Figure 6. Cell-cycle synchronisation and siRNA depletion of cohesin.

For cohesin depletion experiments, cells were transfected with RNAi against SCC1. RNAi against luciferase was used as a control. (A) Plots of FACS sorted cells synchronised in G1- and G2-phase in control and cohesin depleted cells. (B) Western blots showing SCC1 protein levels after RNAi treatment (C). SMC3 ChIP on G2 cells after cohesin depletion showing that cohesin is reduced at the ICR and the CTCF DS sites. (D) Expression levels of IGF2 and H19 relative to β-actin, as determined by real time PCR after cohesin depletion in G1 and G2 phases. IGF2 is significantly upregulated while H19 levels are unaffected. # Denotes significant differences (P<0.05) between control and SCC1 RNAi. (E) DNA methylation levels of individual CpGs in the DMR0 and ICR were determined by pyrosequencing of bisulphite modified DNA from cohesin depleted cells and controls in G2 phase. (F) Average DNA methylation levels at all CpGs detected by pyrosequencing in Figure 6E shows that methylation was not significantly changed after RNAi treatment for SCC1. Box and whisker plots show mean, inter-quartile ranges, max, and min values. Data represent triplicate bisulphite conversion and pyrosequencing reactions from one RNAi and control experiment.