Figure 2. hTAF4-TAFH activity controls conversion of facial dermal fibroblasts to melanocyte-like cells.

(a) Gradual rise in TAFH siRNA concentration from 20 to 100 nM changed the colour of pelleted fibroblasts to dark brown, indicating to the melanin production in these cells upon treatment. (b) Melanin content assay. Fibroblasts were transfected with different amounts of TAFH siRNAs. The relative production of melanin was calculated by measuring cell absorption spectra values at 405 nm in TAFH and control siRNA-treated fibroblasts and normalised to the amount of total protein. For reference control, melanin synthesis was induced by 0.1 mM IBMX (3-isobutyl-1-methylxanthine) in SkMel28 melanoma cells and compared to the value in control siRNA-treated fibroblasts (dashed line). (c) RT-qPCR analysis showed increased expression of melanocyte-specific genes in TAFH siRNA-transfected fibroblasts relatively to the control siRNA-treated cells at 24 h after treatment. Data shown are relatively to the levels of gene expression in terminally differentiated melanocytes, where 0% is the gene expression in primary fibroblasts, and 100% shows the levels of gene expression in primary melanocytes. Data were received from three independent experiments, with p at least < 0.01. (d) Immunofluorescence staining analysis of TAFH or control siRNA-treated fibroblasts at 48 h post-treatment reveals induced expression of MITF in the nuclear compartment of the cells. For positive control, nuclear localisation of MITF expression was verified in SkMel28 and WM266-4 melanoma cells. Scale bar, 40 μm. (e) TAFH siRNA-treatment induced up-regulation of p53 in fibroblasts: phosphorylated p53Ser15 (left) and TP53 mRNAs (right). mRNA data are normalised to GAPDH levels and shown as mean ± SD of three independent experiments with p value < 0.001 (***).