Be12 614f

Mouse BV-2 cells (43 (link)) were cultured in RPMI-1640 medium (R7509, Sigma-Aldrich) supplemented with 2.4 mM L-glutamine (17-605E; Gibco), 10% (v/v) fetal bovine serum (10270-106; Gibco), and 1.2% (v/v) penicillin/streptomycin (15140-122; Gibco). BV-2 cells were scraped off gently from cell culture plates (168381; Thermo Scientific) and collected by centrifugation at room temperature (RT) for 3 min at 800 × g. Cell pellets were resuspended in cell culture medium and plated for maintenance or experiments on cell culture plates or poly-l-lysine (P6282; Sigma-Aldrich) coated glass coverslips. Mouse Neuro-2a (N2a) neuroblastoma cells (ATCC CCL-131; ATCC) were maintained in Dulbecco's Modified Eagle's Medium (BE12-614F; Lonza) containing 2.4 mM L-glutamine, 10% (v/v) fetal bovine serum, and 1.2% (v/v) penicillin/streptomycin. N2a cells were washed once with warm Dulbecco's phosphate-buffered saline (DPBS, 17-512F; Lonza) and incubated in 0.25% trypsin solution (in DPBS, 15090-046; Gibco) for 3–5 min at +37°C. Next, the cell suspension was diluted in the culture medium and centrifuged for 3 min at 800 × g. The supernatant was discarded, and the cells were resuspended in the culture medium and plated for experiments on poly-d-lysine (P6407, Sigma-Aldrich) coated coverslips or on cell culture plates for maintenance.

Hepatocytes were isolated by a non-recirculating collagenase perfusion through the portal vein of anesthetised 8-week-old C57BL/6 WT or Cd14KO male mice fed a normal chow diet. Isolated cells were filtered through a 100 μm pore mesh nylon filter and cultured (2.5 × 10⁶ cells per well) onto 96-well plates in DMEM (BE12-614F; Lonza, Levallois, France) supplemented with 10% (vol./vol.) FCS, 1% (vol./vol.) penicillin/streptomycin and 0.2 nmol/l l-glutamine. After 12 h, the medium was replaced with medium plus industrially purified lipopolysaccharide (LPS; Sigma Aldrich, St Louis, MO, USA) either from the proinflammatory Escherichia coli serotype O55:B5 [22 (link)], or the E. coli strain O111:B4, which stimulates human hepatocytes [23 (link)]. Two doses of LPS were tested: 10 ng/ml (low dose) and 100 ng/ml (high dose), and cells were stimulated for 6 h. Experiments were performed in quadruplicates (control) or pooled duplicates (LPS).

ESCs were grown in feeder-free culture condition and incubated at 37°C under 3% O₂ tension. ESC medium composed of KnockOut DMEM (ThermoFisher, 10829–018), 15% ESC qualified Fetal Bovine Serum (ThermoFisher, 16141–079), 2 mM L-glutamine, 1/500 home-made leukemia inhibitory factor (LIF), 0.1 mM non-essential amino acids, 0,1 mM 2-mercaptoethanol, 50 units/mL penicillin, 50 mg/mL streptomycin supplemented with the two inhibitors (2i); PD 0325901 (1 µM) and CHIR 99021 (3 µM) was used. MEFs were cultured in high glucose DMEM (Lonza, BE12-614F) supplemented with 10% North American FBS, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 50 units/mL penicillin, 1 mM Sodium Pyruvate and 50 mg/mL streptomycin (MEF medium).

Human cancer cell lines HCT-116 (colon carcinoma), U-251 MG (Glioblastoma) and Jurkat (leukaemia) were purchased from the American Type Culture Collection and routinely cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) (Lonza, BE12-614F) supplemented with 10% v/v foetal bovine serum (FBS) (Sigma, F7524, non-USA origin), 2 mM L-glutamine (Lonza, 17-605C) and penicillin/streptomycin (Lonza, DE 17-602E) within a TEB-1000 humidified 5% CO₂ incubator (EBERS Medical Technology) at 37 °C. The R69 lymphoblastoid B cell line was kindly provided by Dr López de Castro, from the “Severo Ochoa” Molecular Biology Centre (CBMSO) and were cultured in RPMI1640 (Sigma, R0883) supplemented with 10% FBS and L-glutamine.

C2C12 cells are from a mouse skeletal muscle cell line (ECACC collection; Sigma-Aldrich, St Louis, MO, USA) and are a useful model to assess the myofilament function [16 (link)]. Cells were kept viable at 37 °C and 5% CO₂. For preparation of the growth medium 1% L-glutamine solution, 1% penicillin-streptomycin (P4333, SIGMA), and a 10% sterile filtered foetal bovine serum previously heated at 55 °C for 30 min were added to 500 mL of 4.5 g/L glucose Dulbecco’s modified Eagle medium (DMEM) (BE12-614F, LONZA). To prepare the differentiation medium, 1% L-glutamine solution, 1% penicillin-streptomycin, and a 2% heat inactivated horse serum (26050-088, Gibco Life Technologies) were added to 500 mL of a 4.5 g/L glucose DMEM. Preparation of the starvation medium involved adding 1% L-glutamine solution and 1% penicillin-streptomycin to 500 mL of a 4.5 g/L glucose DMEM.

All cell lines were obtained from collaborators: MDA-MB-231 breast cancer cell line from Simon Schwartz Navarro (VHIR), HCT116 colorectal cancer cell line from Melinda Halasz (UCD), PhoenixA retroviral packaging cells, and PC3 prostate cancer cells from Bill Keyes (IGBMC) and BJ non-transformed foreskin fibroblasts from Maria Aurelia Ricci (CRG). They were cultured in Dulbecco’s Modified Medium (DMEM, BE12-614F, Lonza, Cultek, Basel, Switzerland) implemented with 10% Fetal Bovine Serum (FBS, 10270106, GIBCO, Termofisher, Waltham, MA, USA) 1% L-Glutamine (BE17-605E, Lonza, Cultek, Basel, Switzerland) and 1% Penicillin/Streptomycin (DE17-602E, Lonza, Cultek, Basel, Switzerland), and kept at 37 °C with 5% CO₂ in a humidified incubator.
Adult Casper fish, obtained from the European Zebrafish Resource Center, were grown at 28.5 ± 1 °C in a 14:10 h light:dark cycle in a recirculating tank system. Embryos were obtained by mating adult fish through standard methods [70 ] and kept in an incubator at 28.5 °C until 2 days in and at 35 °C just after injections until the end of the experiment.
This study was performed under the ethical approval code 10567, provided by the Generalitat of Catalunya.