Journal: Advanced Science

Article Title: CA9‐Targeted PET Imaging for Noninvasive Discrimination of Clear Cell Renal Cell Carcinoma and Associated Tumor Biological Features

doi: 10.1002/advs.76622

Figure Lengend Snippet: Association of CA9 expression with PBRM1 alterations, tumor cell stemness, and clinical outcome in ccRCC. (A) CNV profiles of tumor cells derived from single‐cell RNA sequencing data, with B cells shown as non‐malignant controls. (B) Odds ratios of mutation frequencies for commonly altered genes in ccRCC comparing CA9‐high and CA9‐low groups, assessed using logistic regression analysis. (C) Comparison of CA9 mRNA expression levels between PBRM1 wild‐type and PBRM1‐mutant ccRCC tumors. Data are presented as mean ± SEM. (Mann–Whitney U test, n = 449) (D) Odds ratios comparing the frequencies of PBRM1 loss‐of‐function (LOF) strong and LOF weak mutations between CA9‐high and CA9‐low ccRCC tumors, analyzed using logistic regression. (E) Differences in CA9 mRNA expression among ccRCC tumors harboring different PBRM1 mutation types. Data are presented as mean ± SEM. Kruskal‐Wallis test with Dunn's multiple comparisons test (n = 449). (F) Odds ratios comparing the frequencies of distinct PBRM1 LOF strong mutation subtypes between CA9‐high and CA9‐low ccRCC tumors, assessed using logistic regression. (G) Comparison of CA9 mRNA expression levels among ccRCC tumors with different PBRM1 LOF strong mutation subtypes. Data are presented as mean ± SEM. Kruskal‐Wallis test with Dunn's multiple comparisons test. (n = 449) (H) IHC H‐scores of PBRM1 in ccRCC tissues stratified by CA9 expression level (CA9‐high, n = 15; CA9‐low, n = 14). (I) Correlation analysis of CA9 and PBRM1 expression levels in ccRCC tissue specimens. Representative images from three independent experiments are shown. (J) Comparison of CA9 and PBRM1 protein abundance in the CPTAC proteomic dataset (PDC000127, n = 20). (K) CytoTRACE2‐derived stemness scores of tumor cells stratified by CA9 expression levels. (L) Comparison of CytoTRACE2 stemness scores between CA9‐high (n = 4365) and CA9‐low (n = 4800) tumor cells. Data are presented as mean ± SEM. (Mann‐Whitney U test) (M) Differential expression of stemness‐associated genes (CD44, ITGA4, and KLF4) between CA9‐high and CA9‐low tumor cells. (N, O) Progression‐free interval (PFI) (N) and disease‐specific survival (DSS) (O) of ccRCC patients from the TCGA‐KIRC cohort stratified by CA9 expression. Stratification criteria: For single‐cell analyses in panels A and K‐M, CA9‐high and CA9‐low tumor cells were defined by median CA9 expression, corresponding to the upper and lower 50%, respectively. For TCGA‐KIRC mutation analyses in panels B, D, and F, CA9‐high and CA9‐low tumors were defined as the upper and lower 25% of CA9 TPM expression among samples with available mutation data. For panel H, tissues were stratified by CA9 IHC H‐score using the upper and lower 50%. For panels N–O, patients were stratified by CA9 expression using the top 25% versus bottom 75%. * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies included CA9 (Abclonal, A13682), HIF‐1α (Proteintech, 20960‐1‐AP), HIF‐1β (Proteintech, 14105‐1‐AP), HIF‐2α (Abclonal, A7553), CD70 (Abclonal, A2032), and PBRM1 (Abclonal, A0334).

Techniques: Expressing, Derivative Assay, Single Cell, RNA Sequencing, Mutagenesis, Comparison, MANN-WHITNEY, Quantitative Proteomics

Journal: Diagnostics

Article Title: BAP1 and PBRM1 Loss Is Associated with Aggressive Clinicopathological Features in Clear Cell Renal Cell Carcinoma: Prognostic Implications in a 10-Year Surgical Cohort

doi: 10.3390/diagnostics16121933

Figure Lengend Snippet: Representative immunohistochemical staining (clear cell renal cell carcinoma, 20× magnification). ( A ) Hematoxylin and eosin (H&E) staining showing clear cell morphology. ( B ) BAP1 loss: complete absence of nuclear staining in tumor cells with retained expression in stromal cells and lymphocytes (internal positive controls). ( C ) PBRM1 preserved expression: diffuse and strong nuclear staining in tumor cells.

