Rpmi 1640 medium

Primary satellite cells were isolated from muscle specimens (six patients and six controls), through enzymatic digestion. Briefly the tissue was mechanically fragmented and digested with collagenase/dispase (Roche). The tissue homogenate was then separated through a 40 µm mesh filter and seeded into culture plates. Satellite cells were maintained in RPMI 1640 medium (EuroClone), supplemented with 1% L-glutamine (EuroClone), 1% antibiotics (penicillin 100 IU/ml, streptomycin 100 mg/ml, EuroClone) and 15% fetal bovine serum (Gibco).
The myogenicity of the cell populations was assessed by immunofluorescence using an antibody against desmin (Thermo Fisher Scientific, #PA5-17182), specifically expressed in myogenic cells, and counting the number of desmin-positive cells. Cultures displaying over 70% myogenic cells were selected for further in vitro experiments. To induce the myogenic differentiation, cells were shifted to RPMI 1640 medium with 5% horse serum (EuroClone).

DU-145, 22Rv1 and LNCaP cells were grown in RPMI Medium 1640 (EuroClone) whereas PC3 cells were grown in HAM’s Medium (Euroclone, Milan, Italy), V-CaP cells in DMEM (EuroClone) and HCT116 Dicer^-/- cells in McCOY’s (EuroClone). All the media were completed by adding 10% FBS (Fetal Bovine Serum, EuroClone), 1% penicillin/streptomycin (EuroClone) and 1% L-glutamine (Sigma-Aldrich). The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂.

DU-145 and A-549 cells were grown in RPMI Medium 1640 (EuroClone) whereas PC-3 cells were grown in HAM's medium (EuroClone) and MCF-7 cells in DMEM low glucose (EuroClone). 10% FBS (fetal bovine serum, EuroClone), 1% penicillin/streptomycin (2 mM, EuroClone), and 1% L-glutamine (2 mM, Sigma-Aldrich) were added to the medium. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂.

SW1736 and 8505C cells, derived from human ATC, were cultured in RPMI-1640 medium (Euroclone S.p.A, Milano, Itlay) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 2 mM L-glutamine (Euroclone S.p.A) and 50 mg/mL gentamicin (Gibco; Thermo Fisher Scientific, Inc.). Cells were cultured in a humidified incubator (5% CO₂ and 95% air at 37 °C) (Eppendorf AG, Hamburg, Germany). Both cell lines were validated using short tandem repeat analysis and confirmed to be mycoplasma-free. SW1736 and 8505C cells were treated with DMSO (vehicle; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), DHT (purity ≥ 99%; Selleck Chemicals, Houston, TX, USA) or Paclitaxel (Selleck Chemicals). The DHT was prepared by dissolving 2 mg of powder in 2 mL of DMSO, resulting in a 3.6 mM stocking solution.
To obtain ATC Paclitaxel-resistant cell lines (SW1736-PTX and 8505C-PTX), the two parental cell lines were treated with increasing doses of Paclitaxel arising from 1 nM to 1.5 μM for 6 months. Resistance to pacitaxel was considered to be acquired when a 1.5 μM dose resulted in no effect in terms of cell viability. Resistant cells were cultured in RPMI-1640 medium supplemented with 10%, 2 mM L-glutamine, 50 mg/mL gentamicin and Paclitaxel 1.5 μM.

In this study, we used four different thyroid cell lines: Nthy-ori-3.1, derived from normal thyroid follicular epithelial cells and immortalized by the SV40 large T antigen; BCPAP, derived from papillary thyroid carcinoma; SW1736 and 8505C, from anaplastic thyroid cancer. All cell lines have been validated by short tandem repeat and tested for being mycoplasma-free. Cells were grown in RPMI 1640 medium (EuroClone, Milan, Italy) supplemented with 10% fetal bovine serum (Gibco Invitrogen, Milan, Italy), 2 mM L-glutamine (EuroClone, Milan, Italy), and 50 mg/mL gentamicin (Gibco Invitrogen, Milan, Italy). Cells were maintained in a humidified incubator (5% CO₂, 37 °C).

Human lung (H157),
colon (HCT-15 and
LoVo), and pancreatic (PSN-1) carcinoma cells were obtained from the
American Type Culture Collection (ATCC, Rockville, MD, USA). Human
cervical carcinoma A431 cells were kindly provided by Prof. F. Zunino
(Division of Experimental Oncology B, Istituto Nazionale dei Tumori,
Milan). Human ovarian adenocarcinoma 2008 cells were kindly provided
by Prof. G. Marverti (Dipartimento di Scienze Biomediche, Università
di Modena University, Italy). Human colon cancer cells resistant to
doxorubicin (LoVo MDR) were kindly provided by Prof. M. P. Rigobello
(Dipartimento di Scienze Biomediche, Università di Padova,
Italy). The LoVo-OXP cells were derived, using a standard protocol,
by growing LoVo cells in increasing concentrations of OXP and following
17 months of selection of resistant clones, as previously described.^{23 (link)}Cell lines were maintained in the logarithmic
phase at 37 °C in a 5% carbon dioxide atmosphere using the following
culture media containing 10% fetal calf serum (EuroClone, Milan, Italy),
antibiotics (50 units/mL penicillin and 50 μg/mL streptomycin),
and 2 mM l-glutamine: (i) RPMI-1640 medium (EuroClone) for
A431, PSN-1, H157, HCT-15, and 2008 cells and (ii) Ham’S F-12
(Sigma Chemical Co.) for LoVo, LoVo MDR, and LoVo-OXP cells.