Fig. 5.
The PtdCho biosynthesis pathway is regulated by apoA-I/ABCA1 interaction in fibroblasts. (A, B) Normal and ABCA1 mutant (Q597R) fibroblasts were stimulated with 22OH/9CRA and incubated or not with 20 μg/ml apoA-I for 16 h. Cells were then pulsed with 10 μCi/ml methyl[3H]choline for 1 h, and the distribution of radioactivity among metabolites (Cho, Pcho, and CDP-cho) and PtdCho was determined as described in Experimental Procedures. Values shown are means ± SD of triplicate measures. *P < 0.05 by Student's t-test. (C, D) Stimulated normal and ABCA1 mutant (Q597R) fibroblasts were incubated or not with 20 μg/ml apoA-I for 16 h, followed by mRNA extraction using the RNeasy mini RNA extraction kit (Qiagen). Analysis of mRNA expression of CCTα, CCTβ, CKα, and CKβ was carried by real-time quantitative PCR from 200 ng total RNA as described in Experimental Procedures. Values shown are means ± SD of triplicate experiments. The expression of each gene was normalized to GAPDH expression, and mRNA fold changes relative to controls were determined. *P < 0.05 by Student's t-test.