Fig. 7
MAKV-8 treatment triggers autophagy and double strand breaks. K-562 cells were treated with the indicated concentrations of MAKV-8 at the indicated time points unless otherwise stated. (a) Cell morphology was analyzed after 48h of treatment using modified GIEMSA staining, and pictures were acquired by bright-field microscopy. (b) The appearance of autophagosome-related vesicles was quantified in cells treated with MAKV-8 for 8h. Representative pictures of cells stained with Hoechst in blue and Cyto-ID in green (left panel) and corresponding quantifications (right panel) from three independent experiments are provided. (c) After 8h of treatment, the conversion of LC3-I to LC3-II and expression of p62, two autophagic markers, were evaluated by western blot. Where indicated, bafilomycin A1 was added 2h before the end of treatment. (d) Representative images of electron microscopy analysis in indicated CML cell line: (1) phagophores and (2) autophagolysosomes. (e) The expression level of γH2AX, the earliest marker for DNA damage localized at double strand breaks, was assessed by western blot. Cisplatin (C, 50 µM) was used as a positive control for double strand break induction. Blots used β-actin as the loading control, and pictures are representative of three independent experiments