Fig 1.
Characterization of PBRM1 in ccRCC cells.
(A) Relative transcript expression of PBRM1 in ccRCC cell lines. (B) Protein levels of BRG1 and PBRM1 in ccRCC cell lines. (C) Characterization of deletion in exon 17 of PBRM1 gene in Caki2 cells. (D) Protein levels of PBRM1 in isogenic ccRCC cells (Caki1 with PBRM1 knockdown and Caki2 with PBRM1 re-expression). (D) BRG1 and PBRM1 blotting in BRG1 or PBRM1 immunoprecipitates of Caki2+Vector and Caki2+PBRM1.
Fig 2.
Presence of PBRM1 resulted in decreased proliferation rate of ccRCC cells.
Cellular proliferation rate was monitored in (A) Caki1 and (B) A498 with either control knockdown or PBRM1 knockdown, as well as (C) Caki2 and (D) A704 with either control vectors or PBRM1 re-expression vectors. Both cell lines were treated with ethanol or doxycycline to control for cell line variation or doxycycline effects. n = 8 independent biological replicate experiments. P < 0.0001 (****) (two-way ANOVA test) between PBRM1 re-expression cell lines treated with ethanol (EtOH) or Doxycycline (Dox) at day 11. Error bars represent s.e.m.
Fig 3.
Summary of differentially expressed genes in Caki2 cells upon PBRM1 reexpression.
(A) Hierarchical Cluster Dendrogram of Caki2+Vector and Caki2+PBRM1 samples sequenced. (B) GO Biological Processes enriched by genes upregulated upon PBRM1 reexpression. (C) GO Biological Processes enriched by genes downregulated upon PBRM1 reexpression.
Fig 4.
Downregulation of genes involved in cell proliferation upon PBRM1 re-expression.
(A) Validating relative expression of genes regulating cell cycle (identified in RNA_seq data) in Caki2+Vectorand Caki2+PBRM1 cells by qRTPCR. A designation of * = P < 0.05 (paired Student t-test). n = 3 independent biological replicate experiments. Error bars represent s.e.m. (B) Percentage of cells in different stages of cell cycle (G1, S and G2) determined by flow cytometric analysis. A designation of * = P < 0.05 (paired Student t-test). n = 3 independent biological replicate experiments. Error bars represent s.e.m.
Fig 5.
Alteration of metabolism upon PBRM1 re-expression.
(A) Relative expression of genes regulating hypoxic response in Caki2+Vectorand Caki2+PBRM1 cells subjected to normoxic and hypoxic (0.5% O2) conditions. A designation of * indicates p < 0.05 (Student t-test). n = 3 independent biological replicate experiments. Error bars represent s.e.m. (B) Validating relative expression of genes regulating primary metabolic processes (identified in RNA_seq data) in Caki2+Vectorand Caki2+PBRM1 cells by qRTPCR. (C) Determination of glucose uptake in Caki2+Vectorand Caki2+PBRM1 cells. A designation of **** indicates p < 0.0001 (paired Student t-test). n = 5 independent biological replicate experiments. Error bars represent s.e.m. (D) Immunoblot of IGFBP3, phosphorylated AKT and unphosphorylated AKT in Caki2+Vectorand Caki2+PBRM1 cells. (E) Semi-quantitative estimation of alteration of cholesteryl esters in Caki2+PBRM1 compared to Caki2-Ø. Bars depicted in blue reverse the differences observed during the progression from normal kidney epithelium to ccRCC and bars depicted in tan compound those differences. A designation of * = P < 0.05 (paired Student t-test). n = 3 independent biological replicate experiments. Error bars represent s.e.m.
Fig 6.
Alteration of cell-cell adhesion upon PBRM1-reexpression.
(A) Validating relative expression of genes regulating cell-cell adhesion (identified in RNA_seq data) in Caki2+Vectorand Caki2+PBRM1 cells by qRTPCR. A designation of * indicates p < 0.05 (Student t-test). n = 3 independent biological replicate experiments. Error bars represent s.e.m. (B) Phalloidin staining of F actin (red channel) and DAPI staining of nucleus (blue channel) in Caki2+Vectorand Caki2+PBRM1 cells. A designation of *** indicates p < 0.001 (Fisher’s exact t-test). n = 5 independent biological replicate experiments. Error bars represent s.e.m. (C) In vitro scratch assay illustrating cell migration 24 hours after scratch wound inflection. A designation of * indicates p < 0.05 (Student t-test). n = 8 independent biological replicate experiments. Error bars represent s.e.m.
Fig 7.
Comparative analysis of genes with differential expression in the presence of PBRM1 between ccRCC cells and ccRCC TCGA biospecimen.
(A) GO Biological Processes enriched by genes upregulated in TCGA ccRCC biospecimen with no PBRM1 mutation compared to ccRCC biospecimen with PBRM1 mutations. (B)Venn diagram illustrating the number of differentially expressed genes overlapping between the 2 datasets (Genes with differential expression in 1. Caki2+PBRM1 with respect to Caki2+Vector and 2. ccRCC biospecimen with no PBRM1 mutation with respect to biospecimen with PBRM1 mutations). Inset illustrates GO Biological Processes enriched by upregulated genes in both (1) PBRM1 reexpressed Caki2 cells and (2) TCGA ccRCC biospecimen with no PBRM1 mutation. (C) Box Plot showing the relative expression (log2 RSEM) of genes involved in cell adhesion in TCGA biospecimen.