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. 2020 Sep 13;21(18):6706. doi: 10.3390/ijms21186706

Figure 4.

Figure 4

Cinobufagin suppresses expression of β-catenin/TCF4-induced target genes in melanoma cells. (A) β-catenin/TCF4 target genes decreased by cinobufagin in LEF1-high expressing melanoma cells, but not LEF1-low expressing A549 lung cancer cells. Cells were incubated with cinobufagin for 24 h. After cinobufagin treatment, gene expression levels were measured by qRT-PCR. The values represent the mean ± SD (n = 3); * p < 0.05. (B) β-catenin/TCF4 target genes are not responsive to cinobufagin in LEF1 knocked-down A375 cells. Cinobufagin was treated for 24 h in LEF1 knocked-down A375 cells. β-catenin/TCF4 target genes expression levels were measured by qRT-PCR. The values represent the mean ± SD (n = 3); * p < 0.05 and ns, not significant. (C) LEF1 knock-down efficiency in A375 cells. LEF1 protein levels were measured by Western blotting. (D) Wnt3a-induced β-catenin/TCF4 target genes are decreased by cinobufagin in human melanocytes. Recombinant Wnt3a protein was dissolved in 1X PBS containing 0.1% endotoxin-free HSA. Wnt3a (30 ng/mL) pretreated for 1h prior to cinobufagin treatment, and then cells were incubated for 24 h. β-catenin/TCF4 target genes expression levels were measured by qRT-PCR. The values represent the mean ± SD (n = 3); * p < 0.05 and ** p < 0.01. ns, not significant. Red and blue points, mean value of individual sample.