shRNA induced epigenetic modifications around the target loci. (A) As evaluated by Chip Assay followed by qPCR, significant enrichment of both H3K9Me2 (p = 0.031) and H3K27Me3 (p = 0.023) was found at c-Myc P2 promoter on the 6th day after virosomal delivery of AFPEn–Pr + 2 – myc, whereas its scrambled control did not elicit the same level of enrichment. However, HepG2 cells pre-treated with TSA, did not show significant enrichment of both H3K9Me2 (p = 0.55) and H3K27Me3 (p = 0.37) by AFPEn–Pr + 2 – myc shRNA construct. This indicates that in the presence of TSA, shRNA failed to induce significant heterochromatization around the target site. (B) 6 days post transfection of AFPEn–Pr + 2 – myc in HepG2 cells, acetylation status of the c-Myc P2 promoter was evaluated by utilizing anti-histone 3 acetylated antibodies for ChIP assay followed by quantitative RT-PCR. The acetylation level significantly reduced post shRNA treatment (p = 0.016). However, no decrease in the acetylation level, by shRNA, was observed in the TSA treated HepG2 cells when compared to the scrambled control (p > 0.05). This indicates shRNA mediated possible recruitment of HDACs at the target site causing de-acetylation, which was reversed upon treatment with TSA. (C, D and E) On the 6th day following F-virosomal delivery of AFPEn–Pr + 2 – myc in HepG2 cells, bisulfite PCR products were analyzed for methylation by DNA sequencing. (C) Sequence chromatogram result shows that methylation was induced by test c-Myc shRNA on CpG 8, 9 and 10 of c-Myc P2 promoter. (D) No methylation was induced by control shRNA. (E) Cells pretreated with AZA shows no methylation even by the test shRNA, indicating failure in the recruitment of DNMTs by the shRNA at the target site.