circMYBL2 regulates FLT3-ITD–dependent signaling pathways by modulating the translation of mutant FLT3 kinase. (A) Western blot showing downregulation of FLT3 protein expression upon circMYBL2 knockdown in MOLM-13 and MV4-11 cells. (B) qPCR measuring the expression of FLT3 mRNA upon circMYBL2 knockdown in MOLM-13 and MV4-11 cells. (C) Downregulation of FLT3 protein expression upon circMYBL2 knockdown in FLT3-ITD AML patient samples. (D) qPCR measuring the expression of FLT3 mRNA upon circMYBL2 knockdown in FLT3-ITD AML patient samples. (E-F) Western blot showing the downregulation of STAT5 signaling activation upon circMYBL2 knockdown in MOLM-13 and MV4-11 cells and in AML patient samples. (G) Flow cytometric analysis of apoptosis in MV4-11 cells upon circMYBL2 knockdown or c-MYC overexpression. The numbers indicate the percentages of cells. (H) Western blot showing the decrease in FLT3 protein, p-STAT5, and c-MYC levels upon circMYBL2 knockdown in MOLM-13 and MOLM-13-RQ cells. (I) circMYBL2 knockdown reduced the levels of FLT3 kinase harboring the D835Y mutation in an AML patient sample. (J) Polysomes in cytoplasmic extracts of sh-NC and sh-circMYBL2 MV4-11 cells were fractionated through sucrose gradients, and the relative levels of FLT3 mRNA were analyzed by qRT-PCR in the gradient fractions. ns, not significant.