p re ss human renal cancer cell lines 769 p  (ATCC)

Journal: Cell Death & Disease

Article Title: MAGI3 deficiency unleashes β-catenin conformational change to drive metastatic progression and mTOR inhibitor resistance in ccRCC

doi: 10.1038/s41419-026-08563-x

Figure Lengend Snippet: A Analysis of metastasis-related upregulated gene sets enriched in ccRCC specimens with low MAGI3 expression. Gene expression data from TCGA or GSE73731 were subjected to GSEA to identify pathways associated with metastasis. B Validation of stable MAGI3 overexpression or knockdown in 786-O and 769-P cells using western blotting. C Ectopic expression of MAGI3 suppresses migration in 786-O and 769-P cells. D Knockdown of MAGI3 promotes migration in 786-O and 769-P cells, demonstrated by in vitro scratch assay. E Overexpression of MAGI3 inhibits migration and invasion in 786-O and 769 cells. F Knockdown of MAGI3 promotes migration and invasion in 786-O and 769 cells, assessed by transwell assays. G Knockdown of MAGI3 expression promotes lung metastasis in a ccRCC xenograft model. 786-O/shCtrl or 786-O/shMAGI3 cells were injected into SCID/NOD mice ( n = 15) via tail vein. The mice were sacrificed after 3 months, and the number of pulmonary metastatic nodules was statistically analyzed.

Article Snippet: Human renal cancer cell lines 769-P (CLS Cat# 300106/p490_769-P, RRID: CVCL_1050), 786-O (RRID: CVCL_1051), and 293T (DSMZ Cat# ACC-305, RRID: CVCL_0045) were obtained from ATCC.

Techniques: Expressing, Gene Expression, Biomarker Discovery, Over Expression, Knockdown, Western Blot, Migration, In Vitro, Wound Healing Assay, Injection

Journal: Cell Death & Disease

Article Title: MAGI3 deficiency unleashes β-catenin conformational change to drive metastatic progression and mTOR inhibitor resistance in ccRCC

doi: 10.1038/s41419-026-08563-x

Figure Lengend Snippet: A – C MAGI3 exhibits a negative correlation with β-catenin protein level and Wnt signaling activation in ccRCC specimens. Low MAGI3 level was associated with Wnt signaling activation in ccRCC, as evidenced by enriched gene signatures of Wnt/β-catenin activation in patients with lower MAGI3 levels in TCGA KIRC and GSE73731 datasets ( A ). MAGI3 does not correlate with β-catenin at mRNA levels in clinical specimens ( B ). MAGI3 negatively correlates with β-catenin at protein levels in THPA KIRC datasets ( C ). D , E Overexpression of MAGI3 reduces β-catenin protein level, while knockdown of MAGI3 increased β-catenin protein level in 786-O or 769-P cells. Cells stably transfected with MAGI3 or Ctrl ( D ), or knockdown with shMAGI3 or shCtrl constructs ( E ), were subjected to western blotting analysis to measure the levels of β-catenin, MAGI3 and β-actin protein. F , G Overexpression of MAGI3 decreased the half-lives of β-catenin protein via its interaction with MAGI3. 293T cells were transiently transfected with Flag-β-catenin-wt ( F ) or Flag-β-catenin-T779A ( G ) in absence or presence of GFP-MAGI3 respectively, were treated with CHX (25 μg/mL) for the indicated time before cell harvest for western blotting. Band density was quantified using ImageJ software. Data represent three independent experiments ( n = 3). * p < 0.05; ns, no significant difference ( t test). H Overexpression of MAGI3 promotes β-catenin ubiquitination. I The interaction with β-catenin is essential for MAGI3 to promote β-catenin ubiquitination. 293T cells were transiently transfected with Flag-β-catenin-wt or Flag-β-catenin-Δ773-781 in absence or presence of His-MAGI3 respectively, were treated with MG132 for 10 h. Cell lysates were subjected to IP with anti-Flag antibody-coupled beads. The precipitated complexes were probed with anti-ubiquitin antibody to detect ubiquitinated Flag-β-catenin.

Article Snippet: Human renal cancer cell lines 769-P (CLS Cat# 300106/p490_769-P, RRID: CVCL_1050), 786-O (RRID: CVCL_1051), and 293T (DSMZ Cat# ACC-305, RRID: CVCL_0045) were obtained from ATCC.

