Additional file 7 of Epigenetic reactivation of tumor suppressor genes with CRISPRa technologies as precision therapy for hepatocellular carcinoma

Additional file 7: Figure S6. Related to Fig. 5. Transcriptional reactivation of HHIP gene by novel CRISPRa combinations does not correlate with changes in promoter DNA methylation in transiently transfected Hep3B cells. A Fold change in HHIP mRNA expression evaluated by qRT-PCR 96 h after transient transfection in Hep3B cells. Cells were transfected with SpdCas9-VPR and MS2-MCP-p65-HSF1, or with SpdCas9-TET1-CD and MS2-MCP-p65-HSF1, along with gRNA G4 or with no gRNA (NO G) as control. Relative gene expression was normalized and compared to cells transfected with empty vector control (EV) for statistical analysis. Data presented as means ± SEM (n = 3), and P-values were determined by unpaired t-test (***P = 0.0001, ****P < 0.0001). B Heatmap showing HHIP promoter DNA methylation in Hep3B cells transiently transfected with the novel CRISPRa combinations. Data from Illumina Infinium 850 K methylation EPIC microarrays. For each probe (listed at left), the difference in β-value from the average is shown (at left; green-purple color map) together with the average probe β-value (at center; blue-yellow color map), and the adjusted P-value for differential methylation between gRNA G4 and NO G for the two combinations tested (at right; white-purple color map).