human renal cancer cell lines  (ATCC)

Journal: iScience

Article Title: Versatile roles of annexin A4 in clear cell renal cell carcinoma: Impact on membrane repair, transcriptional signatures, and composition of the tumor microenvironment

doi: 10.1016/j.isci.2025.112198

Figure Lengend Snippet: ANXA4 is upregulated in kidney cancer cell lines by loss of VHL (A) Heatmaps present the expression of VHL and annexin family genes in kidney cancer derived cell lines included in the Cancer Cell Line Encyclopedia database. Z-scores indicate gene expressions relative to the entire cancer cell line database as a reference. Estimated loss of the p-arm of chromosome 3 (chr3p), mutations of the VHL gene, and prediction of oncogenicity (OncoKB) are indicated (colored box – positive, white box – negative for these features; crossed out box – no data available). A498 and 786-O cell lines (red label) were selected for further analysis of ANXA4 . (B) Proteome analysis of A498 and 786-O ctrl. and pVHL re-expression cell lines demonstrated the regulation of ANXA4 in a pVHL dependent manner (two replicates per cell line were analyzed). Heatmap shows relative log 2 fold changes (FC) of pVHL-loss over re-expression for annexin family proteins and marker proteins for pVHL dependent regulation (crossed out box – no ratio could be calculated). (C–E) Western blot analysis for ANXA4 in shRNA mediated A498 ANXA4 knockdown (KD) and 786-O ANXA4 KD or overexpression (oeANXA4) in RPTEC cell lines (TUBA was used as loading control). Scramble shRNAs or Luciferase (oeLUCI) were expressed as controls (ctrl.). Densitometry of western blots shows optical densities of three (D) or four (E) independent experiments. Arbitrary units (a.u.) were normalized to respective controls per experiment. (F–H) Analysis of cell growth curves revealed no differences in ANXA4 KD cell lines and only slight differences in ANXA4 overexpressing (OE) RPTECs (one representative experiment out of several independent experiments is shown; dots indicate mean values and error bars S.E.M. per time point; growth curves indicate fitted growth rates (nonlinear regression) and dotted line indicates ±95% confidence intervals). (I and J) Western blot analysis for ANXA4 in ANXA4 knockout (KO) and pVHL re-expression A498 cells (ANXA2, ANXA3, and TUBA were used as reference). CRISPR/Cas9 generated Ctrl.-1&-2 and KO-1&-2 indicate monoclonal cell lines using independent non-targeting or ANXA4 specific guide RNAs per cell line. Densitometry of western blots confirmed the downregulation of ANXA4 following pVHL re-expression (VHL+) in A498 cells (four independent replicates normalized to respective controls per experiment were analyzed). (K) Cell proliferation analysis by BrdU incorporation in ANXA4 KO and Ctrl. cell lines (12 replicates per genotype out of 4 independent experiments and 3 replicates per experiment; a.u. – arbitrary units). Dot plots show analyzed replicates (error bars indicate mean and S.E.M.; n.s. – not significant; ∗ p < 0.05; ∗∗ p < 0.01 and ∗∗∗∗ <0.0001).

Article Snippet: Human renal cancer cell lines A498 (white female donor, ATCC, HTB-44) and 786-O (white male donor, ATCC, CRL-1932) (both, WT and with VHL re-expression) as well as human renal proximal tubule epithelial cells (RPTEC/TERT1, ATCC, CRL-4031, male donor) were used and obtained as recently described.

Techniques: Expressing, Derivative Assay, Marker, Western Blot, shRNA, Knockdown, Over Expression, Control, Luciferase, Knock-Out, CRISPR, Generated, BrdU Incorporation Assay

Journal: iScience

Article Title: Versatile roles of annexin A4 in clear cell renal cell carcinoma: Impact on membrane repair, transcriptional signatures, and composition of the tumor microenvironment

doi: 10.1016/j.isci.2025.112198

Figure Lengend Snippet: Membrane stress regulates the subcellular localization of ANXA4 (A–J) Influx of Calcium ions induced by Ionomycin and membrane stress by hypoosmolar media or digitonin treatment initiates recruitment of ANXA4 to the nuclear membrane in A498 cells. Representative immunofluorescence images show the subcellular localization of ANXA4 (cells were co-stained by Phalloidin (F-Actin) and Hoechst (Nucleus)). Dashed boxes indicate regions of magnification. Dashed lines indicate positions of correlating line scans. Line scans (B, D, F, H, and J) show mean fluorescence intensities (MFI) as arbitrary units (a.u.) of ANXA4 and F-Actin (red arrows indicate cytoplasm membranes, and black arrows indicate nuclear membranes).

Article Snippet: Human renal cancer cell lines A498 (white female donor, ATCC, HTB-44) and 786-O (white male donor, ATCC, CRL-1932) (both, WT and with VHL re-expression) as well as human renal proximal tubule epithelial cells (RPTEC/TERT1, ATCC, CRL-4031, male donor) were used and obtained as recently described.

