C/EBPb in Chondrocytes Figure 4. C/EBPb affects cell cycle factors. (A) Time course of DNA histograms by a flow cytometric analysis of C3H10T1/2 cells with retroviral transfection of C/EBPb (Rx-C/EBPb) or the control GFP (Rx-GFP) after synchronization at the G2/M phase by nocodazole treatment. The horizontal and vertical axes represent the DNA content and relative frequency, respectively. The blue and red bars indicate the rates of cells in G0/G1 and G2/M phases, respectively. (B) Effects of loss- and gain-of-functions of C/EBPb on relative mRNA levels of cell cycle factors that were identified as possible transcriptional targets of C/EBPb by a microarray analysis (Table S1). The levels were compared by real-time RT-PCR analysis in the cultures between wild-type (WT) and C/EBPb2/2 rib chondrocytes (left), and between WT rib chondrocytes with Rx-C/EBPb and Rx-GFP (right). Data are expressed as means (bars)6SEM (error bars) of 6 samples per group. (C) Double immunofluorescence of p57 (green) and BrdU (red) in the proximal cartilage of tibias of the two genotype embryos (E16.5) and the HE staining (bottom) as a reference. Red, blue, and green bars indicate layers of proliferative zone, hypertrophic zone, and bone area, respectively. Scale bars, 50 mm. doi:10.1371/journal.pone.0004543.g004 p57 signal in the cGKII-GSK-3b action. However, neither cGKII nor GSK-3b overexpression altered at least the activity of the p57 promoter (2150 to +226 bp) with or without induction by C/ EBPb (Figure S3). Moreover, there is a marked difference in the limb cartilage phenotype between cGKII2/2 and C/EBPb2/2 mice. Unlike the cGKII2/2 cartilage characterized by appear- ance of a wide abnormal intermediate layer between the proliferative and hypertrophic zones [35,41], the C/EBPb2/2 PLoS ONE | www.plosone.org 7 February 2009 | Volume 4 | Issue 2 | e4543