Fig. 6. Nuclear HK2 overexpression enhances the DNA-damage response and increases chemoresistance in AML.
a, Levels of γH2AX in NLS1–HK2 and EV control 8227 cells after treatment with 50 nM daunorubicin for 0, 3 and 6 h; n = 2,041 cells from four biological repeats were examined. b, Levels of γH2AX in stem and bulk 8227 cells after treatment with 50 nM daunorubicin for 0, 3 and 6 h; n = 1,786 cells from three biological repeats were examined. c, Levels of 53BP1 in NLS1–HK2 and EV control 8227 cells after treatment with 50 nM daunorubicin for 3 h; n = 645 cells from four biological repeats were examined. d, Levels of 53BP1 levels in stem and bulk 8227 cells after treatment with 50 nM daunorubicin for 3 h; n = 549 cells from three biological repeats were examined. e, Levels of RAD51 in NLS1–HK2 and EV control 8227 cells after treatment with 50 nM daunorubicin for 6 h; n = 551 cells from three biological repeats were examined. f, Levels of RAD51 in stem and bulk 8227 cells after treatment with 50 nM daunorubicin for 6 h; n = 477 cells from two biological repeats were examined. g, Clonogenic growth of NB4 cells transduced with NLS1–HK2 and treated with 50 nM daunorubicin for 3 h before plating. The colonies were counted 6 d after plating; n = 3 biological repeats. h, Comet assay in 8227 cells transduced with NLS1–HK2 before and after incubation with 70 nM daunorubicin for 6 h; n = 1,474 cells from three biological repeats were examined. i, Relative messenger RNA expression levels of genes associated with homologous recombination (XRCC2 and XRCC3) and non-homologous end joining (XRCC5, XRCC6 and PRKDC) in stem and bulk 8227 cells; n = 3 biological repeats. j, GSEA analysis of DNA-repair pathways in primary samples from patients with undifferentiated versus committed AML. The NES and FDR values were analysed using a modified Kolmogorov–Smirnov test. k, Levels of γH2AX in stem and bulk fractions of primary sample from a patient with AML (AML151258) after treatment with 50 nM daunorubicin for 0 and 3 h; n = 225 cells were examined from one of two biological samples. l, Levels of 53BP1 in the stem and bulk fractions of a primary sample from a patient with AML (AML151258) following treatment with 50 nM daunorubicin for 3 h; n = 111 cells were examined from one of two biological samples. a–f,k,l, The protein levels were determined using confocal microscopy. Statistical analyses were performed using a two-tailed unpaired Student’s t-test in all panels except j, where a Fischer’s exact t-test was performed. Data represent the median and interquartile range (a–e,h), the mean (f,k,l) or the mean ± s.e.m. (g,i). Dauno, daunorubicin; a.u., arbitrary units.