Cytotoxic agents were purchased from Sigma-Aldrich. Ara-C was reconstituted in DMSO and daunorubicin or 5′-Aza in distilled H2O, and aliquots were prepared and stored at –80°C. Stocks were diluted in CM immediately prior to use in cytotoxicity assays.
To compare cell proliferation between parental and CRISPR/Cas9-mutated HEL clones, exponentially growing cells were seeded at low density (2 × 104 cells/mL) in CM and counted using a hemocytometer at regular intervals up to 192 hours postseeding. Cell growth at each time point was calculated relative to initial seeding density. Two-way ANOVA was used to test for significant differences in relative cell growth based on TET2 mutation status.
For drug sensitivity experiments, cells were incubated in CM supplemented with appropriate concentrations of cytotoxic agent (5′-Aza, daunorubicin, or Ara-C) or relevant VC for 96 hours, after which viable cells were identified by trypan blue dye exclusion and counted using a hemocytometer. Survival fractions were determined at each drug concentration relative to VC-treated controls. Two-way ANOVA was used to test for significant differences in drug sensitivity based on TET2 mutation status. Inhibition of proliferation in drug-treated cultures was compared with VC-treated cultures and used to calculate the IC50 and IC90 values in GraphPad Prism (6.0.2, GraphPad Software).
For determination of CE, exponentially growing cells were seeded in soft agar (CM supplemented with 0.2% agarose) supplemented with cytotoxic agent (5′-Aza, daunorubicin, or Ara-C) or VC. Macroscopically visible colonies were counted on day 30, and CE was calculated relative to number of cells initially seeded. Student’s t tests (2-tailed) were used to identify significant differences in CE based on TET2 mutation status.
To determine the effect of ABCB1 inhibition on 5′-Aza sensitivity, cells were incubated in CM supplemented with increasing doses of 5′-Aza and an ABCB1 inhibitor (verapamil or tariquidar) or VC. After 96 hours of incubation, viable cells were identified using CellTiter-Glo Luminescent Cell Viability Assay (Promega), and the surviving fraction was determined at each drug concentration relative to VC-treated controls. The resulting dose-response matrix was used to calculate drug synergy using SynergyFinder 2.0. Student’s t test (2-tailed) was used to identify significant differences in synergy scores based on TET2 mutation status.
All assays were performed in triplicate at a minimum and means ± SD were calculated.
To compare cell proliferation between parental and CRISPR/Cas9-mutated HEL clones, exponentially growing cells were seeded at low density (2 × 104 cells/mL) in CM and counted using a hemocytometer at regular intervals up to 192 hours postseeding. Cell growth at each time point was calculated relative to initial seeding density. Two-way ANOVA was used to test for significant differences in relative cell growth based on TET2 mutation status.
For drug sensitivity experiments, cells were incubated in CM supplemented with appropriate concentrations of cytotoxic agent (5′-Aza, daunorubicin, or Ara-C) or relevant VC for 96 hours, after which viable cells were identified by trypan blue dye exclusion and counted using a hemocytometer. Survival fractions were determined at each drug concentration relative to VC-treated controls. Two-way ANOVA was used to test for significant differences in drug sensitivity based on TET2 mutation status. Inhibition of proliferation in drug-treated cultures was compared with VC-treated cultures and used to calculate the IC50 and IC90 values in GraphPad Prism (6.0.2, GraphPad Software).
For determination of CE, exponentially growing cells were seeded in soft agar (CM supplemented with 0.2% agarose) supplemented with cytotoxic agent (5′-Aza, daunorubicin, or Ara-C) or VC. Macroscopically visible colonies were counted on day 30, and CE was calculated relative to number of cells initially seeded. Student’s t tests (2-tailed) were used to identify significant differences in CE based on TET2 mutation status.
To determine the effect of ABCB1 inhibition on 5′-Aza sensitivity, cells were incubated in CM supplemented with increasing doses of 5′-Aza and an ABCB1 inhibitor (verapamil or tariquidar) or VC. After 96 hours of incubation, viable cells were identified using CellTiter-Glo Luminescent Cell Viability Assay (Promega), and the surviving fraction was determined at each drug concentration relative to VC-treated controls. The resulting dose-response matrix was used to calculate drug synergy using SynergyFinder 2.0. Student’s t test (2-tailed) was used to identify significant differences in synergy scores based on TET2 mutation status.
All assays were performed in triplicate at a minimum and means ± SD were calculated.
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