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. 2013 Dec 17;3(1):36–46. doi: 10.1002/cam4.170

Figure 2.

Figure 2

JW74 treatment reduces nuclear active β-catenin levels and inhibits transcription of downstream targets. (A) Cytoplasmic and nuclear fractions extracted from U2OS cells following 48 h treatment with 0.1% DMSO (control) or 10 μmol/L JW74 were analyzed by Western blotting using antibodies against active β-catenin, total β-catenin, ACTIN, or LAMINB1 (loading controls). (B) TCF/LEF reporter assays demonstrate that JW74 inhibits β-catenin mediated activity in U2OS cells. Cells transfected with pTA-Luc-STF and Renilla plasmids were treated with 0.1% DMSO (control) or JW74 (0.1–10 μmol/L) for 48 h. Data are normalized to Renilla. Significantly decreased reporter activity was observed following treatment with 10 μmol/L JW74 (*P = 0.033) and 5 μmol/L JW74 (*P = 0.024). (C) AXIN2 mRNA levels were significantly reduced following JW74 treatments of U2OS cells for 48 h (*5 μmol/L JW74: P = 0.005 and 10 μmol/L JW74: P = 0.042) and 72 h (**5 μmol/L and 10 μmol/L P < 0.001). (D) C-MYC mRNA levels were significantly reduced following incubation of U2OS cells for 48 h (**5 μmol/L and 10 μmol/L P < 0.001). Analyses were performed by qRT-PCR and presented data are normalized to PGK1 and relative to DMSO-treated samples. Error bars represent standard deviation. qRT-PCR, quantitative real-time polymerase chain reaction. TCF/LEF, T-cell factor/lymphoid enhancer-binding factor.