caki 2  (ATCC)

Journal: bioRxiv

Article Title: PBRM1-VHL cooperation rewires lipid and iron metabolism to promote ferroptosis resistance in clear cell renal cell carcinoma

doi: 10.64898/2026.05.02.722429

Figure Lengend Snippet: (A) GPX4 CRISPR dependency (Chronos scores) across tumor lineages from DepMap Public 25Q3+. Individual points represent cell lines; distributions are displayed as violin plots. More negative scores indicate greater dependency on GPX4. Kidney lineage is highlighted in red. (B) Comparison of GPX4 dependency scores for kidney lineage cell lines ( n = 36) versus all other tumor lineages ( n = 822). Statistical significance was determined using a two-sided Wilcoxon rank-sum test (** P ≤ 0.01). (C) Heatmap summarizing median drug response (AUC) across tumor lineages from CTRP for the GPX4 inhibitors ML162, ML210, and RSL3. Lower AUC indicates greater sensitivity. (D) Distribution of CTRP AUC values for ML162, ML210, and RSL3 comparing kidney lineage cell lines ( n = 18) versus all other tumor lineages ( n = 822). Statistical significance was determined using a two-sided Wilcoxon rank-sum test (* P ≤ 0.05; ** P ≤ 0.01). (E) GPX4 dependency scores within kidney-derived lines, comparing renal cell carcinoma ( n = 29) versus non-RCC kidney cell lines ( n = 5). Statistical significance was determined using a two-sided Wilcoxon rank-sum test (* P ≤ 0.05). (F) Kidney cancer cell lines ranked by GPX4 CRISPR dependency score, color-coded by kidney subtype (immortalized embryonic kidney, renal cell carcinoma, renal clear cell carcinoma, renal medullary carcinoma, rhabdoid cancer). Caki-2 is highlighted in red. Dashed line indicates a dependency score of −1. (G) Dose-response viability curves following RSL3 treatment (16 h) in indicated cell lines from breast (MDA-MB-231, BT549), prostate (LNCaP, 22Rv1), brain (A172), kidney cancer (Caki-2, RCC4, SLR25), and normal kidney epithelial (HEK293T) origins. Viability is normalized to DMSO-treated controls. IC₅₀ values were derived from 4-parameter nonlinear regression on n = 3 biological replicates. IC₅₀ values are indicated. (H) Ferrostatin-1 (Fer-1; 1 µM) rescues RSL3 (0.33 µM)- and ML162 (0.33 µM)-induced cytotoxicity in Caki-2 cells following 18 h treatment. Viability is normalized to DMSO-treated controls. (G-H) Data are presented as mean ± SD ( n = 3 biological replicates). Statistical significance was assessed by unpaired two-sided Student’s t-test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).

Article Snippet: Caki-2, HEK293T, MDA-MB-231, BT-549, A172, LNCaP, and 22Rv1 cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: CRISPR, Comparison, Derivative Assay

Journal: bioRxiv

Article Title: PBRM1-VHL cooperation rewires lipid and iron metabolism to promote ferroptosis resistance in clear cell renal cell carcinoma

