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Fig. 5. The functional analysis of fad24 by RNAi. (A) The endogenous expression of the fad24 gene. Total RNA obtained from 3T3-L1 cells transfected with shRNA expression vector for fad24 (white bars) or with scrambled shRNA expression vector as a control (gray bars) at each time point. The expression level of fad24 was determined by Q-PCR, and normalized with 18S rRNA expression determined by Q-PCR. Each column represents the mean±s.d. (n=3). (B) Differentiation of 3T3-L1 cells transfected with shRNA expression vector for fad24. The cells transfected with shRNA expression vector for fad24 (sifad24) or with scrambled shRNA expression vector as a control (Control) were stimulated with inducers. After 8 days of induction, the cells were fixed and stained on six-well plates with Oil red O to detect oil droplets. Bar, 200 µm. (C) The amount of triacylglycerol was measured on 24-well plates. Each column represents the mean±s.d. (n=3). (D) Effect of shRNA expression for fad24 on the expression of various adipogenic genes. Total RNA obtained from sifad24 cells (white bar) or control cells (gray bar) at each time point was subjected to Q-PCR. Expression levels were normalized with ß-actin expression determined by Q-PCR. Each column represents the mean±s.d. (n=3). (E) The expression of C/EBPß and C/EBP at earlier time points of differentiation. Total RNA obtained from sifad24 cells (white bars) or control cells (gray bars) at each time point was subjected to Q-PCR. Expression levels were normalized with 18S rRNA expression determined by Q-PCR. Each column represents the mean±s.d. (n=3).

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