Additional file 8 of Epigenetic reactivation of tumor suppressor genes with CRISPRa technologies as precision therapy for hepatocellular carcinoma

Additional file 8: Figure S7. Related to Fig. 5. Lentiviral transduction of SpdCas9-TET1-CD or SpdCas9-VPR targeting HHIP or MT1M genes in Hep3B cells. A Schematic representation of the “all-in-one” lentiviral vectors, SpdCas9-TET1-CD and SpdCas9-VPR, for the constitutive co-expression of a gRNA and TET1-CD or VPR, C-terminally fused to SpdCas9. B Fold change in HHIP mRNA expression assessed by qRT-PCR at 18, 30, and 40 days in Hep3B cells constitutively co-expressing SpdCas9-TET1-CD and the combination of four gRNAs (MIX 4G) targeting HHIP promoter. C Fold change in MT1M mRNA expression assessed by qRT-PCR at 20 days in Hep3B cells lentivirally co-transduced with SpdCas9-TET1-CD and individual gRNAs (G1, G2, G3, and G4) or the MIX 4G; or with SpdCas9-VPR and the most potent gRNA (G1). Data presented as means ± SEM (n = 3), compared to SpdCas9-TET1-CD NO G, and P-values were determined by two-way ANOVA with Šídák's multiple comparisons test for panel (B), and by two-way ANOVA with Dunnett's multiple comparisons test for panel (C) (****P < 0.0001). SpdCas9 Streptococcus pyogenes deactivated Cas9 protein adopted for epigenome engineering, TET1-CD Ten-Eleven Translocation methylcytosine dioxygenase 1-catalytic domain, VPR VP64, p65, Rta, hU6 RNA polymerase III promoter for human U6 snRNA, hUbC human ubiquitin C promoter, 3xFLAG three tandem FLAG® epitope tags, followed by an enterokinase cleavage site, SV40NLS nuclear localization signal of SV40 (simian virus 40) large T antigen, T2A 2A peptide from Thosea asigna virus capsid protein, cleavable linker, PuroR puromycin N-acetyltransferase gene that confers resistance to puromycin, MIX 4G combination of four gRNAs, ns not significant.