doi: 10.64898/2026.05.02.722429

Figure Lengend Snippet: (A) Mean GPX4 Chronos dependency scores (from DepMap) for renal carcinoma cell lines stratified by PBRM1 and VHL status. Values within each cell are the mean dependency score; n indicates the number of cell lines per genotype group. (B) Effect sizes for the association between genotype class and GPX4 dependency, summarized as point-biserial Pearson’s r with 95% confidence intervals from bootstrap resampling. (C) Immunoblot of PBRM1, VHL, and GPX4 in Caki-2 cells reconstituted with vector, PBRM1, VHL, or PBRM1+VHL. β-actin serves as a loading control. (D-F) Viability of Caki-2 (D), RCC4 (E), and SLR25 (F) reconstituted cells following 18 h treatment with RSL3 (0.33 µM for Caki-2 and SLR25; 0.11 µM for RCC4). Viability is normalized to DMSO-treated controls within each genotype. (G) Ferrostatin-1 (Fer-1; 1 µM) rescue of RSL3 (0.33 µM, 18 h)-induced cytotoxicity in Caki-2 reconstituted cells; viability is normalized to DMSO-treated controls within each genotype. (H) Viability of Caki-2 reconstituted cells following treatment with the independent GPX4 inhibitor ML162 (0.33 µM, 18 h), normalized to DMSO-treated controls. (I) Fer-1 (1 µM) rescue of ML162 (0.33 µM, 18 h)-induced cytotoxicity in Caki-2 reconstituted cells; viability is normalized to DMSO-treated controls within each genotype. (J) Immunoblot validation of CRISPR-mediated disruption of PBRM1 and/or VHL in HEK293T cells (sgControl, sgVHL, sgPBRM1, sgPBRM1+sgVHL). HIF1A accumulation in sgVHL and sgPBRM1+sgVHL lanes serves as a functional readout of VHL disruption. β-actin serves as a loading control; asterisk indicates the VHL band. (K) Viability of HEK293T CRISPR-edited cells following RSL3 treatment (0.33 µM, 18 h), normalized to DMSO-treated controls. (L) Immunoblot validation of HIF1A knockdown by independent shRNAs in Caki-2 vector and PBRM1-reconstituted cells; PCNA serves as a loading control. (M) Viability following GPX4 inhibition (RSL3; 0.33 µM, 18 h) in Caki-2 vector and PBRM1-reconstituted cells with HIF1A knockdown, normalized to DMSO-treated controls. (D-I, K, M) Data are presented as mean ± SD with individual replicates shown ( n = 3 biological replicates). Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple-comparisons test using the indicated reference group (vector for D-I, K; shScramble within each genotype for M). * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: Caki-2, HEK293T, MDA-MB-231, BT-549, A172, LNCaP, and 22Rv1 cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Western Blot, Plasmid Preparation, Control, Biomarker Discovery, CRISPR, Disruption, Functional Assay, Knockdown, Inhibition

Journal: bioRxiv

Article Title: PBRM1-VHL cooperation rewires lipid and iron metabolism to promote ferroptosis resistance in clear cell renal cell carcinoma

doi: 10.64898/2026.05.02.722429

Figure Lengend Snippet: (A) Representative flow cytometry histograms and quantification of BODIPY 581/591 C11 oxidation ratio (oxidized/reduced) in Caki-2 cells expressing vector, PBRM1, VHL, or PBRM1+VHL treated with DMSO or RSL3 (0.33 µM) for 12 hours. (B) As in (A), BODIPY 581/591 C11 oxidation ratio in RCC4 cells reconstituted cells treated DMSO or RSL3 (0.11 µM) for 12 hours. (C) BODIPY 581/591 C11 oxidation ratio in CRISPR-edited HEK293T cells (sgControl, sgPBRM1, sgVHL, sgPBRM1+sgVHL) treated with DMSO or RSL3 (0.33 µM) for 8 hours. (D-E) Representative histograms and mean fluorescence intensity (MFI) quantification of Liperfluo staining in Caki-2 (D) and RCC4 (E) cells following RSL3 treatment as in (A-B). (F) Liperfluo fluorescence as mean fluorescence intensity in CRISPR-edited HEK293T cells following RSL3 treatment as in (C). (G) Representative flow cytometry histograms of BODIPY 581/591 C11 oxidation in Caki-2 reconstituted cells treated with RSL3 (0.33 µM) ± ferrostatin-1 (Fer-1; 1 µM) for 8 h. (H) Schematic illustrating the relationship between the NAD⁺/NADH pool, NADP⁺/NADPH generation via NAD kinase, GSH/GSSG redox cycling, and GPX4-dependent detoxification of lipid peroxides. GSTs: Glutathione S-transferases; Prdxs: Peroxiredoxins. (I) Glutathione redox status (GSH/GSSG ratio) in Caki-2 cells treated with DMSO or RSL3 (0.33 µM) for 8 hours, quantified using the GSH/GSSG-Glo™ Assay. (J) Total NAD (NAD⁺ + NADH) in Caki-2 cells treated with DMSO or RSL3 (0.33 µM) for 8 hours, quantified using the NAD/NADH-Glo™ Assay. Data are presented as mean ± SD with individual replicates shown ( n = 3 biologically independent replicates for panels A, J, I; n = 4 for panels B, C; n = 5 for panels I; n = 6 for panels (D, E, F). For panels comparing ≥3 genotype groups, statistical significance was assessed by one-way ANOVA with Dunnett’s multiple-comparisons test using vector as the reference group. For panels I and J, comparisons between DMSO and RSL3 within each genotype were assessed by unpaired two-sided Student’s t -test. Exact P -values are shown in the figure for comparisons that did not reach the threshold for asterisk annotation. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: Caki-2, HEK293T, MDA-MB-231, BT-549, A172, LNCaP, and 22Rv1 cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Flow Cytometry, Expressing, Plasmid Preparation, CRISPR, Fluorescence, Staining, Glo Assay

