Figure 3.

Expression of key adipogenic and cell cycle (C/EBPα and G0S2) proteins in proliferating human subcutaneous preadipocytes during coculture with monocytes and subsequent terminal differentiation. (a) Gene expression of C/EBPβ, C/EBPα, PPARγ, and G0S2 by real-time PCR in preadipocytes during coculture with monocytes for up to 5 days. The fold change expression of each gene in preadipocytes compares coculture with monocytes to culture with control media after normalization to an internal control (GAPDH) using the 2^−ΔΔCt calculation for relative expression. Data are from one experiment done in duplicate representative of three independent experiments, error bars are means ± s.e.m. (b) Western blot of C/EBPβ, C/EBPα, and G0S2 in preadipocytes at the 5-day time point during coculture with monocytes. The two bands for C/EBPβ represent the two isoforms for this protein at ~36 kDa. PPARγ was probed for but not detected. Densitometry of blots was normalized to actin loading control. (c) Representative phase contrast microscopy images (×40) of preadipocyte confluency after 5 days of coculture with monocytes just prior to hormonal induction of differentiation. (d) Representative phase contrast microscopy images (×40) of Oil Red O-stained adipocytes after 2 weeks of preadipocyte differentiation following 5 days of preadipocyte coculture with monocytes (i.e., after 5 days of coculture with preadipocytes, monocytes were removed and preadipocytes were differentiated in the absence of monocytes). Error bars are means ± s.d. of pixel intensity quantification of four images per condition from one experiment representative of three. *P < 0.05 compared to control media. C/EBPβ, CCAAT/enhancer binding protein (C/EBP), beta; PPARγ, peroxisome proliferator-activated receptor gamma 2; C/EBPα, CCAAT/enhancer binding protein (C/EBP), alpha; G0S2, G0/G1 switch 2.