Article Snippet: PBRM1 immunostaining was performed using a rabbit polyclonal anti-PBRM1 antibody (Bethyl Laboratories, Inc., Montgomery, TX, USA; catalog no. A301-591A) at a dilution of 1:250, incubated for 32 min at 37 °C.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Diagnostics

Article Title: BAP1 and PBRM1 Loss Is Associated with Aggressive Clinicopathological Features in Clear Cell Renal Cell Carcinoma: Prognostic Implications in a 10-Year Surgical Cohort

doi: 10.3390/diagnostics16121933

Figure Lengend Snippet: Representative immunohistochemical staining (clear cell renal cell carcinoma, 20× magnification). ( A ) Hematoxylin and eosin (H&E) staining showing clear cell morphology. ( B ) BAP1 preserved expression: diffuse and strong nuclear staining in tumor cells. ( C ) PBRM1 loss: complete absence of nuclear staining in tumor cells with retained expression in stromal cells and lymphocytes (internal positive controls).

Article Snippet: PBRM1 immunostaining was performed using a rabbit polyclonal anti-PBRM1 antibody (Bethyl Laboratories, Inc., Montgomery, TX, USA; catalog no. A301-591A) at a dilution of 1:250, incubated for 32 min at 37 °C.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: bioRxiv

Article Title: Discrete genetic effects of VHL and PBRM1 inactivation co-operate to disrupt epithelial homeostasis and promote ccRCC

doi: 10.64898/2026.02.18.706657

Figure Lengend Snippet:

Article Snippet: Antigens were detected by overnight incubation at 4°C with primary antibodies diluted 1:1000 in Dako Antibody Diluent Solution (Agilent S3022): tdTomato – anti-RFP (Rockland 600-401-379, RRID:AB_2209751); PBRM1 – anti-PBRM1 (Proteintech 12563-1-AP, RRID:AB_2877865).

Techniques:

Journal: bioRxiv

Article Title: Discrete genetic effects of VHL and PBRM1 inactivation co-operate to disrupt epithelial homeostasis and promote ccRCC

doi: 10.64898/2026.02.18.706657

Figure Lengend Snippet: (a) Pseudo-bulked L2FC plotted against the negative logarithm of the p value (two-tailed Wald test with multiple testing correction by Benjamini-Hochberg method) for genes in VPKO Early versus VKO Early cells. Genes significantly (p < 0.01) and robustly (|L2FC| > 0.5) regulated are colored green. Candidate genes pertaining to lipid and sterol metabolism and epithelial morphogenesis and adhesion are labelled. (b) Gene Ontology terms for biological processes that are significantly over-represented among genes upregulated by Pbrm1 inactivation in Vhl -null cells. GO terms whose over-representation is significant (p < 0.01; one-sided Fisher’s exact test corrected by false discovery rate), and whose member genes together exhibit a net positive average L2FC in VPKO Early versus VKO Early cells are shown. Terms are ordered and tiles are colored by the average L2FC. (c) Pseudo-bulked L2FC in VPKO Early versus VKO Early cells plotted against changes in VPKO Late versus VKO Late cells showing that Pbrm1 -dependent regulation of genes is correlated between the two timepoints. (d) Pseudo-bulked L2FC plotted against the negative logarithm of the p value (two-tailed Wald test with multiple testing correction by Benjamini-Hochberg method) for genes in VPKO Late versus VPKO Early cells, showing that genes significantly regulated after Pbrm1 inactivation at the early timepoint (colored green) are mostly not regulated significantly over time (i.e., have either |L2FC| < 0.5 or p > 0.01 in the VPKO Late versus VPKO Early comparison). (e) Pseudo-bulked L2FC in VPKO Late versus VPKO Early cells plotted against changes in VKO Late versus VKO Early cells showing that time-dependent changes in gene expression are correlated in Vhl- null and Vhl/Pbrm1 -null cells. (f) Expression of sets of genes up- or downregulated over time in Vhl -null cells of any PT identity (‘Adaptive Up’ or ‘Adaptive Down’) in ConKO, VKO, and VPKO cells sequenced either 1-3 weeks (early) or 4-12 months (late) following recombination. Median values and inter-quartile ranges of the data are presented separately for cells from each mouse. Pairwise comparisons have been made between means of the median values for mice of different genotypes using two-tailed two-way ANOVA with multiple testing correction using the Benjamini-Hochberg method. Adaptive upregulation is maintained, and adaptive downregulation is partially accelerated following Pbrm1 inactivation in Vhl -null cells. (g) Pseudo-bulked L2FC in VPKO Early versus VKO Early cells plotted against changes in ConPKO Early versus ConKO Early cells, showing that Pbrm1 -dependent gene regulation is correlated in Vhl -null and Vhl -competent cells. (h) Pseudo-bulked L2FC in VKO Early versus ConKO Early cells plotted against changes in VPKO Early versus ConPKO Early cells, showing that Vhl -dependent gene regulation is correlated between Pbrm1 -competent and Pbrm1 -null cells. (c, e, g, h ) Spearman’s correlation coefficient (ρ) calculated for genes that exhibit significant regulation in either of the depicted comparisons. (i) Oil Red O staining in renal sections from ConKO, ConPKO, VKO, and VPKO mice harvested 4-12 months (late) following recombination, showing the accumulation of lipid droplets (stained red) in PT cells of ConPKO and VPKO mice. Scale bar, 100 μm. 40x magnification.