Journal: bioRxiv

Article Title: PBRM1-VHL cooperation rewires lipid and iron metabolism to promote ferroptosis resistance in clear cell renal cell carcinoma

doi: 10.64898/2026.05.02.722429

Figure Lengend Snippet: (A) Volcano plots of differentially expressed genes (DEGs) in Caki-2 cells expressing vector (Empty), PBRM1, VHL, or PBRM1+VHL following RSL3 treatment (0.33 μM, 4 h; T1). The number of statistically significant DEGs (adjusted P < 0.05) is indicated for each genotype. (B) Gene Set Enrichment Analysis (GSEA) of the GO Biological Process ferroptosis gene set (GO: BP FERROPTOSIS) comparing RSL3 versus DMSO at T2 (10 h) across all four genotypes. Enrichment scores, normalized enrichment scores (NES), nominal P -values, and adjusted P -values are summarized in the accompanying table. (C) Scatter plots of RSL3-induced log2 fold-changes at T2 in PBRM1-, VHL-, and PBRM1+VHL-reconstituted cells compared to Empty vector cells, with Pearson correlation coefficients ( r ) indicated. Each point represents an individual gene. (D) Principal component analysis (PCA) of T2 transcriptomes across all genotypes and treatment conditions (DMSO and RSL3). Each point represents an individual sample; percentage of variance explained by each principal component is indicated on the respective axis. (E) Scatter plots comparing genotype-versus-Empty log2 fold-changes under DMSO and RSL3 conditions at T2 for PBRM1, VHL, and PBRM1+VHL reconstituted cells, with Pearson correlation coefficients ( r ) indicated. Each point represents an individual gene. (F) Scatter plot comparing PBRM1-versus-Empty and VHL-versus-Empty log2 fold-changes at T2 under DMSO conditions. Points are colored by genotype-specificity classification (PBRM1-specific, VHL-specific, or shared). Global Pearson r and shared-gene Pearson r are indicated. (G) Epistasis/additive analysis of combined PBRM1 and VHL transcriptional effects at T2 under DMSO conditions. The predicted log2 fold-change for the PBRM1+VHL state was calculated as the mean of individual PBRM1 and VHL log2 fold-changes and plotted against the observed PBRM1+VHL log2 fold-change. Points are colored by interaction class additive, buffering, or synergy (threshold = 1). Pearson r = 0.78, P < 2.2 × 10⁻¹⁶. (H) Hallmark gene set enrichment analysis (GSEA) bubble plot summarizing pathway-level transcriptional differences for each reconstituted genotype relative to Empty vector under DMSO treatment at T2. Bubble size represents -log10 (adjusted P ) and color represents NES. Only pathways with adjusted P < 0.05 are shown. (I) Pairwise correlation matrix of Hallmark NES profiles between genotypes (PBRM1, VHL, PBRM1+VHL) under DMSO conditions at T2. Color intensity reflects Pearson correlation coefficient. (J) Over-representation analysis (ORA) using EnrichR of ferroptosis-related gene sets from KEGG 2021 Human and WikiPathways 2019 Human databases for each reconstituted genotype under DMSO condition at T2. Bubble size represents -log10(adjusted P value) and fill color represents combined enrichment score. (K) Heatmap of log2 fold-changes for leading-edge ferroptosis-associated genes identified in (J), across genotypes of DMSO conditions at T2.

