Figure 4

Identification of novel EDNRB network components that impact melanogenesis. (a) Genes from EDNRB network are examined for their impact on pigment production in MNT-1 cells. The impact of each siRNA on pigment production is calculated as described in [14] relative to control (c) and tyrosinase (TYR) siRNA treated cells using a normalized percent inhibition calculation. (b) Ten genes are selected from panel (a) that have > 50% NPI, and are not the result of siRNA off-target effects (2/3 of oligos have > 50% inhibition). The efficacy of protein knockdown for 10 pooled siRNAs directed towards these genes is measured by quantitative RT-PCR at both 24 and 48 hours after transfection. The timepoint where maximum inhibition of expression is observed is reported. AGTR1 mRNA level after siRNA treatment is below the detection limit and is not reported. Control siRNAs are depicted with a black bar, and target siRNAs with a white bar. *, p< 0.05. **, p< 0.01. (c) Both EDNRB and selected novel EDNRB network components impact the MITF protein levels in MNT-1 cells. MNT-1 cells are transfected with the indicated siRNAs for 96 hours followed by immunoblotting with MITF and ERK antibody as shown.