Injury-dependent hydrogen peroxide oxidation of IKK-alpha regulates keratinocyte migration through induction of EGF

Hydrogen peroxide is known to promote skin keratinocyte migration, although the mechanism of action is unclear. In an attempt to identify signaling pathways regulated by hydrogen peroxide in the skin, 3 day post fertilized (dpf) zebrafish larvae (nacre strain) were treated with 3mM hydrogen peroxide for 2 hours and subjected to RNA-seq analyses. Pools of about 1000 embryos for each of three biological replicates were derived from 5 independent mating pairs and raised to larval stages until 3 dpf. All larvae were subsequently homogenized in Trizol and total RNA was extracted using a chloroform extraction protocol treated with DNAse. Messenger RNA (mRNA) was subsequently purified from total RNA using biotin-tagged poly dT oligonucleotides and streptavidin-coated magnetic beads, followed by quality control using an Agilent Technologies 2100 Bioanalyzer (values >7 were used for sequencing). The poly(A)-tailed mRNA samples were fragmented and double-stranded cDNA generated by random priming for deep sequencing studies. Overall design: 6 samples total were analyzed. 3 untreated, and 3 hydrogen peroxide treated (3mM, 2hr)