Acute co-ablation of C/EBPa and C/EBPb results in specific molecular defects in squamous differentiation 4OHT-treated IKOab epidermis was analyzed for specific molecular defects in differentiation using IHC staining for molecular markers of specific stages of squamous differentiation. IHC staining in affbff and IKOab epidermis from mice treated with 4OHT were conducted side by side and 3,39-diaminobenzi- dine (DAB) incubation time was standardized to 4OHT-treated affbff epidermis. In 4OHT-treated IKOab epidermis, the basal cell markers, K5 and K14, were no longer restricted to the basal layer, but were aberrantly expressed throughout all suprabasal layers of the epidermis (Fig. 4A). However, K14 and especially K5 IHC staining were significantly less intense in 4OHT-treated IKOab epidermis than in 4OHT-treated affbff epidermis. Immunoblot analysis of epidermal lysates from 4OHT treated affbff and 4OHT-treated IKOab epidermis (Fig. 4B) revealed increased levels of K14 in IKOab epidermis, while K5 showed similar levels to affbff controls substantiating the IHC results showing reduced levels but a greatly expanded K5 compartment (Fig. 4A). K10, an early marker of the spinous layer and of the basal to spinous transition was significantly reduced in 4OHT- Roles of C/EBPs in Adult Skin treated IKOab epidermis (Fig. 4A). K1, another early marker of the spinous layer was delayed in its expression in 4OHT-treated IKOab epidermis and instead of being expressed in the layer adjacent to the basal cell layer, K1 was not expressed until ,2-3 layers of cells above the basal layers (Fig. 4A). Immunoblot analysis for K1 and K10 confirmed the IHC results (Fig. 4B). In general, markers of later stages of differentiation such as involucrin and filaggrin were not decreased. Involucrin, a component of the cornified envelope and a marker of the granular layer, was expressed in the appropriate layers but at higher levels compared to similarly treated affbff epidermis (Fig. 4A-C). Filaggrin, a marker of the granular layer was also expressed at higher levels in 4OHT-treated IKOab epidermis than in similarly treated affbff epidermis (Fig. 4B). In contrast, the expression of loricrin, a component of the cornified envelope, was significantly diminished in 4OHT-treated IKOab epidermis (Fig. 4A-C). In general, mRNA levels of the various differenti- ation markers were in accord with immunoblot and IHC levels (Fig. 4C). Overall the molecular changes in K5, K14, K1, and K10 in the 4OHT-treated IKOab epidermis are consistent with a defect in the basal to spinous transition. Figure 4. Co-ablation of C/EBPa/b results in defects in epidermal squamous differentiation. (A) IHC staining for markers of stratified squamous differentiation in 4OHT-treated controls and 4OHT-treated inducible double knockouts (IKOab). (B) Immunoblot analysis of epidermal lysates from animals treated with 4OHT for markers of stratified squamous differentiation (n = 3 mice/genotype). (C) TaqmanH real time quantitative PCR (K10) and reverse transcriptase semi-quantitative PCR (all others) on mRNA from 4OHT-treated whole skin (n = 5 mice/genotype). Scale bars represent 30 microns. doi:10.1371/journal.pone.0009837.g004 PLoS ONE | www.plosone.org 6 March 2010 | Volume 5 | Issue 3 | e9837