Genome-Wide siRNA-Based Functional Genomics of Pigmentation Identifies Novel Genes and Pathways That Impact Melanogenesis in Human Cells (original) (raw)
Figure 2
Novel, pharmaceutically-tractable melanogenesis gene networks converge on tyrosinase expression.
A) 4 days post transfection with the indicated siRNAs, MNT-1 whole cell lysates were prepared and analyzed by immunoblot for the indicated proteins. A non-targeting siRNA was used as a transfection control (Control). ERK1/2 is shown as a loading control. B) Those siRNAs that inhibited tyrosinase accumulation were examined for consequences on tyrosinase and MITF gene expression by quantitative RT-PCR. 72 hours post transfection with the indicated siRNAs, equal numbers of MNT-1 cells were lysed and cDNA was prepared using a Cells to Ct kit (Ambion). Taqman qRT PCR assays (Applied Biosystems) for tyrosinase and MITF was utilized to identify siRNAs that impacted tyrosinase and MITF expression. C) The indicated siRNAs, targeting novel pigmentation genes identified in the MNT-1 screen, were tested for consequences on tyrosinase accumulation in darkly pigmented and moderately pigmented primary human melanocyte cultures 6 days post-transfection. The results presented here is a venn diagram of the data presented in Figure S5 demonstrating that we have identified pigment regulators that differentially impact pigment production in different genetic backgrounds. D) Pharmacological inhibition of Aldh activity impacts tyrosinase protein accumulation. MNT-1 cells (left panel) and primary melanocyte cultures (right panels) were exposed to 5 µM Aldh inhibitors (cyanamide or Angeli's salt) or the tyrosinase inhibitor hydroquinone [15] for 72 hours as indicated. 24 hours post-treatment, cultures were exposed to UV-B at the doses indicated. Tyrosinase and ERK1/2 levels were assessed by immunoblot. MNT-1: Angeli's salt (5 µM), cyanamide (5 µM), or hydroquinone (5 µM); primary melanocytes: Angeli's salt (50 µM), cyanamide (100 µM), hydroquinone (1 µM). E) Aldh inhibitors impair melanogenesis in primary human melanocytes. Darkly pigmented melanocytes were cultured for seven days in the presence of the indicated dosed of cyanamide (cya), vehicle, or PTU. PTU is the most potent currently known in vitro pigment inhibitor in primary melanocytes [43]. Subsequently, cells were lysed in CellTiter-Glo and the luminescence and absorbance values were used to calculate inhibition of pigmentation as in Figure 1A.