Techniques: Activation Assay, Over Expression, Knockdown, Stable Transfection, Transfection, Construct, Western Blot, Software, Ubiquitin Proteomics

Journal: Cell Death & Disease

Article Title: MAGI3 deficiency unleashes β-catenin conformational change to drive metastatic progression and mTOR inhibitor resistance in ccRCC

doi: 10.1038/s41419-026-08563-x

Figure Lengend Snippet: A , B Overexpression MAGI3 enhanced β-catenin phosphorylation and reduces β-catenin protein level in 786-O or 769-P cells. Knockdown MAGI3 reduces β-catenin phosphorylation and increases β-catenin protein level in 786-O or 769-P cells. Cell stably transfected with MAGI3 or Ctrl ( A ), or knockdown with shMAGI3 or shCtrl ( B ) constructs respectively, were subjected to western blotting analysis. Phosphorylation of β-catenin at Ser33/Ser37/Thr41 was detected with anti-phospho-β-catenin (Ser33/Ser37/Thr41) antibody. C MAGI3 serves as a scaffold protein to facilitate the interaction between GSK3β and β-catenin. D The C-terminus of β-catenin plays an indispensable role in MAGI3-mediated enhancement of the association between GSK3β and β-catenin. 293T cells were transiently transfected with Flag-β-catenin-wt or Flag-β-catenin-Δ773-781 (Flag-β-cat-Δ773-781) along with HA-GSK3β in presence or absence of His-MAGI3. Lysates were precipitated with an anti-Flag antibody and blotted with anti-His, anti-Flag or anti-HA antibody respectively. E Knockdown of MAGI3 inhibits the endogenous interaction between GSK3β and β-catenin, and suppresses the ubiquitination of β-catenin. Cell lysates were subjected to IP with anti-β-catenin antibody-coupled beads. The precipitated complexes were probed with anti-β-catenin, anti-ubiquitin and anti-GSK3β antibody to detect ubiquitinated β-catenin and its interaction with GSK3β. F Intramolecular interaction of β-catenin N-tail (β-cat-N-tail) with its ARM (β-cat-ARM) domain forms a loop structure. β-catenin GST fusion proteins containing the indicated domains were utilized to pull down cell lysates from 293T cells transfected with Flag-β-cat-N-tail, and the pull downed complexes were detected by western blotting. G MAGI3 significantly reduces the binding of extra molecular Flag-β-cat-N-tail with β-catenin. H The C-terminus of β-catenin was indispensable for MAGI3-mediated reduction in the interaction of extra molecular Flag-β-cat-N-tail with β-catenin. β-catenin GST fusion proteins containing the indicated domains were used to pull down cell lysates from 293T transfected with Flag-β-cat-N-tail with or without His-MAGI3, and the pull downed complexes were detected by western blotting.

Article Snippet: Human renal cancer cell lines 769-P (CLS Cat# 300106/p490_769-P, RRID: CVCL_1050), 786-O (RRID: CVCL_1051), and 293T (DSMZ Cat# ACC-305, RRID: CVCL_0045) were obtained from ATCC.

Techniques: Over Expression, Phospho-proteomics, Knockdown, Stable Transfection, Transfection, Construct, Western Blot, Ubiquitin Proteomics, Binding Assay

Journal: Cell Death & Disease

Article Title: MAGI3 deficiency unleashes β-catenin conformational change to drive metastatic progression and mTOR inhibitor resistance in ccRCC

doi: 10.1038/s41419-026-08563-x

Figure Lengend Snippet: A , B IHC analysis of MAGI3 and β-catenin expression in ccRCC specimens from 119 patients. Representative images of MAGI3 and β-catenin staining in patients with low and high MAGI3 expression levels. Scale bars: 200 μm. Magnified views of the wireframe regions are shown in the right panels. Scale bars: 50 μm. C , D Knockdown of β-catenin rescues the increased cell migration and invasion induced by MAGI3 knockdown. 786-O and 769-P cells were transfected with shMAGI3 alone, or in combination with β-catenin siRNA. Migration and invasion were assessed using transwell assays. E , F Blocking Wnt/β-catenin signaling rescues the increased cell migration and invasion induced by MAGI3 knockdown. 786-O and 769-P cells were transfected with shMAGI3 alone, or in combination with IWR-1-endo, an inhibitor of the Wnt/β-catenin pathway. Migration and invasion were assessed using transwell assays ( t test; * p < 0.05; ** p < 0.01; *** p < 0.001; ns, no significant difference; values represent mean ± SD, n = 3).

Article Snippet: Human renal cancer cell lines 769-P (CLS Cat# 300106/p490_769-P, RRID: CVCL_1050), 786-O (RRID: CVCL_1051), and 293T (DSMZ Cat# ACC-305, RRID: CVCL_0045) were obtained from ATCC.