Techniques: Membrane, Immunofluorescence, Staining, Fluorescence

Journal: iScience

Article Title: Versatile roles of annexin A4 in clear cell renal cell carcinoma: Impact on membrane repair, transcriptional signatures, and composition of the tumor microenvironment

doi: 10.1016/j.isci.2025.112198

Figure Lengend Snippet: Loss of ANXA4 results in impaired membrane repair in ccRCC cells (A) Representative fluorescence images show uptake and nuclear staining of Hoechst 33258 (membrane impermeable) in live cells over time after the induction of membrane damage by digitonin. (B) Quantification of one representative experiment for digitonin induced Hoechst 33258 uptake over 35 min ( ANXA4 KD and control A498 cells; dots indicate mean and S.E.M of 12 analyzed region of interests per genotype). (C) Quantification of digitonin induced Hoechst 33258 uptake into ANXA4 KD and control A498 cells for indicated time points ( N = 12 regions of interest per genotype, time point, and experiment and 3 independent experiments (total N = 36) were analyzed). (D–G) Analysis of uptake and nuclear staining of cells with Hoechst 33258 (membrane impermeable) after the induction of mechanical membrane damage by glass beads (or no-beads as negative control). Representative images (d&f) show cells stained with CMFDA cell tracker (green) and cells with damaged membranes stained with Hoechst 33258 (blue). Quantification of membrane damage in media with 2 mM Ca 2+ or without Ca 2+ ( N = 3) and ANXA4 KD or control A498 cells ( N = 5). Dots indicate single experiments. (H and I) ANXA4 localization analysis in ccRCC patients with progressive ( N = 9) or non-progressive ( N = 9) disease. Localization of ANXA4 was quantified into three classes – cytoplasm, nucleus, and plasma membrane (a four tier score was applied: 0 – no staining to 3 strong staining). Representative immunohistochemistry (IHC) images showing the subcellular localization of ANXA4 (brown) in ccRCCs of four representative patients. Nuclei were stained by hematoxylin (blue). All error bars indicate mean and S.E.M.; n.s. – not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Human renal cancer cell lines A498 (white female donor, ATCC, HTB-44) and 786-O (white male donor, ATCC, CRL-1932) (both, WT and with VHL re-expression) as well as human renal proximal tubule epithelial cells (RPTEC/TERT1, ATCC, CRL-4031, male donor) were used and obtained as recently described.

Techniques: Membrane, Fluorescence, Staining, Control, Negative Control, Clinical Proteomics, Immunohistochemistry

Journal: iScience

Article Title: Versatile roles of annexin A4 in clear cell renal cell carcinoma: Impact on membrane repair, transcriptional signatures, and composition of the tumor microenvironment

doi: 10.1016/j.isci.2025.112198

Figure Lengend Snippet: Transcriptome analysis of ANXA4 KO cells reveals altered matrisome and immune signaling (A) Principal component analysis (PCA) of transcriptome analysis (mRNA sequencing) of ANXA4 Ctrl. and KO A498 cell lines. Two replicates of Ctrl.-1&-2 (blue) and KO-1&-2 (red) CRISPR/Cas9 clones were analyzed. (B) Volcano plot of differential expression analysis shows 834 significantly regulated genes (adjusted p -value <0.05; red dots; FC – fold change). Highly altered genes suggest the regulation of extracellular matrix (e.g., LOXL2 , COL12A1 , TNC ), immune signaling (e.g., NFKBIA , IL34 , IL7R ) and epithelial–mesenchymal transition (e.g., ELF3 , CLDN1 , WNT5A ) related mechanisms. (C) Heatmap and clustering of z-scores of the most significant regulated genes (adjusted p -value <0.0001 and absolute log 2 fold change >1) demonstrated the robust regulation of these genes in all monoclonal cell lines and replicates. (D and E) Gene set enrichment analysis shows significant regulation (adj. p -value <0.01) of extracellular matrix, immune signaling, epithelial–mesenchymal transition, chromosomal regulation, and nuclear membrane associated GO-Terms (NES – normalized enrichment score of KO to WT; GS – gene set size). (F) Venn diagram of 834 significantly differentially expressed (DE) genes by loss of ANXA4 and annotation of the transcriptome to databases for immune (Immunome), extracellular matrix (Matrisome, including secreted factors) and epithelial–mesenchymal transition (EMTome) related genes. The Gene list shows the most significant DE genes (adjusted p -value <0.0001 and absolute log 2 fold change >1) annotated to at least one database (colored boxes and gene symbols indicate the annotation of genes to respective databases). (G) Transcription factor (TF) enrichment analysis indicates ELF3 as relevant for ANXA4-dependent regulated genes/gene sets.

