Fig. 1: Analysis of isogenic PBRM1 knockout (KO) cell lines identifies centromere associated protein misregulation as a common feature.

a Workflow for generation of PBRM1 knockouts in a panel of cell lines. Number of independent clones validated for each cell line is indicated at the bottom. b–f Characterisation of PBRM1 knockouts using the hTERT-RPE1 cell line as an example. b Western blotting of whole cell lysates from parental and PBRM1 KO cells for PBRM1. α-tubulin is used as a loading control. c Scaled abundances of PBRM1 in proteomic analyses of whole cell protein extracts. Points correspond to independent biological replicates (n = 2). d Proliferation of RPE1 parental and two PBRM1 KO clones, measured using phase contrast Incucyte images (n = 2). e Cell cycle distribution of RPE1 parental and two PBRM1 KO clones measured using flow cytometry. n = 4, mean ± SEM, data were non-significant (ns) based on a 2way ANOVA using Dunnett’s multiple comparisons test. f Immunofluorescence images of nuclear morphology in RPE1 parental and PBRM1 KO clones. Scale bar corresponds to 40 µm. g–i Protein abundances in PBRM1 knockouts compared to parental cells in hTERT-RPE1 (g), 1BR3-hTERT (h), and U2OS (i) cell lines, detected using LC-MS of whole cell protein extracts. The mean log2 fold change (Log2FC) of protein abundance in PBRM1 knockouts versus parental cells is plotted against the -log p value, calculated using two-sided one-sample t-test. PBRM1 is highlighted in red, while centromere- & pericentromere-associated proteins are highlighted in purple. j Schematic outlining regions of the centromere and pericentric heterochromatin, including the kinetochore in mitosis. k Median Log2FC of annotated centromere- & pericentromere-associated proteins in RPE1 PBRM1 knockouts compared to parental cells. l Transcript levels of annotated centromere- & pericentromere-associated genes corresponding to the proteins in k detected using RNA-seq. Median Log2FC of annotated genes transcribing centromere- & pericentromere-associated proteins in RPE1 PBRM1 knockouts was plotted compared to parental cells. Points in k and l correspond to individual knockout clones from two independent biological replicates. Source data are provided as a source data file.