Rpmi medium

Several human lung carcinoid tumor cell lines (NCI-H727, UMC-11, NCI-H835, and NCI-H720) were provided by ATTC. Cells were maintained at 37 °C in 5% CO₂ and cultured in T75 flasks filled with 10 mL of RPMI medium (EuroClone™, Milan, Italy). Media was supplemented with 10% heat-activated fetal bovine serum (FBS) (EuroClone™, Milan, Italy) and 10⁵ U·L⁻¹ penicillin/streptomycin (EuroClone™, Milan, Italy). Cells were harvested by Trypsinization (Trypsin 0.05% and EDTA 0.02%) (Sigma-Aldrich^® Merck KGaA, Darmstadt, Germany), resuspended in complete medium, then counted through a Leica DM6000 B optical microscope (Leica Microsystems, Wetzlar, Germany) using a standard Beckman Coulter Z2 hemocytometer (Beckman Coulter, Brea, CA, USA) before plating. Cells used in all experiments were below 5 passages.

Freshly isolated CLL B and normal B cells were cultured in direct or indirect (TW) contact with MSCs and HS-5 at a ratio of 20:1. MSCs and HS-5 were cultured in 12 well plate with DMEM until confluence and then placed in RPMI medium (Euroclone), prior the addiction of B lymphocytes. Leukemic cells were collected after 3, 5 and 7 days, leaving intact the adherent layer, and examined for apoptosis status by staining with Annexin V-FITC accordingly to the manufacturer's instructions (Immunostep; Salamanca, Spain). Briefly, aliquots of 5×10⁵ cells were harvested, washed and incubated for 10 minutes in the dark and at RT with anti-CD19 APC (Invitrogen). Then, cells were washed and 100μl of binding buffer plus 5μl of Annexin V-FITC were added for further 10 minutes in the dark and at RT. After the incubation, 100μl of binding buffer were added and cells were analyzed by flow cytometer FACSCalibur. At least 20,000 events were collected using CellQuestPRO software.

U937-Mock cells (the U937 monoblastic cell line transfected with the empty Zn-inducible MT1 promoter vector) were used as the controls; U937-AETO (a zinc-inducible RUNX1/RUNX1T1 model), OCI-AML3 (an AML-M4-derived cell line carrying an NPM1 gene mutation (type A) and the DNMT3A R882C mutation) and OCI-AML2 (an AML-M4-derived cell line carrying the DNMT3A R635W mutation) were kindly provided by Emanuela Colombo, European Institute of Oncology, Milan, Italy. MV4-11 (a biphenotypic B myelomonocytic leukemia carrying the FLT3-ITD mutation and MLL/AF4 translocation) was purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). The cells were cultured in an RPMI medium (Euroclone; Pero, MI, Italy), 10% fetal bovine serum (FBS) (GIBCO-BRL), 20 mM of Hepes, 100 U/mL of penicillin and 100 µg/mL of streptomycin (GIBCO-BRL). The cultures were maintained at 37 °C in a 5% CO₂ humidified incubator.

Human cancer cell lines were obtained from ATCC and have not been cultured for longer than 6 months. H1299 (carcinoma, non-small-cell lung cancer) cells were cultured in Roswell Park Memorial Institute (RPMI) medium (Euroclone) supplemented with 10% fetal bovine serum (Euroclone), 1% L-glutamine (Gibco), and 1% penicillin/streptomycin (Euroclone). U-2 OS (osteosarcoma) cells were cultured in low- glucose Dulbecco’s modified Eagle’s (DMEM) medium (Lonza) with 10% fetal bovine serum (Gibco), 1% L-glutamine (Gibco), and 1% penicillin/streptomycin (Gibco). H1299 cells stably overexpressing FLAG-hTERT and hTR were generated through the transfection of the respective linearized vector and were selected using blasticidin (5 mg/mL) and puromycin (1 mg/mL).

M14 and A375 human melanoma cell lines were purchased from American Type Culture Collection (Manassas, VA). SBCL1 human melanoma cell line was provided by Bruno Giovanella^{53 (link)}. A375 human melanoma cells sensitive (A375S), resistant to Vemurafenib (A375R) or to Dabrafenib and Trametinib (A375DR), were previously characterized^{22 (link)}. HUVEC (PromoCell GmbH, Heidelberg, Germany) were cultured in complete EBM-2 medium (Clonetics Bio Whittaker, now Lonza, Cologne, Germany), containing 2% fetal bovine serum (FBS). All cell lines were grown in RPMI medium (Euroclone, Milan, IT) supplemented with 10% (v/v) FBS, 1% penicillin/streptomycin and 1% L-glutamine (Euroclone) at 37 °C in a balanced air humidified incubator with 5% CO_2. They have been routinely tested for mycoplasma contamination and were recently authenticated (STR profiling) and tested for mycoplasma contamination.

The cell lines used were described in our previous study [21 (link)]. CMT-U309 and CMT-U27 cells were cultured in Roswell Park Memorial Institute (RPMI) medium (Euroclone, Milan, Italy); P114 cells were maintained in Dulbecco’s Modified Eagle’s medium/Nutrient Mixture F-12 Ham (DMEM/F12) (Euroclone); and CF33 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Euroclone). All cell lines were supplemented with 10% fetal bovine serum (Euroclone), 100 IU/mL penicillin/100 μg/mL streptomycin (Euroclone), and 2 mM L-glutamine (Euroclone), and grown in an atmosphere of 5% CO₂ and 95% humidity at 37 °C. All cell lines were routinely tested for mycoplasma contamination.

Murine mes-c-myc A1 (A1) neurons^{74 (link)} were cultured with RPMI medium (EuroClone, Pero (MI), Italy) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine (EuroClone, Pero, Milano, Italy), 5% fetal bovine serum (Gibco-BRL, Milan, Italy), and maintained at 37 °C in humidified 5% CO₂ atmosphere.