Extended Data Fig. 2. Nuclear HK2 over-expression enhances stemness.
(a) cDNA construct of HK2 fused in frame with c-Myc (NLS1) or SV40 (NLS2) Nuclear Localizing Signal (NLS). (b) Representative confocal microscopy images of HK2 (green) or Tom20 (red) in NB4 cells 5 days after transduction with NLS-HK2 or EV. Scale bar= 10µm. Representative images from 3 biologic repeats. (c) Quantification of fluorescence intensity of nuclear HK2 in NB4 cells after transduction with NLS-HK2 or EV. n = 114 cells examined from 3 biologic repeats. (d) NB4 cells were transduced with NLS- HK2 or EV. The level of HK2 in the nucleus and whole-cell lysates was measured after 5 days of transduction using immunoblotting. The densitometry plots of relative HK2 expression were performed in subcellular lysates. n = 3 biologic repeats. (e) Representative confocal microscopy images of HK2 (green) or Tom20 (red) in NLS1-HK2 NB4 cells after transduction with shRNAs targeting the UTR of HK2. Scale bar= 10µm. Representative image from 3 biologic repeats. (f) Growth and viability of NB4 cells at increasing times after transduction with NLS-HK2. n = 5 biologic repeats. (g) Clonogenic growth of NB4 cells transduced with NLS-HK2 and treated with ATRA (100nM). n = 3 biologic repeats. (h) Expression of CD11b in NB4 cells transduced with NLS-HK2 and treated with ATRA (100nM). n = 4 biologic repeats. (i) NB4 cells were treated with ATRA for 72hrs and subcellular fractions were analysed for HK2 by immunoblot. n = 3 biologic repeats. (j) The densitometry plots of relative HK2 expression were performed in subcellular lysates. (k) Representative confocal microscopy images of HK2 (green) and Tom20 (red) in NB4 cells treated with ATRA. Scale bar= 10µm. Representative image from 2 biologic repeats. (l) Representative confocal microscopy images of HK2 (green) in 8227 cells 5 days after transduction with NLS-HK2 or EV. Scale bar= 10µm. Representative image from 3 biologic repeats. (m) Growth and viability of 8227 after transduction with NLS-HK2. n = 2 biologic repeats. (n) Percentage of CD34+CD38- cells in 8227 cells, n = 2 biologic repeats and (o) 130578 cells, n = 2 biologic repeats transduced with NLS-HK2 and treated with ATRA (100nM). (p) TEX cells were transduced with NLS1-HK2 or EV. Subcellular fractions were analysed for HK2 by immunoblot 5 days post transduction. (q) TEX cells transduced with NLS1-HK2 or EV were injected into NSGF mice. Survival of the mice was measured over 75 days (n = 9, EV mice, n = 10, NLS1-HK2 mice). Statistical analyses was performed using a two-tailed unpaired Student’s t-test (d, j) and Ordinary Two-way ANOVA, Tukeys multiple comparison test (g, h). Data from represent mean +/- s.e.m, except (m-o) which represents the mean. A Kaplan Meier curve analysed survival using the gehan-breslow-wilcoxon test.