Immunohistochemistry on phosphorylated FOXO transcription factors in wounded human skin and scratched human primary keratinocytes expressing either a constitutively active form of FOXO3 or shRNA targeting FOXO3.

<p>A) Immunohistochemistry was performed on <i>in vivo</i> wounded human skin four days post wounding as well as on normal skin from the same donor. An anti-pFOXO antibody and a secondary fluorescently labeled antibody was used to detect the levels of phosphorylated FOXO transcription factors in the samples. Notice the increase in fluorescence intensity in the cytoplasm of keratinocytes closest to the front of the migrating epidermal tongue moving in under the wound crust at day 4. B–D) Scratch assays were performed on cultures of primary human keratinocytes transduced with either GFP control or FOXO3 AAA using adenoviral vectors and re-epithelialization was monitored over 24 h. B) Bar graph depicts the percent remaining of the original scratch width after 24 h (n = 9). C) Representative image of scratches from each condition at 0, 12 and 24 h. D) RT-PCR was run on isolated mRNA from scratched wells after 24 h. MMP-9, IL-8 and IL-6 gene expression was normalized using GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) as housekeeping gene. E) Scratch assays were performed on cultures of primary human keratinocytes transduced with either negative shRNA control or shRNA targeting FOXO3 using lentiviral vectors and re-epithelialization was monitored over 48 hours. Bar graph depicts the percent remaining of the original scratch width after 24 h (n = 12).