FIGURE 7.

BRAF activates the MAP2 promoter by attenuating Notch signaling and HES1 expression. A, mBRAF decreases HES1 mRNA in metastatic melanoma cells. RT-PCR analysis was performed using total RNA from WM35 and 451Lu cells transfected with mBRAF. Amplification of a 288-bp HES1 mRNA and actin control is shown. B, overexpression of mBRAF inhibits Notch cleavage and decreases HES1 protein expression in metastatic melanoma cells. Total cell lysates were analyzed by Western blots using the indicated antibodies. Actin levels indicate loading of equal protein. C, Western blot analysis confirmed inhibition of Notch downstream target HES1 by DAPT. The upper band in lanes 1 and 2 appears to be a nonspecific band. The numbers below the bands indicate relative protein levels based on densitometric quantitation and normalization to actin. D, effect of γ-secretase inhibitor DAPT has no effect on MAP2 promoter activity. Melanoma cell lines were transfected in triplicate wells with phMAP2.2 promoter-luciferase plasmid alone or co-transfected with mBRAF and treated with 10 μm DAPT. Forty-eight hours after transfection, cells were harvested, and luciferase activity was measured, and data are represented as -fold change in luciferase activity. Control, MAP2.2 promoter plasmid alone; Inhibitor alone, promoter with DAPT; mBRAF Tx, co-transfection of mBRAF with the promoter; mBRAF Tx + inhibitor, DAPT treatment after co-transfection with MAP2 promoter and mBRAF plasmids. E, γ-secretase inhibition does not enhance mBRAF-induced MAP2 expression. Vector and 451Lu mBRAF cells were treated with 10 μm DAPT or 10 μm U0126, and after 72 h, total RNA was isolated, and qRT-PCR was performed using MAP2 TaqMan® probes and normalized with glyceraldehyde-3-phosphate dehydrogenase VIC-labeled TaqMan® probes.