Fig 2: Src activation and CAF1-depletion both decrease levels of PRC1 subunits.( A) Diagram of the polycomb repressive complexes (PRC1 & PRC2) and their subunits. Subunits in red show significantly decreased abundance after v-Src activation. ( B) Experimental diagram of a SILAC experiment to measure proteome differences between cells treated with non-targeting siRNA (siControl) versus cells treated with siRNA targeting CHAF1A/p150, a critical component of the CAF1 complex. ( C) Plot of –log 10 p-value versus fold change shows downregulation of all PRC1 subunits measured. In contrast, PRC2 subunits remain either unchanged, or slightly increased. ( D) Bar chart showing fold change data from ( C). PRC1 and PRC2 subunits are shaded light and dark grey, respectively. ( E) Immunoblot analysis of PHC3 either (left) comparing -/+ Src induction conditions, or (right) comparing siCTRL versus siRNA targeting CAF1 p150 subunit (sip150). F) qPCR analysis of PHC3 mRNA, comparing same conditions as ( E).

Fig 3: PHC3 depletion alters RING1A localization, cellular motility and invasion.Cells treated with either non-targeting siRNA (siControl), or siRNA targeting PHC3, were analysed by light microscopy for ( A) morphological changes, ( B, C) wound healing assays, and ( D) transwell Matrigel invasion assays. Error bars indicate standard error between three biological replicates. E) A summary of data comparing the ability of full length PHC3 and PHC3 truncation mutants to repress the invasion phenotype induced by v-Src activation. ( F, G, H) Cells were transduced with lentivirus encoding for either ( F) HA-PHC3WT (full length, wild-type), empty ( G), or ( H) HA-PHC3ΔSAM (SAM domain deletion mutant). Cells were then treated either with ethanol (control), or 4-OHT, and immunostained for HA tag (green) and RING1A (red). Arrows indicate RING1A nuclear foci. Representative images of three replicates. Scale bar: 5 µm. ( I) Immunoblot analysis of H2AK119ub -/+ Src activation. Note that the loading control shown is identical to Figure 15E.