Enhanced expression of ATG4B and induced autophagic flux in CD34⁺ CML cells upon IM treatment. (A) Quantitative real-time PCR analysis of transcript levels of ATG4 family members in CD34⁺ CML cells from 3 patient samples in the presence and absence of IM (5 µM) after in vitro incubation of cells for 6 hours. Values shown are the mean ± SEM of triplicate measurements of each transcript normalized to β2M and relative to uncultured CD34⁺ cells. (B) Western blotting analysis of ATG4B protein expression in the same cells upon IM treatment in vitro. Protein expression of ATG4B relative to actin was compared. Values shown are the mean ± SEM of measurements. (C) Western blotting analysis of LC3-II levels in CD34⁺ CML cells from 3 patient samples treated with IM (5 µM), DA, (150 nM), NL (5 µM), CQ (10 µM), or TKI + CQ for 5 hours. LC3 antibody used preferentially recognized LC3-II. Protein expression of LC3-II relative to actin was compared, and relative levels of LC3-II protein expression in treated cells compared with control cells (no treatment) are shown. (D) A representative sample of the FACS profiles of intracellular Cyto-ID green detection dye in CD34⁺ CML cells in the presence or absence of IM (5 µM), CQ (10 µM), or IM + CQ for 5 hours (left). Detection of the differences in mean fluorescence intensity of intracellular Cyto-ID green detection dye obtained from IM nonresponders in the presence or absence of indicated inhibitors (n = 4, right). The increase in fluorescence intensity in the IM + CQ–treated samples relative to IM or CQ treatment is indicative of autophagic flux. Values shown are the mean ± SEM of measurements from 4 CML patient samples.