Roles of C/EBPs in Adult Skin Acute co-ablation of C/EBPa and C/EBPb results in hyperproliferative basal and suprabasal keratinocytes Cell proliferation analysis was conducted using a 1 h pulse label with BrdU, followed by IHC analysis for BrdU positive S-phase cells. 4OHT-treated IKOab epidermis demonstrated a significant increase in the numbers of BrdU positive S-phase cells in the IFE ff ff and ORS keratinocytes (Fig. 5 A&B) over 4OHT-treated a b epidermis. In 4OHT-treated IKOab IFE, there was a 4.7 fold increase in BrdU positive basal S-phase keratinocytes compared to ff ff mice (Fig. 5C). In normal epidermis, similarly treated a b suprabasal proliferating keratinocytes are infrequent as proliferat- ing keratinocytes are restricted to the basal layer. Analysis of 4OHT-treated IKOab epidermis revealed suprabasal BrdU positive S-phase keratinocytes were greatly increased and were observed at a ,27 fold increase over 4OHT-treated affbff epidermis (Fig. 5B&C). Collectively, these results demonstrate hyperproliferative basal and suprabasal keratinocytes and suggest an impairment of basal keratinocyte cell cycle withdrawal in mice missing C/EBPa and C/EBPb. Acute co-ablation of C/EBPa and C/EBPb results in molecular, morphological and differentiation defects in sebocytes In untreated mouse skin, sebaceous glands are associated with the upper follicle and these glands contained numerous sebocytes with a large light staining, finely vacuolated, clear translucent cytoplasm and a distinct round nucleus. Small reserve cells, (proliferative undifferentiated sebocytes) were less numerous and were peripheral in the glands (Fig. 6A). In the skin of 4OHT- treated IKOab mice, hair follicles contained distinct sebaceous gland lobules in the typical location on the hair follicle (Fig. 6B); however, these glands contained unusual looking sebocytes that appeared undifferentiated, had reduced cytoplasmic volume and lacked clear fine vacuolation supportive of lipid accumulation. Affected glands had an increased cellular density with more closely apposed nuclei and the cells were morphologically reminiscent of the reserve undifferentiated cells. To determine whether other sebaceous type glands were similarly affected, we examine the Meibomian and preputial glands for C/EBPa and C/EBPb Figure 5. Co-ablation of C/EBPa/b results in hyperproliferative basal and suprabasal keratinocytes. (A) IHC staining for BrdU-positive ff ff and 4OHT-treated IKOab epidermis. (B) Suprabasal BrdU-positive S-phase keratinocytes in keratinocytes in HF-ORS and IFE in 4OHT-treated a b ff ff and 4OHT-treated IKOab 4OHT-treated IKOab mice. (C) Quantification of BrdU-positive basal and suprabasal IFE keratinocytes in 4OHT-treated a b mice (n = 3 mice/genotype, 3 strips per mouse; *p,0.05). Scale bars represent 30 microns. doi:10.1371/journal.pone.0009837.g005 PLoS ONE | www.plosone.org 7 March 2010 | Volume 5 | Issue 3 | e9837