Figure 4

Qantitative-PCR based assay for selective fatty acid, cholesterol biosynthetic pathway and regulatory genes following IGF-1 treatment. The relative abundance of mRNA as compared to untreated control samples was assayed by q-PCR and plotted on a histogram for 6 genes involved in the cholesterol biosynthetic pathway, namely for HMG CoA synthase 1 (Hmgcs1), HMG CoA reductase (Hmgcr), (Mevalonate kinase (Mk/Mvk1), Cytochrome P450 51 (Cyp51), Lanosterol synthetase (Lss), and Dehydrocholestrol reductase (7Dhcr) (Fig. 4 A–F); 2 genes involved in fatty acid biosynthesis, namely Fatty Acid synthase (Fas) and stearoyl-Coenzyme A desaturase 1 (Scd1)(Fig. 4G and 4H); and 3 regulatory genes involved in fatty acid and cholesterol biosynthesis, namely SREBP-1 and -2, and Sterol Cleavage Activator Protein (SCAP) (Fig. 4J, 4K and 4L). The beta-Actin mRNA was assayed here as a control (Fig. 4I). In addition, 6 other genes unrelated to the fatty acid or cholesterol pathways were assayed; namely Chac1, Klf10, Ier2, Socs3, Shh and Dusp1 (Fig. 4 M-R, respectively) and the data show that the expression profile for all of these genes is similar to patterns obtained from our microarray experiments. The pattern obtained is in general agreement with the Affymetrix GeneChip data for these genes, with the exception of SREBP-1 where no change was detected with the microarray study. The extent of induction obtained with q-PCR, however, is greater than the microarray hybridization signals, probably due to the greater dynamic range observed with PCR amplification.