Skip to main content
. 2006 Nov;169(5):1652–1662. doi: 10.2353/ajpath.2006.050769

Figure 4.

Figure 4

Melanocyte sensitivity to 4-TBP is increased in the presence of factors that promote MITF expression. a: Melanocyte cultures, established from three normally pigmented individuals (HM), varied 6.8-fold in melanin content (ie, between 31 and 211 μg/mg protein). Cells were transferred to basal culture medium for 5 days and then to either complete medium or basal medium supplemented with either 0.1 nmol/L ET-1 or 1 μmol/L forskolin. After 5 days, cells were either harvested for protein extraction or dosed with 300 μmol/L 4-TBP. Cell lysates were prepared and normalized for protein content, then subjected to Western blot analysis to detect MITF and actin. MITF expression was increased in cell cultured in forskolin, ET-1, and complete medium. Viability was determined after 72 hours for cells dosed with 4-TBP. Viability is shown relative to viability of melanocytes, cultured in the appropriate medium, and treated with vehicle alone. The toxicity of 4-TBP did not correlate with pigment status but was significantly increased (*P < 0.05) in the presence of forskolin, ET-1, and complete medium (which contains bovine pituitary extract as a source of MSH). b: To confirm that melanocytes derived from adult breast skin are similarly sensitized to 4-TBP by MSH, whereas melanocytes that lack functional receptors for MSH do not, we determined the sensitivity of five melanocyte lines: one adult, breast-derived; and four neonatal foreskin-derived—two normally pigmented and two with loss-of-function MC1R mutations. All normal lines were from type IV skin (darkly pigmented). Melanocyte viability was determined in the absence and presence of either 1 μmol/L forskolin or 10 nmol/L MSH as described above. As with neonatal melanocytes, adult melanocytes were more sensitive to 4-TBP in the presence of MSH than in basal media and most sensitive in the presence of forskolin. Melanocytes that lack functional receptors for MSH showed equal sensitivity to 4-TBP in the absence and presence of MSH but were sensitized by forskolin. Bars indicate SD of triplicates within a representative experiment.