Article Snippet: Antigens were detected by overnight incubation at 4°C with primary antibodies diluted 1:1000 in Dako Antibody Diluent Solution (Agilent S3022): tdTomato – anti-RFP (Rockland 600-401-379, RRID:AB_2209751); PBRM1 – anti-PBRM1 (Proteintech 12563-1-AP, RRID:AB_2877865).

Techniques: Two Tailed Test, Comparison, Gene Expression, Expressing, Staining

Journal: bioRxiv

Article Title: Discrete genetic effects of VHL and PBRM1 inactivation co-operate to disrupt epithelial homeostasis and promote ccRCC

doi: 10.64898/2026.02.18.706657

Figure Lengend Snippet: (a) Pseudo-bulked L2FC in VPKO Late versus ConKO Late cells plotted against ‘predicted’ changes calculated as addition of the effects of Vhl inactivation (VKO Late vs ConKO Late) and early effects of Pbrm1 inactivation (VPKO Early vs VKO Early). Genes that exhibited significant regulation in either of the depicted comparisons are plotted. Genes whose regulation in VPKO Late versus ConKO Late cells exhibit significant (p < 0.01; two-sided Wald z-test) deviation from the additive effects of Vhl and Pbrm1 inactivation are colored green. Spearman’s correlation coefficient (ρ). (b) Cells from ConKO, VKO, and VPKO mice projected onto UMAP space and clusters defined for VPKO Late cells (left panel), and the proportion of cells in each cluster derived from mice of each of the genotypes and timepoints (right panel), showing differential occupancy of Clusters 12, 9, and 2 by VPKO Late cells. (c-e) Proportion of cells from VPKO Late mice or mice from other genotypes and timepoints in Clusters 12 (c) , 9 (d ), and 2 (e ). Pairwise comparisons by Wilcoxon test. Error bars indicate the median and the inter-quartile range. (f) Expression of candidate genes that are part of the integrated stress response (ISR), and expression score for an ISR gene module derived from Han et al., 2023, in cells from Clusters 9, 12, or other clusters, showing that Clusters 12 and 9 are both characterized by increased expression of ISR genes. (g) Proportion of cells with detected reads for candidate genes downregulated in cells from Cluster 2 compared to those in the remaining clusters, showing that Cluster 2 cells are characterized by reduced expression of several PT differentiation transcription factors and adhesion molecules.

Article Snippet: Antigens were detected by overnight incubation at 4°C with primary antibodies diluted 1:1000 in Dako Antibody Diluent Solution (Agilent S3022): tdTomato – anti-RFP (Rockland 600-401-379, RRID:AB_2209751); PBRM1 – anti-PBRM1 (Proteintech 12563-1-AP, RRID:AB_2877865).

Techniques: Derivative Assay, Expressing

Journal: bioRxiv

Article Title: Discrete genetic effects of VHL and PBRM1 inactivation co-operate to disrupt epithelial homeostasis and promote ccRCC

doi: 10.64898/2026.02.18.706657

Figure Lengend Snippet:

Article Snippet: Primary antibodies: tdTomato (Rockland 600-401-379, RRID:AB_2209751) and PBRM1 (Proteintech 12563-1-AP, RRID:AB_2877865) were diluted in blocking buffer (1:1,000).

Techniques:

Journal: bioRxiv

Article Title: Discrete genetic effects of VHL and PBRM1 inactivation co-operate to disrupt epithelial homeostasis and promote ccRCC

