Obatoclax mesylate

Obatoclax Mesylate (GX15-070) (Selleck chemicals, Texas, USA) was resuspended in DMSO to prepare a stock solution of 20 mM, aliquoted and stored at −20°C. It was brought to the desired working concentration by appropriately diluting in culture medium. 0.001% DMSO was used as vehicle control across all experiments. The pan-caspase inhibitor Z-VAD-FMK (Abcam, Cambridge, UK), Chloroquine and Necrostatin-1 (Calbiochem, USA) were used at 50 μM concentration. MTT reagent was dissolved in phosphate buffered saline (PBS) to a final concentration of 5 mg/ml. Changes in the mitochondrial network architecture were studied by using MitoTracker Green, MitoTracker Red CMXRos and MitoSOX Red dyes (Molecular probes, Invitrogen, USA). DAPI (4′,6-diamidino-2-phenylindole) or Hoechst 33342 (BD Biosciences, New Jersey, USA) were used to visualize nuclei. All standard chemicals were purchased from Sigma unless otherwise indicated. pBABE-puro mCherry-EGFP-LC3B construct was a gift from Jayanta Debnath (Addgene plasmid #22418). Control/Scrambled siRNA, siGLO, siRNA against human MCL-1 and ATG5 were obtained from Dharmacon (CO, USA). siRNA transfections were performed using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions.

Ponatinib was purchased from LC Laboratories (Woburn, MA, USA). Trabectedin was obtained from PharmaMar (Colmenar Viejo, Spain). Cisplatin was kindly provided by the Institute of Inorganic Chemistry, University of Vienna. Ponatinib, dasatinib, obatoclax mesylate, venetoclax, vincristine, PD173074, and stattic were obtained from Selleckchem (EUBIO, ANDREAS KÖCK e.U., Vienna, Austria). Nintedanib and imatinib were purchased from LC Laboratories (Woburn, MA, USA). Doxorubicin was acquired from Sigma-Aldrich (St. Louis, MO, USA). Paclitaxel was procured from Bristol Myers Squibb (New York City, NY, USA).

Obatoclax mesylate (OBAT) was purchased from Selleck Chemicals (Munich, Germany). The OBAT was dissolved in DMSO and stored as 5 mM stock solutions at −20 °C. RPMI 1640 medium and fetal calf serum were obtained from GIBCO BRL Life Technologies (Gaithersburg, MD, USA). L-glutamine, antibiotic antimycotic solution (AAS), dimethyl sulfoxide (DMSO), NBT, phorbol 12-myristate 13-acetate (PMA) and anti-β-actin antibody (cat. #A5316) were purchased from Sigma Aldrich (St. Louis, MO, USA). The FITC Annexin V Apoptosis Detection Kit I, propidium iodide (PI)/RNase staining buffer and anti-CD-11b were obtained from BD Biosciences (San Jose, CA, USA). The CellEvent™ Caspase 3/7 Green Flow Cytometry Assay kit was purchased from Molecular Probes (Eugene, OR, USA). The Hemacolor® Rapid Staining kit was obtained from Merck Millipore (Darmstadt, Germany). The antibody against Bcl-2 (cat. #2876) and horseradish peroxidase-conjugated secondary antibody (cat. #7074) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Obatoclax mesylate and ABT-737 were purchased from Selleck Chemicals (Munich, Germany), dissolved in DMSO and stored as 5 mM stock solutions at −20 °C. Mafosfamide cyclohexylamine salt was kindly provided by Dr Ulf Niemeyer (NIOMECH, Bielefeld, Germany) and dissolved in aqua pro injectione (Polpharma, Poland) directly before treatment of cells. Daunorubicin was purchased from Sigma Aldrich (St. Louis, MO, USA) and stored as 0.5 mM stock solutions in aqua pro injectione at −20 °C. RPMI 1640 medium and fetal calf serum were from GIBCO BRL Life Technologies (Gaithersburg, MD, USA). L-glutamine, antibiotic antimycotic solution (AAS), dimethyl sulfoxide (DMSO), (4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and acridine orange were purchased from Sigma Aldrich. FITC Annexin V Apoptosis Detection Kit I was obtained from BD Pharmingen (BD Biosciences, San Diego, CA).

Reagents used for treatments included GSK1120212 (Selleck), Obatoclax mesylate (Selleck), ABT-263 (Selleck), Doxorubicin (Selleck), Z-VAD-FMK (R&D Systems), Necrostatin-1 (Tocris), L-glutamine (Gibco), Methyl-pyruvate (Sigma), 3-MA (Sigma), Concanamycin C (ThermoFisher) and Bafilomycin A1 (Sigma).