Article Snippet: Caki-2, HEK293T, MDA-MB-231, BT-549, A172, LNCaP, and 22Rv1 cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: PBRM1-VHL cooperation rewires lipid and iron metabolism to promote ferroptosis resistance in clear cell renal cell carcinoma

doi: 10.64898/2026.05.02.722429

Figure Lengend Snippet: (A-B) Volcano plots of DEGs in Caki-2 cells expressing vector (Empty), PBRM1, VHL, or PBRM1+VHL under DMSO (A) or RSL3 (0.33 μM, 10 h; T2) (B) conditions. Statistically significant DEG counts (adjusted P < 0.05) are indicated for each genotype. (C) Hallmark GSEA bubble plot summarizing pathway-level transcriptional differences for each reconstituted genotype relative to Empty vector under RSL3 treatment at T2. Bubble size represents −log10(adjusted P ) and color represents NES. Only pathways with adjusted P < 0.05 are shown. (D) GSEA enrichment of two partial EMT gene sets — Type 2 (fibrotic/stiffness-related) and Type 3 (metastatic/migratory) — for each reconstituted genotype relative to Empty vector under DMSO conditions at T2. Bubble size represents −log10(adjusted P ) and color represents NES. (E) Heatmap of log2 fold-changes for leading-edge genes driving Type 2 fibrotic EMT enrichment across PBRM1, VHL, and PBRM1+VHL reconstituted genotypes relative to Empty vector under DMSO conditions at T2. Color scale represents log2 fold-change relative to Empty vector. (F) Distribution of average log2 fold-changes for the full Type 2 fibrotic EMT gene set across PBRM1, VHL, and PBRM1+VHL reconstituted genotypes relative to Empty vector under DMSO conditions at T2. Vertical lines indicate the mean.

Article Snippet: Caki-2, HEK293T, MDA-MB-231, BT-549, A172, LNCaP, and 22Rv1 cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: PBRM1-VHL cooperation rewires lipid and iron metabolism to promote ferroptosis resistance in clear cell renal cell carcinoma

doi: 10.64898/2026.05.02.722429

Figure Lengend Snippet: (A) GSEA bubble plot of the top enriched iron-related gene sets across PBRM1, VHL, and PBRM1+VHL reconstituted Caki-2 cells relative to Empty vector under DMSO conditions at T2. Gene sets were drawn from GO Biological Process, GO Molecular Function, Human Phenotype Ontology, WikiPathways, and Hallmark collections. Bubble size represents −log10( P value) and fill color represents NES. (B) Heatmap of log2 fold-changes for iron-handling genes across PBRM1, VHL, and PBRM1+VHL reconstituted cells relative to Empty vector under DMSO treatment at T2. Genes are grouped by functional iron-handling category. Color scale represents log2 fold-change. (C) Immunoblot of ferritin light chain (FTL) protein levels in Caki-2 cells expressing Empty vector, VHL, PBRM1, or PBRM1+VHL. PCNA serves as a loading control. (D) Representative flow cytometry histograms (YL1-A channel) and quantification of FerroOrange mean fluorescence intensity (MFI) in Caki-2 cells expressing Empty vector, PBRM1, VHL, or PBRM1+VHL, treated with DMSO or RSL3 (0.33 µM) for 8 h. MFI is normalized to Empty vector within each treatment condition. (E) Total cellular iron content (Fe²⁺ + Fe³⁺) in Caki-2 reconstituted cells under DMSO and RSL3 treatment (0.33 µM, 8 h), quantified by colorimetric iron assay. Values are normalized to Empty vector within each treatment condition. (F) Cell viability in Caki-2 reconstituted cells following RSL3 treatment (0.33 µM, 18 h) in the presence of vehicle (DMSO), ferrous iron supplementation, or the iron chelator deferoxamine (DFO) (100 µM). Viability is expressed relative to vehicle-treated controls within each genotype. Data are presented as mean ± SD with individual replicates shown ( n = 3 biologically independent replicates). Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple-comparisons test using Empty vector as the reference group. Exact P -values are shown in the figure for comparisons that did not reach the threshold for asterisk annotation. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: Caki-2, HEK293T, MDA-MB-231, BT-549, A172, LNCaP, and 22Rv1 cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Plasmid Preparation, Functional Assay, Western Blot, Expressing, Control, Flow Cytometry, Fluorescence, Iron Assay