doi: 10.64898/2026.02.18.706657

Figure Lengend Snippet: (a) Pseudo-bulked L2FC plotted against the negative logarithm of the p value (two-tailed Wald test with multiple testing correction by Benjamini-Hochberg method) for genes in VPKO Early versus VKO Early cells. Genes significantly (p < 0.01) and robustly (|L2FC| > 0.5) regulated are colored green. Candidate genes pertaining to lipid and sterol metabolism and epithelial morphogenesis and adhesion are labelled. (b) Gene Ontology terms for biological processes that are significantly over-represented among genes upregulated by Pbrm1 inactivation in Vhl -null cells. GO terms whose over-representation is significant (p < 0.01; one-sided Fisher’s exact test corrected by false discovery rate), and whose member genes together exhibit a net positive average L2FC in VPKO Early versus VKO Early cells are shown. Terms are ordered and tiles are colored by the average L2FC. (c) Pseudo-bulked L2FC in VPKO Early versus VKO Early cells plotted against changes in VPKO Late versus VKO Late cells showing that Pbrm1 -dependent regulation of genes is correlated between the two timepoints. (d) Pseudo-bulked L2FC plotted against the negative logarithm of the p value (two-tailed Wald test with multiple testing correction by Benjamini-Hochberg method) for genes in VPKO Late versus VPKO Early cells, showing that genes significantly regulated after Pbrm1 inactivation at the early timepoint (colored green) are mostly not regulated significantly over time (i.e., have either |L2FC| < 0.5 or p > 0.01 in the VPKO Late versus VPKO Early comparison). (e) Pseudo-bulked L2FC in VPKO Late versus VPKO Early cells plotted against changes in VKO Late versus VKO Early cells showing that time-dependent changes in gene expression are correlated in Vhl- null and Vhl/Pbrm1 -null cells. (f) Expression of sets of genes up- or downregulated over time in Vhl -null cells of any PT identity (‘Adaptive Up’ or ‘Adaptive Down’) in ConKO, VKO, and VPKO cells sequenced either 1-3 weeks (early) or 4-12 months (late) following recombination. Median values and inter-quartile ranges of the data are presented separately for cells from each mouse. Pairwise comparisons have been made between means of the median values for mice of different genotypes using two-tailed two-way ANOVA with multiple testing correction using the Benjamini-Hochberg method. Adaptive upregulation is maintained, and adaptive downregulation is partially accelerated following Pbrm1 inactivation in Vhl -null cells. (g) Pseudo-bulked L2FC in VPKO Early versus VKO Early cells plotted against changes in ConPKO Early versus ConKO Early cells, showing that Pbrm1 -dependent gene regulation is correlated in Vhl -null and Vhl -competent cells. (h) Pseudo-bulked L2FC in VKO Early versus ConKO Early cells plotted against changes in VPKO Early versus ConPKO Early cells, showing that Vhl -dependent gene regulation is correlated between Pbrm1 -competent and Pbrm1 -null cells. (c, e, g, h ) Spearman’s correlation coefficient (ρ) calculated for genes that exhibit significant regulation in either of the depicted comparisons. (i) Oil Red O staining in renal sections from ConKO, ConPKO, VKO, and VPKO mice harvested 4-12 months (late) following recombination, showing the accumulation of lipid droplets (stained red) in PT cells of ConPKO and VPKO mice. Scale bar, 100 μm. 40x magnification.

Article Snippet: Primary antibodies: tdTomato (Rockland 600-401-379, RRID:AB_2209751) and PBRM1 (Proteintech 12563-1-AP, RRID:AB_2877865) were diluted in blocking buffer (1:1,000).

Techniques: Two Tailed Test, Comparison, Gene Expression, Expressing, Staining

Journal: bioRxiv

Article Title: Discrete genetic effects of VHL and PBRM1 inactivation co-operate to disrupt epithelial homeostasis and promote ccRCC

doi: 10.64898/2026.02.18.706657

Figure Lengend Snippet: (a) Pseudo-bulked L2FC in VPKO Late versus ConKO Late cells plotted against ‘predicted’ changes calculated as addition of the effects of Vhl inactivation (VKO Late vs ConKO Late) and early effects of Pbrm1 inactivation (VPKO Early vs VKO Early). Genes that exhibited significant regulation in either of the depicted comparisons are plotted. Genes whose regulation in VPKO Late versus ConKO Late cells exhibit significant (p < 0.01; two-sided Wald z-test) deviation from the additive effects of Vhl and Pbrm1 inactivation are colored green. Spearman’s correlation coefficient (ρ). (b) Cells from ConKO, VKO, and VPKO mice projected onto UMAP space and clusters defined for VPKO Late cells (left panel), and the proportion of cells in each cluster derived from mice of each of the genotypes and timepoints (right panel), showing differential occupancy of Clusters 12, 9, and 2 by VPKO Late cells. (c-e) Proportion of cells from VPKO Late mice or mice from other genotypes and timepoints in Clusters 12 (c) , 9 (d ), and 2 (e ). Pairwise comparisons by Wilcoxon test. Error bars indicate the median and the inter-quartile range. (f) Expression of candidate genes that are part of the integrated stress response (ISR), and expression score for an ISR gene module derived from Han et al., 2023, in cells from Clusters 9, 12, or other clusters, showing that Clusters 12 and 9 are both characterized by increased expression of ISR genes. (g) Proportion of cells with detected reads for candidate genes downregulated in cells from Cluster 2 compared to those in the remaining clusters, showing that Cluster 2 cells are characterized by reduced expression of several PT differentiation transcription factors and adhesion molecules.

Article Snippet: Primary antibodies: tdTomato (Rockland 600-401-379, RRID:AB_2209751) and PBRM1 (Proteintech 12563-1-AP, RRID:AB_2877865) were diluted in blocking buffer (1:1,000).

Techniques: Derivative Assay, Expressing