Fig. 1(A) Sequences of siRNAs, wild-type melanocortin-1 receptor (WT-Mc1r), and mutant (MT)-Mc1r. (B) B16F10 cells treated with siRNAs were analyzed via transwell migration assay. Transfection of WT-Mc1r at 50 nM led to a significant reduction in migration of the cells compared to transfection of MT-Mc1r at 50 nM. (C) Confirmation of Mc1r down-regulation by reverse transcription polymerase chain reaction (RT-PCR). Two control genes (Ald1a and Ctbp1) were used to confirm the specificity of the siRNAs, and glyceraldehyde 3-phosphate dehydrogenase expression levels were used for normalization. Values represent the average of three independent real-time PCR experiments, each carried out in duplicate, and error bars represent standard deviation. aSignificant difference with a P value of 0.05.
Fig. 2(A) Confirmation of microarray data by reverse transcription polymerase chain reaction (RT-PCR). A subset of genes that showed down-regulation of 2.5-fold or higher by wild-type melanocortin-1 receptor (WT-Mc1r) compared to mutant (MT)-Mc1r in microarray assay and a nontarget gene (Ald1a) whose expression level was unchanged were used to validate the results from the microarray assay. Glyceraldehyde 3-phosphate dehydrogenase expression levels were used for normalization. Values represent the average of three independent real-time PCR experiments, each carried out in duplicate, and error bars represent standard deviation. (B) Immunoblotting for Srxn1 shows down-regulation by WT-Mc1r. β-Actin was used as the loading control. aSignificant difference with a P value of 0.05.
Table 1List of Real-Time Reverse Transcription Polymerase Chain Reaction Primers
Table 2Genes Up-Regulated More than 2.5-Fold by Mc1r siRNA
Table 3Genes Down-Regulated by Mc1r siRNA by More than 2.5-Fold