Snippet: FOXC2 promotes VM in ccRCC cells by transcriptionally regulating LAMA4. (A) Western blot analysis of LAMA4 protein expression upon FOXC2 knockdown. (B) qPCR detection of LAMA4 mRNA levels following FOXC2 knockdown (n=3; ** p < 0.01, *** p < 0.001). (C) Western blot analysis of FOXC2, LAMA4, VE‑cadherin, and the EMT markers E‑cadherin, N‑cadherin, and Vimentin in FOXC2‑overexpressing (OE‐ FOXC2) and LAMA4‑knockdown (sh‐LAMA4) cell models. (D) Tube formation assays demonstrate that FOXC2 overexpression significantly enhances VM capacity in renal cancer cells, while LAMA4 knockdown partially reverses this effect (n=3; *** p < 0.001). (E) Transwell invasion assays reveal that FOXC2 overexpression promotes cancer cell invasion, which is suppressed by LAMA4 knockdown (n=3; ** p < 0.01, *** p < 0.001). (F) Wound healing assays confirm that FOXC2 overexpression increases cell migration, an effect attenuated by LAMA4 knockdown (n=3; *** p < 0.001). 0.001). (G) Statistical analysis was performed on the following metrics: tube formation (number of tubes, branches, junctions, meshes, and total length), wound healing (wound healing percentage), and Transwell (number of migrated cells). ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. (H) Luciferase reporter assays using LAMA4 promoter truncations transfected into renal cancer cells. FOXC2 regulates promoter activity across distinct LAMA4 promoter regions (n=3; ns p > 0.05, *** p < 0.001). (I) ChIP with FOXC2 antibody followed by qPCR amplification of LAMA4 promoter fragments (site1–site4) (n=3; ns p > 0.05, ** p < 0.01). (J) Luciferase assays comparing wild‐type and site2‐mutated LAMA4 promoter activity in renal cancer cells. FOXC2 selectively regulates wild‐type promoter activity (n=3; ns p > 0.05, *** p < 0.001).

Article Snippet: In parallel, immature BMDMs were divided into three groups and treated for 24 h: an untreated control, a positive control stimulated with 25 ng/mL IL‐4 (SinoBiological, 51084‐MNAE), and an experimental group treated with 25 ng/mL LAMA4 (Origene, TP724250).

Techniques: Western Blot, Expressing, Knockdown, Over Expression, Migration, Luciferase, Transfection, Activity Assay, Amplification

Journal: Advanced Science

Article Title: The FOXC2‐LAMA4 Axis Orchestrates Vasculogenic Mimicry and Immunosuppressive Niche Formation to Drive Metastatic Cascade in Renal Cell Carcinoma

doi: 10.1002/advs.202516382

Figure Lengend Snippet: FOXC2‐LAMA4 remodels the metastatic microenvironment by promoting TREM2 + CD206 + MAM polarization in pulmonary metastases. (A) Experimental design and grouping for orthotopic renal carcinoma xenografts. (B) In vivo imaging system dynamically monitors renal orthotopic tumor growth (days 7, 14, 21, 28) and lung metastasis formation (terminal imaging at day 28) across groups. (C) Comparison of renal tumor volumes at the experimental endpoint among groups. (D) Lung metastasis imaging and H&E staining validation in the Foxc2 overexpression group (Foxc2 OE Lama4 NC ) vs. Foxc2 overexpression + LAMA4 knockdown group (Foxc2 OE Lama4 KD ). (E) Dimensionality reduction and cell type annotation of scRNA data from lung tissues of Foxc2 OE Lama4 NC and Foxc2 OE Lama4 KD mice. (F) Heatmap of MAMs signature genes showing upregulated immunosuppressive markers (e.g., Trem2, Arg1) (log2FC >1, * p < 0.05). (G) Tissue preference analysis (R/oe score) highlights significant enrichment of MAMs (red arrow) in the Foxc2 OE Lama4 NC group. (H),(I) Flow cytometry analysis of the effect of LAMA4 on inducing immunosuppressive polarization. Shown is the MFI of CD206 (H) within the CD68 + population for human THP1‑derived macrophages and (I) within the CD11b + F4/80 + population for mouse BMDMs following treatment with LAMA4. Untreated cells and IL‑4‑treated cells served as negative and positive controls, respectively (n = 3, **** p < 0.0001). (J) scRNA data from ccRCC lung metastases show high LAMA4 expression and elevated Trem2 + macrophage markers (TREM2, C1QC, APOE, CD163) in GPNMB‐Hi and FOLR2‐Hi macrophages. (K) scRNA of murine lung metastases: Macrophages in the oeFoxc2 ncLama4 group exhibit higher M2 markers, while T cells display pronounced exhaustion markers. (L),(M) Flow cytometry analysis of immune cell phenotypes in lung metastases from the orthotopic kidney cancer model. (L) Proportion of exhausted (CD8a + PD‑1 + ) CD8 + T cells. (M) MFI of TREM2 on macrophages. Comparisons are between the FOXC2 OE LAMA4 NC and FOXC2 OE LAMA4 KD groups (n = 5). ( ** p < 0.01, *** p < 0.001).

Article Snippet: In parallel, immature BMDMs were divided into three groups and treated for 24 h: an untreated control, a positive control stimulated with 25 ng/mL IL‐4 (SinoBiological, 51084‐MNAE), and an experimental group treated with 25 ng/mL LAMA4 (Origene, TP724250).

