RASSF9 Regulated Epidermal Homeostasis Figure 12. RASSF9 affects keratinocyte proliferation and differentiation. (A, B) RASSF9-mediated proliferation measured by BrdU incorporation using a Cell Proliferation Biotrak ELISA kit (Amersham) and BrdU immunofluorescence. The BrdU incorporation rate was normalized to the amount of input protein or nuclei. Values are shown as fold changes versus the control group or percentage of nuclei presented in top panel and bottom panel, respectively. (A) BrdU incoporation rate was performed in mouse primary keratinocytes after two-day incubation under growth medium. Mean 6 SD (n = 3). *, P,0.05. (B) BrdU incoporation rate was evaluated from RASSF92/2 keratinocytes expressing HA-RASSF9 versus GFP control by adenoviral post transduction on 1 day and then subjected to growth medium for 2 days. Mean 6 SD (n = 2). *, P,0.05. (C, D) The terminal differentiation of keratinocytes was analyzed by immunoblotting with specific antibodies against filaggrin and loricrin. K14 was used as loading control. Total cell extracts were prepared from mouse primary keratinocytes incubated for the indicated time under condition for growth (0.06 mM calcium) or differentiation (2 mM calcium). The intensity of protein expression was determined as the density of the relevant band normalized to that of the K14 loading control, as determined by ImageQuant 5.1. The resultant values were further normalized to baseline (lane 1 of same blotting) and shown below the images. Similar results were obtained from three independent experiments. (C) Primary RASSF92/2 and WT mouse keratinocytes incubated in the indicated medium for 4 days. (D) Primary RASSF92/2 keratinocytes transduced with Adv/HA-RASSF9 or a GFP-expressing control virus and then subjected to differentiation induction for 3 and 4 days. Panel B and D: +/+, wild type; 2/2, RASSF92/2. doi:10.1371/journal.pone.0017867.g012 replication by associating with cyclin-CDK and binding to PCNA via two distinct functional domains [28,29]. Reduced p21Cip1 expression, observed in RASSF92/2 keratinocytes and skin, is thus likely to contribute to their hyperproliferation. Consistent with this hypothesis, we found that the endogenous levels of RASSF9 mRNA were initially induced when keratinocytes were exposed to a calcium concentration of 2 mM (differentiation- inducing conditions) for 1 day, but were thereafter reduced as differentiation progressed (Figure S10). Additional attempts to determine endogenous RASSF9 protein expression in keratino- PLoS ONE | www.plosone.org 16 March 2011 | Volume 6 | Issue 3 | e17867