Lentiviral-mediated knockdown of ATG4B in K562 and K562 IM-resistant cells impairs autophagy and reduces viability and growth of these cells. (A-B) Western blot analysis of cell lysates from K562 cells (A) and IM-resistant cells (IMR K562) (B) transduced with either a nontargeting shRNA control sequence (SHC) or constructs containing each of 3 different shRNA targeting sequences against human ATG4B (shATG4B). Antibodies used are indicated. The LC3-II/LC3-I ratios measured in transduced control and shATG4B cells are shown. Accumulation of the autophagy markers LC3-II and p62 in ATG4B knockdown cells indicates a block in autophagy. (C-D) Viability of transduced K562 (C) and IM-resistant cells (D) was measured ±IM after 48 hours and apoptosis of these cells determined by Annexin V staining and FACS analysis. (E-F) Growth of transduced K562 (E) and IM-resistant cells (F) was measured every 24 hours for 72 hours. (G-H) Numbers of colonies produced from transduced K562 (G) and IM-resistant cells (H) in semisolid culture ±IM or DMSO (control). Colony numbers for large (>500 cells), medium (50-500 cells), and small (<50 cells) are indicated. Data shown are the mean ± SEM of measurements from 3 (K562) or 2 (IMR K562) independent experiments. Asterisk indicates significant difference between control and ATG4B knockdown cells at *P < .05, **P < .01, and ***P < .001.