Techniques: Expressing, Staining, Knockdown, Migration, Transfection, Blocking Assay

Journal: Cell Death & Disease

Article Title: MAGI3 deficiency unleashes β-catenin conformational change to drive metastatic progression and mTOR inhibitor resistance in ccRCC

doi: 10.1038/s41419-026-08563-x

Figure Lengend Snippet: A Knockdown of MAGI3 increased the IC 50 of Everolimus in 786-O and 769-P cells. Dose-response survival curves of MAGI3-knockdown 786-O and 769-P cells exposed to increasing concentrations of Everolimus for 48 h. Mean ± SD; n = 3; two-way ANOVA. B The reduced drug sensitivity caused by MAGI3 knockdown is restored by treatment with XAV-939, an inhibitor of the Wnt/β-catenin pathway. Cell viability of 786-O and 769-P cells was determined using CCK-8 assays. C , D MAGI3 knockdown promotes clonogenic formation in Everolimus-treated 786-O and 769-P cells, while concomitant treatment with XAV-939 and Everolimus markedly inhibits clonogenic formation. Colony formation assays were conducted in 786-O and 769-P cells treated with Everolimus in the absence or presence of XAV-939 for 14 days. Quantification analysis of clone formation assays. E Combination treatment with XAV939 and Everolimus synergistically inhibits cell invasion of ccRCC. Transwell invasion assays were conducted in 786-O and 769-P cells treated with Everolimus in the absence or presence of XAV-939 for 24 h. ( t test; * p < 0.05; ** p < 0.01; *** p < 0.001; ns, no significant difference; values represent mean ± SD, n = 3).

Article Snippet: Human renal cancer cell lines 769-P (CLS Cat# 300106/p490_769-P, RRID: CVCL_1050), 786-O (RRID: CVCL_1051), and 293T (DSMZ Cat# ACC-305, RRID: CVCL_0045) were obtained from ATCC.

Techniques: Knockdown, CCK-8 Assay

Journal: bioRxiv

Article Title: Fibroblast-derived Collagen VI shapes the structure and function of the tumor-immune microenvironment in clear cell renal cell carcinoma

doi: 10.64898/2026.03.19.712351

Figure Lengend Snippet: (A) Schematic depicting cancer cell reseeding on TK173-synthesized CDMs and further functional workup (Created in BioRender. Schell, C. (2026) https://BioRender.com/x5sqb3y ). (B) Volcano plot of RNA sequencing analysis depicting differentially expressed genes of A498 cancer cells after reseeding on TK173-synthesized CDMs (COL6-containing CDM or depleted CDM; red dots indicate significantly regulated genes with adjusted p<0.05 and log 2 fold change (FC) > |0.5|). (C) Gene ontology (GO) gene set enrichment analysis of GO-terms of the molecular function category. (D) Volcano plot analysis for filtered matrisome genes. (E) IF staining for Nucleus (blue), F-actin (white), FN (green) and COL6 (violet) of A498 cells reseeded on shCTRL- and shCOL6-CDMs. (F) Scanning electron microscopy of A498 cells interacting with CDMs (yellow dotted boxes highlight areas of zoom in). (G&H) Morphometric analysis of reseeded cells (cell area and perimeter; Violin plots of individual cells of N=3 independent experiments, Mann-WhitneyLULtest). (I) Quantification of confluence of A498 cells growing on shCTRL- and shCOL6-CDMs (N=4 independent experiments, unpaired t test). (J&K) Representative phase-contrast images with DNA staining (blue) of A498 cells after cultivation for 3 days. Dot plot depicting quantification of A498 cells per cm² grown on CDMs (N=4 independent experiments, unpaired t test). (L&M) Representative IF staining of ccRCC patient samples for DNA (blue), PAX8 (red), KI-67 (green) and COL6 (violet) was used to classify and mask (bottom image) for proliferating tumor cells (orange), non-proliferative tumor cells (grey), as well as COL6 (violet). Violin plot depicting quantification of the distance of non-proliferative and proliferating tumor cells to COL6 (Mann-WhitneyLULtest). (N) Heatmap depicting Spearman’s correlation analysis of indicated proliferative cell state marker genes with COL6 , SERPINE1 and SERPINE2 genes in the ccRCC TCGA and CPTAC cohorts. (O&P) Spatial transcriptomics analysis of ccRCCs for spatial co-occurrence of COL6 , SERPINE1 , SERPINE2 and proliferative cell state marker genes. (O) Exemplary spatial mapping of the co-occurrence score on one sample (PD47512) and spatial mapping for the COL6, SERPINE and proliferative gene scores. (P) Correlation matrix of the spatial correlation between the COL6, SERPINE and proliferative gene sets (N=5 patients, Spearman’s correlation). Bars indicate mean and S.E.M in dot plots or median and quartiles in violin plots; ∗ – p < 0.05, ∗∗ – p < 0.01 and ∗∗∗∗ – p < 0.0001.

Article Snippet: Human renal cancer cell lines A498 (HTB-44, ATCC,) and 786-O (CRL-1932, ATCC) as well as human renal fibroblasts TK173 (RRID: CVCL_C8FA) and human renal proximal tubule epithelial cells (RPTEC/TERT1, ATCC, CRL-4031) were used and obtained as recently described .

Techniques: Synthesized, Functional Assay, RNA Sequencing, Staining, Electron Microscopy, Marker, Spatial Transcriptomics