Dulbecco s modified eagle s glutamax medium

Total nucleated cells were isolated from the washout and filters used for bone marrow collection (n = 3). Cells were counted and plated at 100,000 cells/cm² in low glucose Dulbecco’s modified Eagle’s medium + glutamax (Gibco Invitrogen, Carlsbad, CA, USA) containing penicillin/streptomycin (Sigma-Aldrich, Saint Louis, Missouri, USA) and heparin 30 U/ml (Hospira Italia, Napoli, I) supplemented with 10% FBS (Gibco Invitrogen, Carlsbad, CA, USA) or 7.5% platelet derivatives. Cells were incubated at 37 °C and 5% CO₂ concentration. After 3–4 days in culture, non-adherent cells were removed and fresh medium was added (P0). The resulting plastic adherent cells were expanded until 80–90% confluence and then harvested with TrypLE™ Select (Gibco Invitrogen, Carlsbad, CA, USA) and counted (P0).
BM-MSC were thawed and expanded in presence of 10% FBS or 5, 7.5 and 10% PL or PR-SRGF. At each sub-cultivation, the population doubling (PD) was calculated as follows: PD = log10(N)/log10(2); where N is the number of cells harvested-the number of cells initially seeded. The cumulative PD (cPD) was calculated adding to the PD of the passage under analysis the PDs of the previous passages.

COS7 cells were cultured in Dulbecco’s modified Eagle’s medium/GlutaMAX (Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen) and 100 units/ml penicillin/streptomycin (Invitrogen) under 5% CO₂ at 37 °C. The cells were transiently transfected with HA-tagged TMEM30A or Flag-tagged ATP8A2. After 2 days, the transfected cells were fixed with 4% paraformaldehyde, and the TMEM30A proteins were visualized using rat anti-HA antibody (Roche, Redwood City, CA, USA). The ER marker proteins were labeled with a rabbit anti-Calnexin antibody (Cell Signaling Technology, CA, USA), the Golgi markers were labeled with a rabbit anti-GM130 antibody (BD Biosciences, Mississauga, ON). The early endosomes were labeled with an antibody against the specific marker Rab11a (BD Biosciences, Heidelberg, Germany). The secondary antibodies used included goat anti-rabbit Alexa 488, goat anti-rabbit Alexa 594, goat anti-mouse Alexa 488, goat anti-mouse Alexa 594, donkey anti-rat Alexa 488 and donkey anti-rat Alexa 633 (Invitrogen). The images were captured under a Zeiss LSM 800 confocal scanning microscope.

HeLa (kind gift of C. Passananti), H1299 (kind gift of G. Blandino), U2OS (kind gift of F. Moretti), hTERT-immortalized HF [56 (link)], HCT116 parental or DICER-defective (kind gift of B. Vogelstein) were cultured at 37 °C and 5% CO₂ in Dulbecco’s modified Eagle’s medium GlutaMAX supplemented with 10% fetal bovine serum, penicillin/streptomycin (Life Technologies) and routinely tested for mycoplasma contamination. Cells were transfected using Lipofectamine LTX and PLUS reagent for plasmid DNA and RNAiMAX for small interfering RNAs (siRNAs; Life Technologies). HIPK2-specific RNA interference was performed as described [19 (link)]. Kinase inhibitor treatments were performed on unsynchronized, proliferating cells. Hesperadin (Selleckchem) and ZM-447439 (Signalchem) were used to inhibit Aurora-B and Bi 2536 (AbMole) to inhibit PLK1. Solvent dimethylsulfoxide (DMSO; Sigma-Aldrich) was used as control.