Journal: bioRxiv

Article Title: PBRM1-VHL cooperation rewires lipid and iron metabolism to promote ferroptosis resistance in clear cell renal cell carcinoma

doi: 10.64898/2026.05.02.722429

Figure Lengend Snippet: (A) CUT&RUN genome browser tracks at the FTL locus (chr19:49,467,374–49,471,955) in Caki-2 cells. Tracks show H3K4me3, IgG, PBRM1, and PHF10 (PBAF-specific complex subunit) occupancy. Gene structure is shown below. (B) CUT&RUN genome browser tracks at the SLC40A1 locus (chr2:190,424,572–190,446,813) in Caki-2 cells. Tracks are displayed as in (A). (C) Immunoblot of ferritin heavy chain (FTH1) in Caki-2 cells expressing Empty vector, VHL, PBRM1, or PBRM1+VHL. PCNA serves as a loading control. (D) Immunoblot of transferrin receptor (TFRC) in Caki-2 cells expressing Empty vector, VHL, PBRM1, or PBRM1+VHL. PCNA serves as a loading control. (E) Representative flow cytometry histograms (YL1-A channel) and quantification of FerroOrange mean fluorescence intensity (MFI) in Caki-2 reconstituted cells treated with RSL3 (0.33 µM, 8 h). MFI is normalized to Empty vector. (F) Total cellular iron content (Fe²⁺ + Fe³⁺) in Caki-2 reconstituted cells under RSL3 treatment (0.33 µM, 8 h), quantified by colorimetric iron assay and normalized to Empty vector. (E-F) Data are presented as mean ± SD with individual replicates shown ( n = 3 biologically independent replicates). Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple-comparisons test using Empty vector as the reference group. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: Caki-2, HEK293T, MDA-MB-231, BT-549, A172, LNCaP, and 22Rv1 cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Western Blot, Expressing, Plasmid Preparation, Control, Flow Cytometry, Fluorescence, Iron Assay

Journal: bioRxiv

Article Title: PBRM1-VHL cooperation rewires lipid and iron metabolism to promote ferroptosis resistance in clear cell renal cell carcinoma

