Fig. 3.

WNK4 may be involved in the cell cycle regulation of 3T3-L1 adipocyte. (a) CHIP assay of C/EBPβ on PPARγ2 promotor. The 3T3-L1 cells expressing si-Nega or si-WNK4 were induced to differentiate for 2 days. Cells were subjected to chromatin immunoprecipitation (ChIP) with antibody specific to C/EBPβ, and the immunoprecipitated samples were normalized to the input internal control DNA (actin gene). The values are expressed as mean ± 95%CI (n = 3) relative to si-Nega-transfected cells. IgG immunoprecipitated samples only showed a signal < 10% that of the C/EBPβ-antibody immunoprecipitated samples. Similar results were obtained in three separate experiments. (b) Cellular localization of WNK4. In differentiated 3T3-L1 cells on day 2, WNK4 protein was detected in the cytosolic and nucleic fractions. The asterisks (*) and sharps (#) show nonspecific bands and WNK4, respectively. (c) Inhibition of cyclins during 3T3-L1 differentiation. In the si-WNK4 group, time-dependent increases of cyclin D1, D3, A2, and B1 after MDI stimulation were significantly inhibited. (d) BrdU assay during MCE. Representative image of a BrdU assay 24 h after induction of adipogenic differentiation in si-Nega- or si-WNK4-transfected 3T3-L1 cells; BrdU (green), Hoechst (blue). Scale bar is 100 μM. (e) Quantification analysis of a BrdU assay in 3T3-L1 cells. Values are expressed as mean ± 95%CI. *P < 0.05; **P < 0.01.