Article Snippet: Human renal cancer cell lines A498 (white female donor, ATCC, HTB-44) and 786-O (white male donor, ATCC, CRL-1932) (both, WT and with VHL re-expression) as well as human renal proximal tubule epithelial cells (RPTEC/TERT1, ATCC, CRL-4031, male donor) were used and obtained as recently described.

Techniques: Sequencing, CRISPR, Clone Assay, Quantitative Proteomics, Membrane

Journal: iScience

Article Title: Versatile roles of annexin A4 in clear cell renal cell carcinoma: Impact on membrane repair, transcriptional signatures, and composition of the tumor microenvironment

doi: 10.1016/j.isci.2025.112198

Figure Lengend Snippet: ELF3 mediates ANXA4 associated phenotypes in ccRCC (A and B) Western blot analysis for ELF3 and CLDN1 confirms the upregulation of ELF3 and CLDN1 in ANXA4 KO cells (ANXA4 and TUBA are shown as reference). Densitometry of western blots shows optical densities (OD) as arbitrary units (a.u.). Three independent experiments were analyzed, and ODs were normalized to the mean OD of Ctrl-1&-2 per experiment. (C) Western blot analysis for ANXA4 and CLDN1 in ELF3 overexpression (oeLEF3, Myc-DDK tagged) or control (luciferase overexpression (oeLuci)) cell lines treated with TGF-β or control shows inverse regulation of ANXA4 and ELF3 in TGF-β treated cells. (D) Analysis of transwell migration of ANXA4 ctrl. and KO cells (Ctrl.-1&-2 and KO-1&2 cells were combined for statistical analysis; two replicates per genotype of four independent experiments were analyzed (total N = 8 per genotype); a.u. – arbitrary units relative to control). (E and F) Analysis of the cell invasion of ANXA4 ctrl. and KO cells (Ctrl.-1&-2 and KO-1&2 cells were combined for statistical analysis; four replicates per genotype out of four independent experiments were analyzed). Representative fluorescence images of DAPI stained cell nuclei show transmigrated (invasive) cells after 16 h (G) Analysis of transwell migration of ELF3 overexpression (oeLEF3, Myc-DDK tagged) or control (luciferase overexpression (oeLuci)) cells (three independent experiments and 4 replicates per genotype per experiments were analyzed; a.u. – arbitrary units relative to control). (H and I) Analysis of the cell invasion of Luciferase (control) and ELF3 overexpression A498 cells ( n = 3). Representative fluorescence images of DAPI stained cell nuclei show transmigrated (invasive) cells after 16 h (J and K) Gap closure assay for Luciferase (control) and ELF3 overexpression (oeELF3) A498 cells. Representative phase contrast images show gap width after 8 h. Quantification shows mean values (dots) of three independent experiments at indicated time points (gap width was normalized to initial gap width per experiment for statistical analysis). (I) Venn diagram of 4123 with ELF3 significantly co-expressed genes in the TCGA ccRCC cohort (adjusted p -value <0.0001) and annotation to databases of immune (Immunome), extracellular matrix (Matrisome, including secreted factors), and epithelial–mesenchymal transition (EMTome) related genes. Selected gene list shows secreted factors that are significantly co-expressed with ELF3 . Colored boxes indicate (co-) annotation of genes to respective databases. Dot plots show independent replicates analyzed (error bars indicate mean and S.E.M.; n.s. – not significant; ∗ p < 0.05; ∗∗ p < 0.01 and ∗∗∗ <0.001).

Article Snippet: Human renal cancer cell lines A498 (white female donor, ATCC, HTB-44) and 786-O (white male donor, ATCC, CRL-1932) (both, WT and with VHL re-expression) as well as human renal proximal tubule epithelial cells (RPTEC/TERT1, ATCC, CRL-4031, male donor) were used and obtained as recently described.

Techniques: Western Blot, Over Expression, Control, Luciferase, Migration, Fluorescence, Staining

Journal: iScience

Article Title: Versatile roles of annexin A4 in clear cell renal cell carcinoma: Impact on membrane repair, transcriptional signatures, and composition of the tumor microenvironment

doi: 10.1016/j.isci.2025.112198

Figure Lengend Snippet:

Article Snippet: Human renal cancer cell lines A498 (white female donor, ATCC, HTB-44) and 786-O (white male donor, ATCC, CRL-1932) (both, WT and with VHL re-expression) as well as human renal proximal tubule epithelial cells (RPTEC/TERT1, ATCC, CRL-4031, male donor) were used and obtained as recently described.

Techniques: Virus, Generated, Recombinant, Membrane, Electron Microscopy, Saline, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Western Blot, RNA Sequencing, Gene Expression, Imaging, Plasmid Preparation, shRNA, Control, Luciferase, Cloning, Expressing, Software, Cell Analysis, Microscopy, Cell Culture