doi: 10.64898/2026.05.02.722429

Figure Lengend Snippet: (A) GSEA bubble plot of lipid metabolism-related gene sets from the MSigDB C2 curated and C5 ontology collections in PBRM1, VHL, and PBRM1+VHL reconstituted Caki-2 cells relative to Empty vector under DMSO conditions at T2. Bubble size represents −log10( P adj) and fill color represents NES. Only gene sets with adjusted P < 0.05 are shown. (B) Heatmap of log2 fold-changes for a curated panel of 119 lipid metabolism genes organized by functional category in PBRM1, VHL, and PBRM1+VHL reconstituted cells relative to Empty vector under DMSO conditions at T2. Color scale represents log2 fold-change relative to Empty vector. (C) Schematic of the integrative DepMap pharmacogenomic analysis pipeline. Basal gene expression (TPM) and ferroptosis inducer drug sensitivity (CTRP AUC; RSL3, ML162, ML210, erastin) were correlated by Pearson correlation for lipid metabolism differentially expressed genes and Z-scored across the full compound library. CRISPR gene effect scores (Chronos) were separately correlated with GPX4 inhibitor AUC and Z -scored across all tested genes. Both scores were used to prioritize candidate ferroptosis resistance and sensitizer genes. (D) Venn diagrams showing the intersection of three evidence layers for candidate resistance genes (left, red) and candidate sensitizer genes (right, blue): RNA-seq differential expression in reconstituted cells, expression-AUC pharmacogenomic correlation, and CRISPR dependency-AUC correlation. Numbers indicate genes falling within each overlap region. (E) Integrated summary of validated dual candidates meeting both expression and CRISPR thresholds. Left bars show the expression–AUC correlation Z -score (averaged across RSL3, ML162, ML210, erastin); middle bars show the CRISPR dependency–AUC correlation Z -score (averaged across RSL3, ML162, ML210); right heatmap shows log2 fold-changes from RNA-seq at T2. Genes are grouped into resistance (upper) and sensitizer (lower) categories and annotated by metabolic pathway. (F) Violin plots of the global low-to-high unsaturation lipid species ratio (double bonds ≤ 1 versus ≥ 2) across Empty vector, PBRM1, VHL, and PBRM1+VHL Caki-2 cells measured by untargeted lipidomic. Higher ratios indicate a more MUFA-enriched membrane lipid composition. (G) Violin plots of the average number of double bonds per phospholipid acyl chain across genotypes, as a continuous membrane unsaturation index. Lower values reflect a less peroxidation-prone membrane composition. (H) Low-to-high unsaturation ratio as in (F), resolved by phospholipid class (PC, PE, PI, PS, PG) across all four genotypes. (I) Violin plots of the PC/PE molar ratio across genotypes. An elevated PC/PE ratio in reconstituted cells is consistent with a relative reduction of PE species, which constitute the proximal substrates of ferroptotic lipid peroxidation. (J) Log2 fold-changes of individual lipid species in PBRM1, VHL, and PBRM1+VHL cells relative to Empty vector, shown across two independent lipidomics batches. Lipid abundances were log2-transformed and normalized within each batch by subtracting the mean log2 value of Empty vector samples, centering Empty at zero while preserving biological variability. Individual biological replicates are shown with batch identity indicated by point shape; genotype means are shown as mean ± SEM. (K) Donut charts of neutral lipid class composition for each genotype. Darker/teal segments represent the cholesteryl ester (CE) fraction; yellow/pale segments represent the triacylglycerol (TAG) fraction. The percentage of CE within total neutral lipids is indicated for each genotype. (L) Relative abundance of CE 18:1 and CE 18:2 species (left y-axis, bars) and the CE 18:1/CE 18:2 ratio (right y-axis, line) across Empty vector, PBRM1, VHL, and PBRM1+VHL cells. Elevated CE 18:1 relative to CE 18:2 in reconstituted cells reflects preferential accumulation of oleate-esterified cholesteryl species and a reduction in oxidizable linoleate-containing neutral lipids. (M) CE saturation state across renal carcinoma cell lines in DepMap, stratified by genotype class as in . CE saturation was calculated as the ratio of low-unsaturation to high-unsaturation CE species. Box plots display the median with interquartile range. (N) Cross-dataset validation of CE saturation state. CE saturation Z-scores derived from DepMap kidney cancer cell line lipidomics and in-house Caki-2 lipidomics are plotted side by side across genotypes. Concordance between datasets supports the generalizability of the CE 18:1-enriched neutral lipid phenotype in PBRM1/VHL-reconstituted cells. (F, G, I) Lipidomics data are from batch 2 ( n = 4 biological replicates per genotype, except VHL, n = 3; see Methods). Statistical significance was assessed by unpaired two-sided Welch’s t -test relative to Empty vector. (H) Lipidomics data from batch 2 as in (F, G, I); Welch’s t -test within each phospholipid class relative to Empty vector. (J) Data from both independent lipidomics batches (batch 1: n = 4 per genotype; batch 2: n = 4 per genotype except VHL, n = 3). Batch identity is shown by point shape; genotype means are mean ± SEM. Statistical comparisons were performed within each batch using Welch’s t -test on log₂-transformed values; batch-specific log₂ fold-change estimates were then combined using fixed-effect meta-analysis. (K, L) Lipidomics data from batch 2 as in (F, G, I). (L) Data are mean ± SEM; statistical significance was assessed by unpaired two-sided Welch’s t -test relative to Empty vector. (M, N) DepMap kidney cancer cell-line lipidomics data (publicly available); no new experimental replicates. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: Caki-2, HEK293T, MDA-MB-231, BT-549, A172, LNCaP, and 22Rv1 cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Plasmid Preparation, Functional Assay, Gene Expression, Drug discovery, CRISPR, RNA Sequencing, Quantitative Proteomics, Expressing, Membrane, Transformation Assay, Preserving, Biomarker Discovery, Derivative Assay

Journal: bioRxiv

Article Title: PBRM1-VHL cooperation rewires lipid and iron metabolism to promote ferroptosis resistance in clear cell renal cell carcinoma

