Figure 6. Correction of P53 hypermethylation improves ischemic wound closure.

(A) Topical delivery of 5-azacytidine to murine ischemic bipedicle wounds. (B) Representative IHC and intensity analysis (right panel) of 5-methylcytosine in ischemic wounds treated with either vehicle control or 5-azacytidine. (Scale bar: 50 μm; n = 5; *P < 0.05, Student’s t test). (C) Wound closure determined by digital planimetry (top). Data presented as percentage of wound area (bottom). n = 7, 8, *P < 0.05 (Student’s t test). Data represented as mean ± SEM. (D) Vector components used for targeted demethylation of P53 promoter in keratinocytes using CRISPR/dCas9 approach. Keratinocyte targeting was achieved by KRT14 promoter–driven guide RNAs. (E) The mouse P53 promoter locus used for demethylation events. The locations of the targets (1–3) for sgRNAs are indicated by red pointers. (F) Topical delivery of TET1 CD and targeted sgRNAs to the ischemic wound employing tissue nanotransfection (TNT2.0) technology. (G) Schematic diagram of the TNT process. (H) Western blot analysis showing the expression of P53 in bipedicle ischemic wounds of mice nanotransfected with TET1CD and peptide repeat in presence or absence of KRT14 promoter–driven P53 gRNA targets. Data expressed as fold-change using β-actin as loading control. (n = 6; *P < 0.05, Student’s t test). (I) Demethylation activity was measured by bisulfite sequencing of murine P53 promoter region (mm10_chr11:69,578,954-69,579,215). (J) Wound closure was monitored at different days after wounding in bipedicle ischemic wounds of mice subjected to TNT by digital planimetry (left). Data presented as percentage of wound area (right). n = 8, *P < 0.05 (Student’s t test). (K) Representative IHC analysis of P53 in ischemic wounds subjected to TNT. (L) Intensity analysis of the images. (Scale bar: 100 μm; n = 6; *P < 0.05, Student’s t test). nairing, hair-removal technique using a depilatory agent (Nair, Church and Dwight).