Effect of Nf1 knockdown on Wnt pathway in MSC80 cells.

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Effect of Nf1 knockdown on Wnt pathway in MSC80 cells. Effect of Nf1 knockdown on Wnt pathway in MSC80 cells. A, MSC80 cells were transfected with either siRNA-targeting NF1 or nontargeting siRNA (NT). Western blots using anti-neurofibromin antibodies were perfomed to verify Nf1 inactivation at protein level in MSC80 cells. GAPDH was used to normalize the Western blots. Results were reproduced in 3 independent experiments, and figures represent a typical experiment. B, MSC80 cells were transiently cotransfected by the SuperTOP-Flash Luciferase or SuperFOP-Flash Luciferase plasmids with either nontargeting (NT) siRNA or siRNA against NF1. Luciferase activity was then analyzed. *, P < 0.05, and **, P < 0.01 by Tukey post hoc tests after one-way ANOVA when compared with control. C, MSC80 cells were transfected with either NT or a siRNA against Nf1. Total RNA was extracted and RT-qPCR was performed using specific primer recognizing Axin2. D, Western blots were performed using anti-active (dephosphorylated β-catenin) and anti-total β-catenin antibodies. GAPDH was used to normalize the Western blots. The Western blots were quantified using Image J software (rsb.info.nih.gov). Armelle Luscan et al. Clin Cancer Res 2014;20:358-371 ©2014 by American Association for Cancer Research