skin phenotype in the C/EBPa and C/EBPb knockout mouse is that functional redundancies between the transcription factors mask their roles in the single knockouts. In support of this notion are studies demonstrating that the knockin of C/EBPb into the C/ EBPa locus can rescue C/EBPa2/2 mice from perinatal lethality [27] and restore granulocytic differentiation and glycogen metabolism [27]. A recent study in developing mouse skin in which C/EBPa and C/EBPb were co-deleted during epidermal development produced mice that were only viable for a few hours after birth, and the analysis of epidermis from these mice revealed hyperplasia and decreased expression of spinous and granular markers of differentiation [28]. Thus, while C/EBPa and C/EBPb are critical for epidermal development, their roles in stratified squamous differentiation in the adult epidermis remain poorly understood. Sebaceous glands extend off of the upper hair follicle and are formed from a sebocyte progenitor that expresses Blimp 1, keratin 14 (K14) and keratin 5 (K5) [29] and these cells produce a proliferative population of sebocytes which in turn differentiate to produce the lipid/sebum producing cells [30]. These differentiated sebocytes lyse and release their contents of lipids/sebum into the hair follicle canal and make their way to surface of the skin where they prevent drying of the skin and hair [31]. cMyc [30,32], hedgehog signaling [33] and AP-2a/AP-2c [21] regulate seba- ceous gland development/differentiation. C/EBPa and C/EBPb are abundantly expressed in sebocytes of the sebaceous glands [34,35], however, no function for these transcription factors in sebocytes has been described and no sebaceous gland phenotype was reported in C/EBPa or C/EBPb knockout mice. To address possible functional redundancies and to reveal the functional roles of C/EBPa and C/EBPb in the adult epidermis and sebaceous gland, mouse models were developed in which either family member could be ablated alone or together in the epidermis and sebaceous glands of adult mice. Our results demonstrate C/EBPa and C/EBPb are essential for sebocyte differentiation and epidermal squamous differentiation involving the basal to spinous transition. Methods Ethics statement All animal work described in the study involving animal husbandry, experimentation, and care/welfare have been con- ducted according to NIH guidelines and approved by NCSU Institutional IACUC committee. Animals and treatments K14-CreERtam homozygous mice [36] (CD1) were obtained from Jackson Labs (#005107) and crossed with either floxed-a (B6.129)[37], floxed-b (B6.129)[38] or floxed-ab (B6.129). Floxed- a and floxed-b mice were genotyped as previously described [37,38]. Mice were genotyped for K14-CreERtam using the following primers: forward, CGATGCAACGAGTGATGAG- GTTC; reverse, GCACGTTCACCGGCATCAAC. Mice 6–8 weeks of age in telogen were treated topically to clipped skin with 4OHT (Sigma catalog # T176-50MG) 1.0 mg/day for days 1–5 and again on days 13–17 dissolved in 95% ethanol (1.0 mg/ 200 ul) and skin collected for IHC on day 21. Preparation of epidermal homogenates Mice were killed by cervical dislocation and dorsal skin was removed and subjected to 15 seconds heat shock in 65uC deionized water followed by 15 seconds in ice water. Water was removed from skin by blotting between paper towels and Roles of C/EBPs in Adult Skin epidermis was scraped from dermis. For C/EBPa and C/EBPb proteins, epidermal scrapes were homogenized on ice in RIPA buffer (1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1.5 mM sodium orthovanadate, 1.5 mM phenylmethylsufonyl fluoride, 1.5 mM dithiothreitol, and 1x protease inhibitor cocktail (Roche Diagnostics GmbH ref 11 836 001) in PBS), and centrifugated at 14,000 g for 20 minutes at 4uC. Supernatants were stored at 280uC prior to use. Protein quantification was determined by Bio-Rad Protein Assay reagent (Bio-Rad, Cat# 500–0006). For keratins and cornified envelope precursors, epidermal scrapes were homogenized in 20% 2-mercaptoethanol and 5% SDS, boiled 5 minutes, allowed to cool and centrifugated at 14,000 g for 10 minutes. Immunoblot analysis Equal amounts of protein were denatured in sample buffer, loaded onto 12% tris-glycine gels (Invitrogen, EC6005), separated by gel electrophoresis and transferred to PVDF membrane. Membranes were blocked for one hour (PBS with 0.1% tween/ 5.0% milk/1.0% BSA), and incubated overnight at 4uC (PBS with 0.1% tween/1.0% BSA) with one of the following rabbit polyclonal antibodies: C/EBPa (Santa Cruz, SC-61, 1:2000), C/ EBPb (Santa Cruz, SC-150, 1:2000), K1 (Covance, 1:10,000), K5 (Covance, 1:10,000), K10 (Covance, 1:10,000), K14 (Covance, 1:10,000), involucrin (Covance, 1:10,000), loricrin (Covance, 1:10,000), filaggrin (Covance, 1:10,000). Membranes were then rinsed one hour in PBS-T (0.1% tween) and subjected to one hour incubation (PBS-T, 1.0% BSA) with anti-rabbit IgG, horseradish peroxidase-linked secondary (GE Healthcare, NA934V, 1:2500), incubated one minute with Western Lightening Plus-ECL (PerkinElmer NEL105001EA) and subsequently exposed to film. Each blot represents n$3 mice per genotype. Membranes were stripped and re-probed for b-actin (Sigma A-5441, 1:25,000, mouse) for one hour followed by one hour incubation anti-mouse horseradish peroxidase-linked secondary (GE Healthcare, NXA931, 1:25,000), incubated one minute with Western Light- ening Plus-ECL (PerkinElmer NEL105001EA) and subsequently exposed to film. Cell proliferation analysis Mice were injected intraperitoneally with bromodeoxyuridine (BrdU, Sigma B5002, 100 mg/kg in PBS) and killed 1 hour later. Skin was fixed in 10% neutral buffered formalin phosphate (NBF) for 20 h and moved to 70% ethanol and subsequently embedded in paraffin. Immunohistochemical (IHC) staining for BrdU was performed as previously described [23] on 5 mM sections with anti-BrdU (BD Biosciences 27644, 1:25). Immunohistochemical (IHC) staining Tissues were fixed in 10% NBF for 20 h, switched to 70% ethanol and embedded in paraffin. Tissue sections (5 mM) were stained for H&E or specific IHC as described [23,26,39] with the following antibodies: C/EBPa (Santa-Cruz SC-61, 1:1000), C/ EBPb (Santa-Cruz SC-7962, 1:1000), K5 (Covance PRB-160P, 1:2000), K10 (Covance PRB-159P, 1:2000), involucrin (Covance PRB-140C, 1:2000), loricrin (Covance PRB-145P, 1:2000), K6 (Covance PRB-169P, 1:2000) and fatty acid synthase (FAS- N)(Santa-Cruz SC-20140, 1:1000). Oil Red O staining Fresh unfixed epidermis sections were frozen on liquid nitrogen in Tissue-TekH O.C.T (Sakura Finetek, #4583) compound and stored at 280uC. Frozen sections (10 microns) were washed PLoS ONE | www.plosone.org 2 March 2010 | Volume 5 | Issue 3 | e9837