Fig. 6. Functional analysis of fad24 using FAD24-overexpressing NIH-3T3 cells. (A) The exogenous expression of the fad24 gene was determined by northern blot analysis. Total RNA (25 µg) obtained from a stable transformant was subjected to northern blot analysis for fad24. The retroviral exogenous gene expression and endogenous gene expression are indicated. (B) Differentiation of FAD24-overexpressing NIH-3T3 cells in the presence of BRL49653, a ligand for PPAR. NIH-3T3 cells stably expressing fad24 or control cells (infected with empty vector) were treated with inducers containing BRL49653. After 8 days of induction, the cells were fixed and stained with Oil Red O to detect oil droplets. Bar, 100 µm. (C) Northern blot analyses of adipocyte marker genes during the differentiation of FAD24-overexpressing NIH-3T3 cells. Total RNA from cells after the induction was isolated and 25 µg per lane was subjected to northern blot analysis for each adipocyte marker gene. Staining with EtBr for ribosomal RNA is shown as a control. (D) Expression of C/EBPß and C/EBP at earlier time points during the differentiation of FAD24-overexpressing NIH-3T3 cells. Total RNA obtained from FAD24-overexpressing NIH-3T3 cells (white bars) or control cells (gray bars) at each time point was subjected to Q-PCR. Expression level was normalized with 18S rRNA expression determined by Q-PCR. Each column represents the mean±s.d. (n=3).