![Inhibition of histone methylation and MLL-fusion target gene expression by EPZ-5676. (A) Immunoblot analysis of H3K79me2 levels in histones extracted from MV4-11 cells treated for 4 days with increasing concentrations of EPZ-5676. (B) Concentration-dependent inhibition of H3K79 methylation in MV4-11 (left) and HL60 (right) cells following 4-day EPZ-5676 treatment as measured by quantitative ELISA assay for H3K79me2. (C) Immunoblot analysis of histones extracted from MV4-11 cells treated for 4 days with either 1 µM EPZ-5676 or vehicle control and probed with a panel of specific methyl-lysine and methyl-arginine antibodies. Total H3 and H4 antibodies were used as the loading controls. (D) Time course of cellular H3K79me2 depletion in MV4-11 cells incubated in the presence of 1 µM EPZ-5676 (left). The diminution of intracellular H3K79me2 as a function of time after dosing with EPZ-5676 was fit to a simple exponential decay function that included a non-zero limit at infinite time, as described.39 Time course of recovery of H3K79me2 levels in MV4-11 cells upon removal of EPZ-5676 from the culture medium following a 4-day incubation (right). In both plots, H3K79me2 levels are normalized to total H3 and expressed as a percent of DMSO-treated (control) cells at each time point. Recovery of intracellular H3K79me2 levels following compound washout displayed a significant lag phase before semilinear recovery. These data were fit to a modified version of the expolinear function for crop growth as a function of photon interception and leaf area.40 The amount of H3K79me2 at any given time after compound washout (y) was fit as y = ymin +[(v/a)ln{1 + exp(a(t-tlag))}], where ymin is the non-zero limit of H3K79me2 at the start of the washout experiment, v is the velocity of the linear phase of recovery, and a is a constant of proportionality for our purposes. (E) Concentration dependent inhibition of HOXA9 and MEIS1 transcription by EPZ-5676 as measured by qRT-PCR in MV4-11 cells following 6 days of treatment (left; n = 2, mean ± SD are shown). Time course of inhibition of HOXA9 and MEIS1 mRNA expression as measured by qRT-PCR in MV4-11 cells incubated in the presence of 1 µM EPZ-5676 (right). Relative mRNA expression is plotted as a percentage of those at day 0 (n = 2; mean values ± SD are shown).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/122/6/10.1182_blood-2013-04-497644/4/1017f2.jpeg?Expires=1720283129&Signature=OpXSoKPeajE-ngUcaWzVI6afSFKoFqrsqFwfVv07yGssj0oSx-ZCyoQ4OaKdGmiIVkWWjvh6eAjOYYkHJ0QGYrHSYfzrji6oV0DbixxvjAS-G~z5jdUFxoRErsxgUvCNM~yiEoubur9hcTFtQxMnt4e0Inj5~GofsHs8RuNix~gP3zxY7vVaO~a9ObiHdalf-NRDbi0HNUQBt9tNJedEiHRMjjdsLhBFCSp28RMSwMaHx1E6EZG-HQAJowjUE~SAgQUMXqo4OvhTkKb8q2jcGJmAqjv7N5qOWMk7hkaGQD4Gooad355NOA44ZXz~3LGcoEOpOrArPpQylLpMYFJSOA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Inhibition of histone methylation and MLL-fusion target gene expression by EPZ-5676. (A) Immunoblot analysis of H3K79me2 levels in histones extracted from MV4-11 cells treated for 4 days with increasing concentrations of EPZ-5676. (B) Concentration-dependent inhibition of H3K79 methylation in MV4-11 (left) and HL60 (right) cells following 4-day EPZ-5676 treatment as measured by quantitative ELISA assay for H3K79me2. (C) Immunoblot analysis of histones extracted from MV4-11 cells treated for 4 days with either 1 µM EPZ-5676 or vehicle control and probed with a panel of specific methyl-lysine and methyl-arginine antibodies. Total H3 and H4 antibodies were used as the loading controls. (D) Time course of cellular H3K79me2 depletion in MV4-11 cells incubated in the presence of 1 µM EPZ-5676 (left). The diminution of intracellular H3K79me2 as a function of time after dosing with EPZ-5676 was fit to a simple exponential decay function that included a non-zero limit at infinite time, as described.39 Time course of recovery of H3K79me2 levels in MV4-11 cells upon removal of EPZ-5676 from the culture medium following a 4-day incubation (right). In both plots, H3K79me2 levels are normalized to total H3 and expressed as a percent of DMSO-treated (control) cells at each time point. Recovery of intracellular H3K79me2 levels following compound washout displayed a significant lag phase before semilinear recovery. These data were fit to a modified version of the expolinear function for crop growth as a function of photon interception and leaf area.40 The amount of H3K79me2 at any given time after compound washout (y) was fit as y = ymin +[(v/a)ln{1 + exp(a(t-tlag))}], where ymin is the non-zero limit of H3K79me2 at the start of the washout experiment, v is the velocity of the linear phase of recovery, and a is a constant of proportionality for our purposes. (E) Concentration dependent inhibition of HOXA9 and MEIS1 transcription by EPZ-5676 as measured by qRT-PCR in MV4-11 cells following 6 days of treatment (left; n = 2, mean ± SD are shown). Time course of inhibition of HOXA9 and MEIS1 mRNA expression as measured by qRT-PCR in MV4-11 cells incubated in the presence of 1 µM EPZ-5676 (right). Relative mRNA expression is plotted as a percentage of those at day 0 (n = 2; mean values ± SD are shown).
