Lentiviral-mediated knockdown of ATG4B blocks autophagy, impairs the survival of CD34⁺ CML stem/progenitor cells and sensitizes these cells to IM. (A) Western blot analysis of cell lysates obtained from CD34⁺ cells of 3 CML patient samples transduced with either a nontargeting shRNA control (SHC) or constructs containing one of 3 different shRNA targeting sequences against human ATG4B (shATG4B). Antibodies used are indicated. (B) The same transduced cells were stained with an anti-LC3B antibody and analyzed by confocal microscopy. Representative images at maximum projection are shown. An accumulation of endogenous LC3B puncta upon ATG4B knockdown is observed. (C) Viability of the same transduced cells was measured ±IM after 72 hours and apoptosis of these cells determined by Annexin V staining and FACS analysis. (D) Growth of the same transduced CD34⁺ CML cells was measured every 24 hours for 72 hours. (E) Numbers and types of colonies produced from the same transduced cells in semisolid culture ±IM (5 µM) or DMSO (control). (F) LTC-initiating cell-derived colony numbers (LTC-IC-derived CFCs) were measured in transduced CD34⁺ cells from 2 CML patient samples ±IM (5 µM) or DMSO (control). Data shown are the mean ± SEM of all 3 CML patient samples or technical duplicates for each CML patient sample. Asterisk indicates significant difference between control and ATG4B knockdown cells at *P < .05, **P < .01, and ***P < .001.