Figure 3

The expression of CCDC3 is well correlated with TAp63γ levels upon different drug treatment and during adipocyte differentiation. (a,b) MCF-7 cells treated with TNFα or DMSO at 20 ng/ml for 20 min and harvested for qRT-PCR (a) and WB (b) analyses to detect CCDC3 level. (c) HepG2 cells treated with 1 mM metformin at the time points as indicated and harvested for WB analysis to determine protein levels with indicated antibodies. Densitometry analysis is labeled on top of each band. (d) 3T3-L1 preadipocytes undergo differentiation to adipocytes. After incubation in differentiation media for 8 days, 3T3-L1 adipocytes were stained with Oil Red O. The absorbance of the extracted Oil Red O was spectrophotometrically determined at 570 nm to measure triglyceride (TG) accumulation. The error bars represent the standard error of the mean. (e) Cells after incubation in differentiation media for different days as shown in panel d were harvested for WB analysis with indicated antibodies. (f) ChIP assays of undifferentiated (F8) and differentiated (A4.5) adipocytes using the indicated antibodies followed by q-PCR for CCDC3 promoter sequences or negative control sequences (3′-UTR). Data are presented as means ± S.D., n = 3. (g) 3T3-L1cells infected with shTAp63 lentivirus and control virus at 2.5 days after differentiation. At 2 days after infection (Day 4.5 after differentiation), cells harvested for WB analysis to determine protein levels with indicated antibodies.