Fig. 4

Apatinib negatively regulated the β-catenin signaling in esophageal cancer. a Heatmap for β-catenin signaling in esophageal cancer tissues compared to that in normal tissues using TCGA data. *P < 0.05, **P < 0.01. b KYSE30 cells were treated with apatinib (10 μM) for 24 h. Western blotting was performed to detect β-catenin expression in whole-cell lysates, as well as in the cytoplasmic and nuclear fractions. c KYSE30 cells were pretreated with MG132 (10 μM) and exposed to apatinib (10 μM) for 24 h. Whole cellular extracts were prepared for coimmunoprecipitation assay with anti-β-catenin followed by immunoblotting with anti-Ub antibody. KYSE30 cells were incubated with DMSO or apatinib (10 μM) for 24 h. d β-catenin-TCF/LEF transcriptional activity was detected by luciferase reporter assays. P values were determined by Student’s t test. **P < 0.01. e The mRNA expression levels of Myc, Jun, Wisp1 and Cyclin D1 were examined by qPCR. P values were determined by Student’s t test. *P < 0.05, **P < 0.01. f Representative images showing double immunofluorescence staining for β-catenin (green) and nuclei (DAPI, blue). Scale bar, 25 μm. Data are representative of three independent experiments (mean and SEM in d, e)