Hela cells

HeLa cells (Leibniz Institute DSMZ‐German Collection of Microorganisms and Cell Cultures GmbH, DSMZ no. ACC 57) were cultured at 37°C and 5% CO₂ in high glucose Dulbecco's Modified Eagle Medium with GlutaMAX™ Supplement (DMEM; Gibco) supplemented with 1 mM sodium pyruvate (Gibco), 10% foetal calf serum (Linaris) and 1% Penicillin–Streptomycin (Gibco). Cells were passaged when they were 80–90% confluent. Cells tested negative for mycoplasma contamination.

Materials for cell culture were all purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA) unless indicated otherwise. A549 cells (human lung adenocarcinoma cells; ATCC^® Manassas, VA, USA), CaCo-2 cells (human epithelial colorectal adenocarcinoma cells, HTB-37; ATCC, Manassas, VA, USA) and J774A.1 cells (murine macrophage-like cells; DSMZ, Braunschweig, Germany) were cultivated in DMEM, Vero cells (African green monkey kidney cells; DSMZ, Braunschweig, Germany) and HeLa cells (cervical carcinoma cells; DSMZ, Braunschweig, Germany) were cultivated in MEM. All media were supplemented with 10% FCS, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids and 100 U/mL penicillin and 100 g/mL streptomycin. CHO-K1 cells (Chinese hamster ovary cells strain K1; DSMZ, Braunschweig, Germany) were cultivated in DMEM and HAM’s F12 (1:1), supplemented with 5% FCS, 1 mM sodium pyruvate, 0.05 mM non-essential amino acids and 100 U/mL penicillin and 100 g/mL streptomycin. Cells were cultivated under humidified conditions at 37 °C with 5% CO₂ and trypsinized and reseeded every two to three days for at most 25 times. For intoxication experiments, cells were seeded in culture dishes one day before and treated in FCS-free media with PT and the respective compounds.

HeLa cells, purchased from DSMZ GmbH (Leibniz, Germany), and an ME-180 (HTB-33) cell line, purchased from ATCC (Manassas, VA, USA), were cultured in RPMI 1640 (Gibco, ThermoFischer, Waltham, MA, USA) and McCoy’s 5A (Lonza, Switzerland) supplemented with 2 mM L-glutamine, 10% fetal bovine serum (FBS) (Gibco, ThermoFischer, Waltham, MA, USA) in a humidified atmosphere of 5% CO₂ at 37 °C. Cisplatin (Santa Cruz Biotechnology, Dallas, TX, USA) treatments were carried out in triplicates essentially as described previously [12 (link)]. 0.1% (v/v) DMSO was used as a negative control for Cisplatin. Treated cells were trypsinized by 1X Trypsin-EDTA (Gibco, ThermoFischer, Waltham, MA, USA) and washed in 1X cold PBS (Gibco, ThermoFischer, Waltham, MA, USA). Cell pellets were stained with Annexin V-PE (BD Biosciences, Franklin Lakes, NJ, USA) and 7AAD (BD Biosciences, Franklin Lakes, NJ, USA) in the presence of 1X Annexin binding buffer (BD Biosciences, Franklin Lakes, NJ, USA) followed by incubation at room temperature for 15 min in the dark. The rates of Annexin V and/or 7AAD-positive cells were quantified by a FACSCanto flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

VCR-R CEM and CCRF-CEM cells were obtained from Prof. Maria Kavallaris (University of New South Wales, Sydney, Australia) [21 (link)] and were cultured in RPMI 1640 (PAN Biotech, Aidenbach, Germany) containing 10% FCS (PAN Biotech). VCR-R CEM cells are a multidrug-resistant subline of CCRF-CEM cells [22 (link)], generated by long-term exposure to increasing concentrations of vincristine [23 (link)]. Jurkat cells were bought from ATCC and cultured in RPMI 1640 containing 10% FCS and pyruvate. Cell lines were typically passaged twice to thrice a week. HeLa cells were purchased from DSMZ (Braunschweig, Germany) and cultured in DMEM (PAN Biotech) containing 10% FCS. The model of ALL patients’ leukemia cells growing in mice has been described previously [24 (link)]. In the present study, patient-derived xenograft (PDX) cells were engrafted and freshly isolated from the bone marrow or spleen of NSG mice (The Jackson Laboratory, Bar Harbour, ME, USA) and subsequently cultured in RPMI 1640 supplemented with 20% FCS and P/S. Peripheral blood mononuclear cells (PBMC) were obtained from ATCC and cultured in RPMI 1640 supplemented with 20% FCS and P/S.
Cell line STR profiling was performed. None of the cell lines used are listed in the database of commonly misidentified cell lines maintained by ICLAC. All cells are proven to be mycoplasma-free quarterly.

HeLa cells (DSMZ, Germany) were cultured in growth medium (D-MEM, 1% MEM, 10% heat-inactivated fetal calf serum and 1% penicillin/streptomycin; Gibco, Germany) in T-75 cm² culture flasks (Greiner, Frickenhausen) and incubated at 37°C in a 5% CO₂ incubator. HFF cells were also grown in the same culture medium like HeLa cells but devoid of 1% MEM. Cells were allowed to grow to 80 - 90% confluence and were sub-cultured as follows: culture medium was discarded and cells were washed briefly two times with pre-warmed PBS, pH 7.4. Thereafter, cells were trypsinized with 1 ml Tryple Express per flask (Gibco, Germany) and incubated for 3 min at 37°C and 5% CO₂. Cell detachment from the culture flasks with Tryple Express was halted by adding 10 ml of culture medium and the cell suspension was transferred to a 15 ml centrifuge tube. Centrifugation was done for 5 min at 200 × g and the cell pellet was re-suspended in 3–5 ml culture medium.

Cell line sources are as follows: HeLa cells for single-molecule imaging were from the German Collection of Microorganisms and Cell Cultures GmbH (ACC 57); Expi293F cells (Thermo Fisher); Expi293F GnTI- (Thermo Fisher); IL-17C HEK-Blue NF-kB Reporter cells (Invivogen). Cell lines were authenticated as follows: HeLa cells (German Collection of Microorganisms and Cell Cultures GmbH), Expi293F cells (Thermo Fisher), Expi293F GnTI⁻ (Thermo Fisher) and IL-17C HEK-Blue NF-κB reporter cells (Invivogen) were guaranteed by the suppliers and no additional authentication was performed by the authors of this study. HeLa cells for single-molecule imaging tested negatively for mycoplasma (PCR). Expi293F cells (Thermo Fisher) and Expi293F GnTI⁻ cells (Thermo Fisher) used for protein production were not tested for mycoplasma contamination by the authors of this study. The IL-17C HEK-Blue NF-κB reporter cells (Invivogen) were tested for mycoplasma contamination by the manufacturer and no additional testing was performed by the authors of this study.