Streptomycin

Human myelomonocytic THP-1 cells were cultured in RPMI 1640 medium (Aurogene, Rome, Italy) supplemented with fetal bovine serum (10%; Aurogene), L-glutamine (2 mM; Aurogene), penicillin (100 IU/mL; Aurogene) and streptomycin (100 mg/mL; Aurogene). Cell culture medium was replaced every 2−3 days, and the cultures were maintained at 37 °C and 5% CO₂ in a fully humidified incubator. The day before each experiment, cells were plated in 48-well culture plates (90.000 cells/well) and were differentiated by treatment with PMA (50 nM, 24 h; Sigma-Aldrich). PMA-differentiated THP-1 cells were washed twice with phosphate-buffered saline (PBS) and primed with LPS (10 μg/mL, 4 h; Sigma-Aldrich) in serum-free medium. Cells were then incubated with compounds dissolved in medium containing 0.1% DMSO for 1 h and cell death was triggered with ATP (5 mM, 90 min; Sigma-Aldrich). MCC950 (Sigma-Aldrich batch #45216 and batch #85021 and from Crysdot (product n. CD31002496; OS05876-18070932) was used in the experiments.

A murine monocyte/macrophage J774 cell
line was obtained from the American Type Culture Collection (ATTC
TIB 67). The cell line was grown in adhesion in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with glutamine
(2 mM, Aurogene Rome, Italy), Hepes (25 mM, Aurogene Rome, Italy),
penicillin (100 U/mL, Aurogene Rome, Italy), streptomycin (100 μg/mL,
Aurogene Rome, Italy), fetal bovine serum (FBS, 10%, Aurogene Rome,
Italy), and sodium pyruvate (1.2%, Aurogene Rome, Italy) (DMEM completed).
The cells were plated at a density of 1 × 10⁶ cells
in 75 cm² culture flasks and maintained at 37 °C under
5% CO₂ in a humidified incubator until 90% confluence.
The culture medium was changed every 2 days. Before a confluent monolayer
appeared, the subculturing cell process was carried out.

Murine monocyte/macrophage
J774 cell line was obtained from the American Type Culture Collection
(ATTC TIB 67). The cell line was grown in adhesion in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with glutamine
(2 mM, Aurogene Rome, Italy), Hepes (25 mM, Aurogene Rome, Italy),
penicillin (100 U/mL, Aurogene Rome, Italy), streptomycin (100 μg/mL,
Aurogene Rome, Italy), fetal bovine serum (FBS, 10%, Aurogene Rome,
Italy), and sodium pyruvate (1.2%, Aurogene Rome, Italy) (DMEM completed).
The cells were plated at a density of 1 × 10⁶ cells
in 75 cm² culture flasks and maintained at 37 °C under
5% CO₂ in a humidified incubator until 90% confluence.
The culture medium was changed every 2 days. Before a confluent monolayer
appeared, subculturing cell process was carried out. Cells (0.5 ×
10⁶ cells/mL) were pretreated for 2 h in the absence or
presence of 28 (0.1–10 μM) and then stimulated,
for 24 h, with lipopolysaccharide (LPS) from E. coli, Serotype 0111:B4, (10 μg/mL; 100 μL in DMEM completed
with FBS, Sigma-Aldrich, Milan, Italy).^{42 (link)} The supernatants were collected for the measurement of PGE₂ levels by ELISA assay (Cayman Chemical, Vinci-Biochem, Vinci, Italy).

Upon collection, biopsies from four control, four FSHD STIR− and five FSHD STIR+ muscles were mechanically and enzymatically digested to isolate cells. Muscle specimens were washed in phosphate-buffered saline (PBS, Aurogene, Rome, Italy) supplemented with 1% antibiotics (penicillin 100 IU/ml, streptomycin 100 mg/ml, Aurogene), initially processed by manual fragmentation with scissors and subsequently digested with 2 mg of collagenase/dispase (Merck, Darmstadt, Germany) at 37 °C for 30 min. The tissue homogenates were filtered through a 40 µm cell strainer and the cells pelleted after centrifugation were resuspended in CYTO-GROW medium (Resnova, Rome, Italy). Cells were plated in a 25 cm² flask coated with 0.1% Gelatin (Stem cell Technologies, Vancouver, Canada) and incubated in a humidified atmosphere at 37 °C and 5% CO₂.