Fig. 4

ZFP57 upstream region is a methylation-dependent regulator of transcription at this locus. a Schematic as in Fig. 3 above but showing difference in methylation (Δβ) between HCT116 WT cells vs HCT116 DKO cells. The intron/exon structure and positions of the forward (FW) and reverse (RV) primers for RT-(q)PCR on the ZFP57 gene are also shown. b Methylation levels at individual CpG covered by the pyrosequencing assay in WT (HCT116) and knockout (DKO) cells. Values are shown as mean +/− SD for each site: *p < 0.05; **p < 0.01; ***p < 0.001. c RT-PCR showing upregulation using the primers indicated in a, key as above. CTRL, positive control (human reference total RNA); NTC, negative control (no template control); 100 bp, size standards ladder; ACTB, β-actin loading control. d Confirmation of upregulation by RT-qPCR using the same primers, values normalized to HPRT; FC, fold change. e Methylation levels using pyroassay as in B but in 5-aza-dC treated SH-SY5Y cells (5-aza-dC), as compared to untreated (UT). f RT-PCR for 5-aza-dC treated cells from e. g RT-qPCR confirmation of ZFP57 upregulation in 5-aza-dC-treated SH-SY5Y cells