TWIST1 regulates MMP-1 expression in melanoma cell lines. (A) WM793TR cells were engineered to inducibly express TWIST1 shRNA #3 and either a LacZ gene cassette or a shRNA-resistant TWIST1 gene. Cells were left untreated or induced with doxycycline for 96 hours. After treatment, western blots/qRT-PCR were performed. In addition, induction was performed for the final 24 hours in serum-free media for detection of secreted MMP-1. Columns, average of three independent experiments; bars, SD; **p<0.01. (B) Sbcl2 cells overexpressing either LacZ or TWIST1 were harvested at approximately 70% confluency and analyzed as above. Columns, average of three independent experiments; bars, SD; *p<0.05. (C) WM793TR cells were induced with doxycycline to overexpress either LacZ or TWIST1. At the same time, those cells were either treated with DMSO or 10µM U0126. Lysates and mRNA were harvested and analyzed as above. Columns, average of three independent experiments; bars, SD; *p<0.05; **p<0.01.