Figure 5.

Autophagy inhibitor Spautin-1 aggravates apoptosis induced by D4476, and CK1α inhibits autophagy by targeting the p53/AMPK/mTOR signaling pathway downstream of MDM2 in AML cells. (A) After treatment with 40 µM D4476 in the presence or absence of 10 µM autophagy inhibitor Spautin-1 for 48 h, the cell viability was measured by CCK-8 assay. Data are expressed as mean ± SD of at least three independent experiments, and statistical analysis was performed by one-way ANOVA with Tukey test. (B) After treatment with 40 µM D4476 in the presence or absence of 10 µM Spautin-1 for 48 h, apoptosis was determined by Annexin V-FITC/PI. (C) Co-immunoprecipitation showed that CK1α interacted with MDM2 and p53 in HEL cells. Ten percent whole cell lysate was used as input samples. (D) Treatment with D4476 upregulated the level of p53 and MDM2 and phosphorylated AMPK, and inhibited the phosphorylation of mTOR in both HL-60 and HEL cells. (E) Treatment with D4476 did not significantly alter the level of MDM2 when CK1α was genetically inhibited in HEL cells. Images representing at least three independent experiments are shown. CK1α, casein kinase 1α; AML, acute myeloid leukemia; MDM2, murine double minute 2; mTOR, mammalian target of rapamycin; AMPK, 5′ AMP-activated protein kinase.