Fig. 8

Bufalin inhibits CAMKK2 by inhibiting ICC proliferation and migration via ANXA2 and promoting mitochondrial dysfunction. A Ca²⁺ detection showed that after overexpression of CAMKK2, Ca²⁺ content increased, and after treatment with bufalin, Ca²⁺ content decreased. B Western blot analysis of expression of metastasis related proteins ANXA2 in QBC-939 cells exposed to bufalin or control. C The quantification of western blot. Protein levels were normalized to tubulin. D Mitochondrial protein and cytoplasmic protein were isolated, and the expression of ANXA2 in mitochondria and cytoplasm was detected. E Mitochondrial morphological changes after overexpression of CAMKK2 and treatment with bufalin were detected by transmission electron microscopy. F Expression of Wnt/ β-catenin signaling pathway related proteins after ANXA2 is knocked down in QBC-939 cells. G-H After overexpression of CAMKK2 in QBC-939 cells, ANXA2 is knocked down and the cell migration ability changes. I After overexpression of CAMKK2, ANXA2 is knocked down and Wnt/ β-catenin signaling pathway related proteins are expressed in QBC-939 cells. J Statistical analysis of protein level of I. All results were presented as the mean ± SD (n = 3). ns: p ≥ 0.05, *p < 0.05, **p < 0.01