doi: 10.64898/2026.05.02.722429

Figure Lengend Snippet: (A) Box plots of expression-AUC Z-scores for the 13 ferroptosis resistance candidate genes across DepMap cancer cell lines. For each gene, the Pearson correlation between basal expression and ferroptosis inducer AUC was Z-scored across all compounds in the CTRP library. Individual points represent correlations for RSL3 (red circles), ML162 (blue triangles), ML210 (purple squares), and erastin (green diamonds). Dotted lines indicate the threshold of Z = ±0.5 (adjusted P < 0.05). Positive Z-scores indicate that higher gene expression correlates with ferroptosis resistance across cell lines. (B) Box plots of expression-AUC Z-scores for the six ferroptosis sensitizer candidate genes, formatted as in (A). Negative Z-scores indicate that lower gene expression correlates with ferroptosis resistance across cell lines. (C) Box plots of CRISPR-AUC Z-scores for the 13 resistance candidate genes. For each gene, Pearson correlations between CRISPR gene effect scores (Chronos) and GPX4 inhibitor AUC (RSL3, ML162, ML210) were Z-scored across all genes. Individual points represent correlations for each drug as in (A). Dotted lines indicate the threshold of Z = ±0.3 (adjusted P < 0.05). Negative Z-scores indicate that gene loss is disproportionately lethal in ferroptosis-resistant cell lines, reflecting functional dependency of the resistant state on those genes. (D) Box plots of CRISPR-AUC Z-scores for the six sensitizer candidate genes, formatted as in (B). Positive Z-scores indicate that gene loss correlates with increased ferroptosis sensitivity, consistent with a role in sustaining the ferroptosis-susceptible state. (E) Scatter plot of expression-AUC correlation (r, x-axis) versus CRISPR dependency-AUC correlation (r, y-axis) for candidate genes relative to canonical ferroptosis regulators. Points are colored by category: anchor genes (green), resistance candidates (red), and sensitizer candidates (blue). GPX4 and ACSL4 serve as positive and negative reference anchor points for expected resistance and sensitivity signals, respectively. (F) Cell viability of Caki-2 reconstituted cells following co-treatment with RSL3 (0.33 µM, 18 h) and the SCD inhibitor CAY10566 (SCDi; 0.5 µM). Viability is normalized to DMSO-treated controls within each genotype. data are mean ± SD ( n = 3 biologically independent replicates). (G) CUT&RUN genome browser tracks at the SCD locus (chr10:102,105,143–102,126,035) in Caki-2 cells. Tracks show H3K4me3, IgG, PBRM1, and PHF10 (PBAF-specific complex subunit) occupancy. Gene structure is shown below. (H) CUT&RUN genome browser tracks at the SREBF1 locus (chr17:17,714,907–17,714,724) in Caki-2 cells. Tracks are displayed as in (G). (I) Species-level heatmap of the top remodeled phosphatidylcholine (PC) lipid species across individual biological replicates for each genotype (Empty, PBRM1, VHL, PBRM1+VHL). Each row represents one lipid species annotated at sum-composition level. Rows are colored by unsaturation category: green indicates low-unsaturation species (≤1 double bond; resistance-associated) and red indicates high-unsaturation species (≥2 double bonds; sensitivity-associated). Color scale represents log2 abundance relative to Empty vector mean within each batch. (J) Species-level heatmap of the top remodeled phosphatidylethanolamine (PE) lipid species across individual biological replicates, formatted as in (G). (K) TAG low-to-high unsaturation ratio (double bonds ≤ 1 versus ≥ 2) across Empty vector, PBRM1, VHL, and PBRM1+VHL cells. Data are from lipidomics batch 2 ( n = 4 per genotype, except VHL, n = 3; see legend and Methods). Individual replicates are shown with mean ± SEM. Statistical significance was assessed by unpaired two-sided Welch’s t -test relative to Empty vector. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: Caki-2, HEK293T, MDA-MB-231, BT-549, A172, LNCaP, and 22Rv1 cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Gene Expression, CRISPR, Functional Assay, Plasmid Preparation

Journal: Journal of Cannabis Research

Article Title: Design, synthesis, and biological profiling of fluorinated cannabidiol and cannabigerol derivatives as promising therapeutic agents

doi: 10.1186/s42238-026-00403-1

Figure Lengend Snippet: Inhibitory effects of CBD ( A ) and CBG derivatives ( B ) on human renal cancer cells. A-498 and CAKI-2 cell lines plated with 6 × 10 3 density were treated with 10, 30 and 60 µM of the examined compounds, respectively, and fluorescence intensities of living cells were measured to determine the cytotxic effect of the compounds. The intensity of treated samples was normalized to the untreated control samples. Value of 0.05 was set as the threshold for statistical significance. The figure represents mean ± SD

Article Snippet: Human renal carcinoma cell lines, A-498 and CAKI-2 obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% Fetal Bovine Serum (FBS) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) and maintained at 37 °C in a humidified atmosphere under 5% CO 2 /95% air.

Techniques: Fluorescence, Control