Fig 2: Western blot analysis showing the expression of BMI1, RING1A, RING1B, EZH2, ?-H2AX, Ub-H2A, p-CHK2, p-ATM, RNF8, RNF168, MEL18, p53BP, BRCA1 proteins in MDAMB-231cells transfected with miR-15a, miR-16 or both miR-15a/16 under etoposide-induced DNA damage conditions. Actin served as a gel loading control. Blots were cropped to enhance the representation (A,B). Immunofluorescence data showing accumulation of BMI1, ?-H2AX (C) RING1B (D) and Ub-H2A (E) p53BP (F) in MDAMB-231 cells transfected with miR-15a miR-16 or both miR-15a/16 under etoposide- treated conditions. Bar indicates 200 µm.

Fig 3: miR-200a, miR-200b, miR-15a, miR-429, miR-203 regulate PRC1 proteins in MDAMB-231 cells.Expression of BMI1, RING1A, RING1B and Ub-H2A in MDAMB-231(A) and BT549 (B) cells having overexpression of miR-200a, miR-200b, miR-15a, miR-429, miR-203. Expression of BMI1, Ub-H2A protein in MDAMB-231cells transfected with Anti-miR- 200a, Anti-miR-200b, Anti-miR-15a, Anti-miR-449, Anti-miR-203 (C) BMI1, RING1A localization in MDAMB-231 cells having overexpressed miR-200a, miR-200b, miR-15a, miR-449, miR-203under confocal microscopy (D, E). Cells transfected with Scramble Vector were used as controls. Bar indicates 200µm.

Fig 4: Loss of PRC1-mediated transcriptional repression in high-risk UM.a Unbiased hierarchical clustering of UM cell lines, 92.1 and MP38, in biological triplicates, based on normalized FPKM values of the GEP 12-discriminant geneset19. b Western blot of BAP1 and PRC1 core ligases RING1 and RNF2 relative to actin in 92.1 and MP38 cells. Data representative of biological triplicates. c Immunofluorescence of ubiquitinated H2A (green) and DAPI (blue) in MSK-UM03 (greatest proportion of GEP1 tumor cells in the cohort) and MSK-UM01 (greatest proportion of GEP2 tumor cells in the cohort). Representative images from six biological specimens. d Heatmaps representing CUT&RUN intensities of H2AK119Ub normalized to IgG in 92.1 and MP38. Data representative of biological duplicates. e Expression of H2AK119Ub target genes for individual tumor cells ranked by average imputed expression of the GEP2 gene signature (gene signatures annotated in Supplementary Data File 1) in ascending order from left to right. For each gene, imputed expression was z-normalized across all cells and smoothed using a 20-cell moving average window. Top, filled area plot showing average expression of GEP2 signature genes across ranked tumor cells. f Expression of H2AK119Ub target genes across the 4 molecular TCGA subtypes. Statistical significance tested using one-way ANOVA; p = 7.3 × 10-6; n = 80. Bars, mean of average expression; error bars, standard error of the mean. g Overall survival of (n = 80) TCGA-UM patients with primary tumors stratified by high (top 50th percentile, n = 40) and low (bottom 50th percentile, n = 40) expression of the ‘H2AK119Ub targets’ geneset. Statistical significance tested using two-sided log-rank test.

Fig 5: Increased chromosome segregation errors upon PRC1 inhibition.a Volcano plots showing the top differentially expressed pathways in low-risk UM cells (92.1) treated with PRT vs DMSO for 24 h; evaluated across biological triplicates. Normalized enrichment score (NES) for selected genesets shown as a function of significance, -log10(FDRq); FDRq, Bonferroni corrected p value. An unfiltered list of all significant gene signatures is provided in Supplementary Data File 5. b Examples of UM cells in anaphase stained for DAPI (blue) and centromeres (red); demonstrating different patterns of chromosome segregation errors. Additional patterns of missegregation are shown in Supplementary Fig. 9b. Representative images from biological triplicates. c Abundance of chromosome segregation error patterns during anaphase in low-risk UM cells (92.1) upon PRT-treatment as a function of time. Statistical significance tested using two-sided unpaired Student’s t test. Source data are provided as a Source Data file. d Abundance of chromosome segregation error patterns during anaphase across UM cell lines, arranged from left to right based on their GEP2 score. Source data are provided as a Source Data file. e Western blot of H2AK119Ub, STING, RING1, and RNF2 relative to Actin in Mel285 cells upon RING1 and RNF2 knockout. Data representative of biological triplicates. f Abundance of chromosome segregation error patterns during anaphase in UM cells (Mel285.Cas9) upon RNF2 knockout. Statistical significance tested using two-sided unpaired Student’s t test. Source data are provided as a Source Data file. Stacked bars, mean of each missegregation pattern; error bars, standard error of the mean across experimental triplicates (c–f).