Techniques: In Vivo Imaging, Imaging, Comparison, Staining, Biomarker Discovery, Over Expression, Knockdown, Flow Cytometry, Expressing

Journal: Advanced Science

Article Title: The FOXC2‐LAMA4 Axis Orchestrates Vasculogenic Mimicry and Immunosuppressive Niche Formation to Drive Metastatic Cascade in Renal Cell Carcinoma

doi: 10.1002/advs.202516382

Figure Lengend Snippet: LAMA4‐ITGA6 binding activates STAT6 phosphorylation to drive GATA3‐dependent TREM2 + CD206 + MAM polarization, promoting metastatic outgrowth. (A) Schematic representation of the experimental setup to investigate the role of LAMA4 in macrophage polarization. THP‐1 cells were treated with PMA to differentiate into macrophages, followed by stimulation with LAMA4 (5 ng/mL). (B) Volcano plot comparing gene expression profiles between control and LAMA4‐treated THP‐1 macrophages. Red dots indicate upregulated genes; blue dots indicate downregulated genes. GATA3 was highly expressed in the LAMA4 treatment group. (C) Heatmap showing DEGs between control and LAMA4‐treated THP‐1 macrophages. Upregulated and downregulated genes are represented in red and blue, respectively. CXCL2, CXCL1, and TNF (inflammatory factors) exhibited elevated expression in controls, whereas ARG1, GATA3, and HES3 (immunosuppressive markers) showed significant upregulation in LAMA4‐treated macrophages. (D) GSEA demonstrating LAMA4‐mediated upregulation of the Fatty Acid Metabolic signaling pathway and downregulation of the TNF/NF‐κB signaling signaling pathway. (E) Pseudotime trajectory constructed by Monocle revealed that MAMs are derived from monocytes/macrophages (Mono/Mac) in mouse lung metastatic niches. (F) SCENIC analysis revealed heightened Gata3 regulon activity score (Gata3 RAS) enriched in MAMs, with concurrent elevation of Gata3 activity in Mono/Mac from oeFOXC2_ncLama4 mice group. (G) Cell‐cell communication analysis indicated that tumor cells in the oeFOXC2_ncLama4 group release enhanced LAMININ signals, which significantly activate downstream pathways in monocytes/macrophages (Mono/Mac), suggesting LAMA4‐mediated LAMININ signaling drives Mono/Mac differentiation into MAMs via specific receptor engagement. (H) Western blot analysis confirmed significant downregulation of GATA3 protein following siRNA‐mediated silencing, with concomitant reduction in TREM2 and CD206 expression. (I) Western blot analysis demonstrated that escalating LAMA4 concentrations (0, 1, 5, 25 ng/mL) induced progressive upregulation of immunosuppressive markers CD206 and TREM2 in macrophages. (J) Molecular docking of human and murine LAMA4 with ITGA6 reveals conserved binding capacity. Structures depict ITGA6 (cyan cartoon; extracellular domain in yellow) and LAMA4 (blue cartoon). (K) Comparative analysis of Itga6 expression in Mono/Mac and MAM populations within lung metastases revealed significantly elevated levels in oeFoxc2_ncLama4 vs. oeFoxc2_kdLama4 cohorts, suggesting enhanced responsiveness to LAMA4‐mediated signaling. (L) Validation of the interaction between LAMA4 and ITGA6. Lysates from co‐cultures of 786‐O renal carcinoma cells and THP‐1 macrophages were subjected to Co‐IP using an anti‐LAMA4 antibody, followed by immunoblotting with an anti‐ITGA6 antibody to confirm their direct binding.(M) FOXC2 knockdown attenuates the LAMA4‐ITGA6 interaction. Co‐IP was performed on lysates from co‐cultures of THP‐1 macrophages with 786‐O cells stably expressing either sh‐Ctrl or sh‐FOXC2, using an anti‐LAMA4 antibody. Western blot analysis for ITGA6 shows reduced complex formation upon FOXC2 knockdown. (N) STAT6‐neutralizing antibody (anti‐ ITGA6 Ab, 5 µg/mL) treatment significantly blocked LAMA4 (25 ng/mL)‐induced upregulation of p‐STAT6, GATA3, TREM2, and CD206 proteins in Western blot analysis. (O) In vivo imaging of orthotopic Renca‐luciferase Foxc2‐overexpressing (Renca‐luc Foxc2 oe ) renal tumors in BALB/c mice treated with ITGA6‐neutralizing antibody (10 mg/kg, i.v., weekly) vs. Rat‐IgG control, showing differential tumor progression and metastatic burden at days 7, 14, and 28 post‐implantations.

Article Snippet: In parallel, immature BMDMs were divided into three groups and treated for 24 h: an untreated control, a positive control stimulated with 25 ng/mL IL‐4 (SinoBiological, 51084‐MNAE), and an experimental group treated with 25 ng/mL LAMA4 (Origene, TP724250).

Techniques: Binding Assay, Phospho-proteomics, Gene Expression, Control, Expressing, Construct, Derivative Assay, Activity Assay, Western Blot, Biomarker Discovery, Co-Immunoprecipitation Assay, Knockdown, Stable Transfection, In Vivo Imaging, Luciferase