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. Author manuscript; available in PMC: 2013 Dec 15.
Published in final edited form as: Cancer Res. 2012 Dec 7;72(24):6382–6392. doi: 10.1158/0008-5472.CAN-12-1033

Figure 1.

Figure 1

TWIST1 is up-regulated in melanoma cell lines, particularly in VGP, downstream of oncogenic B-RAF and is positively regulated by active ERK1/2 signaling. (A) Western blot analysis of melanoma cell lines,organized by genotype and tumor stage, or neonatal human epidermal melanocytes (NHEM) was performed to confirm up-regulation of TWIST1 protein in melanoma cells. WM793 cells appear in both panels for comparison; RGP (Radial Growth Phase), VGP (Vertical Growth Phase), MM (Metastatic Melanoma). (B) WM115 or WM793 cells were transfected with a control siRNA or siRNA directed against total or mutant B-RAF. After 72 hours, lysates were harvested and western blots performed. (C) WM793TR (TR= Tet Repressor) cells were transduced with an inducible B-RAF shRNA, and either an inducible GFP or an shRNA-resistant B-RAFV600E gene cassette. After 7 days of doxycycline treatment, lysates were harvested for western blot. (D) Western blot analysis was conducted on lysates from mutant B-RAF cells treated with PLX4720 (1µM) or U0126 (10µM) for 24 hours. (E) Metastatic melanoma tumor #1 and #2 were placed in short-term culture after surgical resection and treated for 24 hours with U0126 (20µM) and AZD6244 (3.3µM), respectively. Lysates were generated and western blot analysis performed. (F) WM115 cells and WM793 cells were transfected with control siRNA, ERK1 siRNA and/or ERK2 siRNA. After 72 hours, lysates were harvested and western blots performed. (G) Quantitative RT-PCR was performed from WM793 and 1205Lu cells (treated with 1µM PLX4720, 24 hours), from WM115, WM9, and A375 cells (treated with 1µM PLX4032, 24 hours), and from WM793 cells treated with U0126 (10µM, 24 hours). Columns, average of three independent experiments; bars, SD; *p<0.05, **p<0.01.