Niclosamide and pyrvinium attenuate the activities of Wnt/Axin2/Snail axis. (A) Axin2 and Snail mRNA level in MG-63 and SAOS-2 cells. The transcript abundance was determined by quantitative RT-PCR. (B) Western blot analysis of osteosarcoma cells treated with 0.25 µM niclosamide and 0.03 µM pyrvinium. Tubulin served as a loading control. Representative images are shown from at least two independent experiments. (C) Relative luciferase activity representing β-catenin/TCF-dependent transcription in treated cells. Osteosarcoma cells were transiently transfected with the TOPFlash luciferase construct and SV40 Renilla-expression vector in each well. After 24 h of incubation, cells were treated with 0.25 µM niclosamide and 0.03 µM pyrvinium. Luciferase activity was measured 24 h later with normalization to the activity of SV40 Renilla. (D) mRNA levels of β-catenin target genes by quantitative RT-PCR. The value of sample was normalized to the levels of GAPDH mRNA. Quantitative RT-PCR experiments were carried out in triplicate (error bars, ±SD. * p < 0.05, ** p < 0.01 versus DMSO, n.s., not significant).