Methocult 4230

Colony‐forming assays were performed [22]. Cells were plated into MethoCult 4230 (StemCell Technologies), supplemented with 100 ng/ml SCF, TPO, granulocyte colony‐stimulating factor (G‐CSF; Amgen, Thousand Oaks, CA, http://www.amgen.com), granulocyte macrophage colony‐stimulating factor, interleukin‐3 (IL‐3), IL‐6, and 4 U/ml erythropoietin (all from Peprotech), and scored 10 days later.

Colony formation capacity was evaluated out in semisolid methylcellulose medium (2.5 × 10³ cells/mL for SET2 cells; 1 × 10³ cells/mL for HEL cells; MethoCult 4230; StemCell Technologies Inc., Vancouver, BC, Canada) after reversine exposure for 36 hours (equal number of viable cells were plated for each condition). Additionally, long-term exposure was also evaluated. Colonies were detected after 10 days of culture by adding 1 mg/mL of MTT reagent and scored by Image J quantification software (U.S. National Institutes of Health, Bethesda, MD, USA).

Cell viability was measured by methylthiazole tetrazolium (MTT) assay for HEL and U937 cells. A total of 5×10⁴ ShControl or shIRS2 cells per well were plated in a 96-well plate in RPMI 10% FBS at different concentrations of ruxolitinib (100 or 300nM) or with DMSO for 48h. Briefly, 10μL of a 5mg/mL solution of MTT was added to the wells and incubated at 37°C for 4h. The reaction was stopped by using 100μL of 0.1 N HCl in anhydrous isopropanol and was evaluated by measuring the absorbance at 570nm using an automated plate reader. Colony formation was carried out in semisolid methyl cellulose medium (0.5×10³ cell/mL; MethoCult 4230; StemCell Technologies Inc., Vancouver, BC, Canada). Colonies were detected after 8 days of culture by adding 1mg/mL of MTT reagent and scored by Image J quantification software (US National Institutes of Health, Bethesda, MD, USA). For HEL and U937 cell lines, shControl and shIRS2 cells were submitted to colony formation assay in the presence of ruxolitinib in two different concentrations (100 or 300nM) or DMSO for 8 days.

Colony formation was carried out in semisolid methyl cellulose medium (1×10³ cell/mL; MethoCult 4230; StemCell Technologies Inc., Vancouver, BC, Canada). Colonies were detected after 10 days of culture by adding 1 mg/mL of MTT reagent and scored by Image J quantification software (U.S. National Institutes of Health, Bethesda, MD, USA).

Colony formation capacity was evaluated out in semisolid methylcellulose medium (1.5 × 10³ cells/ml; MethoCult 4230; StemCell Technologies Inc., Canada). Clonogenic assays were performed in the presence of medium alone (without serum), vehicle (DMSO control 0.01%) and ATRA (0.1 and 0.5 µM). Colonies were detected after 10 days of culture by adding 1 mg/ml of MTT reagent and scored with the Image J quantification software (US National Institutes of Health, USA).

Colony formation was conducted in a semi-solid methylcellulose medium (0.5 × 10³ cells/mL; MethoCult 4230; StemCell Technologies Inc., Vancouver, BC, Canada). The NB4, NB4-R2, MOLM3, OCI-AML3, Jurkat, and Namalwa cells were seeded in the presence of a vehicle or eribulin (0.12, 0.25, 0.5, or 1 nM) for eight days. The colonies were detected by adding 100 µL (5 mg/mL) of MTT reagent, and they were scored using the Image J quantification software (U.S. National Institutes of Health, Bethesda, MD, USA).