Figure 6. Defects in adipogenesis due to 14-3-3ζ depletion are due to cell cycle arrest and up-regulation of Cdkn1b/p27^Kip1.

(a–d) Analysis by flow cytometry (a) of control or si14-3-3ζ-transfected 3T3-L1 cells at different stages of the cell cycle at 0 (b), 24 (c) and 48 (d) hours following differentiation (n=4 independent experiments; *P<0.05 when compared with siCon-transfected cells, assessed by Student's t-test). (e) Analysis of various cell cycle regulatory genes by RNA-Seq from siCon- or si14-3-3ζ transfected 3T3-L1 adipocytes (n=4 per goup, *P<0.05 when compared with siCon cells at the same time point, assessed by Student's t-test). (f) Immunoblotting of p27^Kip1 from lysates of differentiating of siCon- or si14-3-3ζ-3T3-L1 adipocytes (n=4 experiments). (g,h) Co-transfection of siRNA against 14-3-3ζ and p27^Kip1 was used to examine whether knockdown of both proteins could restore differentiation, as determined by Pparγ expression (g) or Oil Red-O staining (h). (n=4 independent experiments). Error bars represent s.e.m.