Gentamicin

Immobilization of gentamicin (Sigma-Aldrich, USA) was performed by simple soaking of graft pieces (pristine and polycatecholamine-coated ones) in 1 mg/mL gentamicin solution in 0.1 M Britton-Robinson buffer pH 8.5 for 24 h, typically at 25 °C, 37 °C and 50 °C, depending on the particular experiment. Then the samples were washed twice in distilled water and dried at 37 °C. The amount of immobilized drug was calculated from gentamicin concentrations in the buffer before and after the process and evaluated quantitatively after drug derivatization with phthaldialdehyde (Sigma-Aldrich), as described elsewhere [21 (link)]. For comparison, the reference method of gentamicin immobilization on protein-sealed vascular prostheses using 0.5–2.5% glutaraldehyde (Chempur, Piekary Śląskie, Poland) was performed on FlowNit Bioseal^® collagen-sealed polyester knitted prosthesis, according to a procedure described elsewhere [5 (link)].
Samples of grafts obtained in optimized coating conditions and with immobilized gentamicin were selected for further experiments. They are listed in Table 1.

MDA-MB-231 (MDA231), MDA436, BT549, and SUM149PT human breast tumor cell lines, HEK293 human embryonic kidney and NIH3T3 mouse fibroblast cells were purchased from the ATCC (Virginia, USA) and ASTERAND BIOSCIENCE. MDA231, MDA436, and BT549 human tumor cell lines were cultured in DMEM:F12 HAM media (1:1), and SUM149PT cells in F12 HAM media, supplemented with 10% heat inactivated fetal bovine serum (FBS) (Sigma), 10 μg/ml insulin (Roche), 1% Gentamicin (Sigma), and 1% amphotericin (Sigma). HEK293 cells were cultured in DMEM supplemented with 10% heat inactivated FBS (Sigma) and 1% Gentamicin (Sigma) 1% amphotericin (Sigma). NIH3T3 cells were cultured in DMEM supplemented with 10% heat inactivated calf new-born serum (Sigma), 1% amphotericin (Sigma) and 1% Gentamicin (Sigma). Cells were kept at 37 °C and 5% CO₂, and the media was replaced every 2/3 days. HEK293 cells were passaged when they reached 80% confluency 1:10 every 48 h, while BT549 and MDA231, MDA436, and SUM149PT cells were passaged when reached 80% confluency 1:5 every 72 h. Cells were discarded after up to five consecutive passages and replaced by freshly thawed stocks. All cell lines were tested and confirmed negative for mycoplasma on a monthly bases at the host institution. All the cell lines were authenticated using STR profile by the Genetic Analysis Service at Miguel Hernandez University, Spain.

C2C12 cells were grown in Dulbecco’s modified Eagle medium supplemented with 20% fetal bovine serum (Sigma-Aldrich), 2 mM glutamine (Sigma-Aldrich), 50 μg/ml gentamicin (Sigma-Aldrich) (GM). After one day, the medium was replaced with differentiation medium (DM) made of Dulbecco’s modified Eagle medium supplemented with 2% horse serum (Sigma-Aldrich), 2 mM glutamine (Sigma-Aldrich), 50 μg/ml gentamicin (Sigma-Aldrich).
Satellite cells were plated on 0,01% collagen (Sigma-Aldrich)-coated dishes with Dulbecco’s modified Eagle medium supplemented with 20% horse serum (Sigma-Aldrich),100 U/ml penicillin (Sigma-Aldrich), 100 μg/ml streptomycin (Sigma- Aldrich), 50 μg/ml gentamicin (Sigma-Aldrich), 3% of chicken embryo extract as growing medium (GM). After 3 days, the medium was replaced with differentiation medium (DM) (GM diluted 1:10).
To obtain HDAC4 deletion in vitro, satellite cells derived from HDAC4^fl/fl Pax7^CE/+Cre+ mice were treated with 0.4 µM 4 OH-TMX (Calbiochem) or vehicle (methanol) as controls, for 72 hours, as described in^{35 (link)}.
To inhibit P21 expression, satellite cells were treated with 10 µM UC2288 (trans-1-(4-chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)-urea; Calbiochem) or vehicle (DMSO, Sigma-Aldrich), for 24 h hours, as described in^{51 (link)}.

MPCs were expanded in Skeletal Muscle Cell Growth Medium until reaching the
desired number of cells. The pharmacological treatments started at the last 2
days in Skeletal Muscle Cell Growth Medium and continued during the 5 days of
secondary differentiation. MPCs were treated with 10 μM SB-431542
(Tocris, 1614) as in (31 (link)). After testing a range of gentamicin
(Sigma-Aldrich, G1397) concentrations (10 to 600 μM), 200 μM
gentamicin was used in the 96-well human neuromuscular circuit coculture assay,
as 600 μM gentamicin caused cytotoxicity. PTC124 (Generon, A8553) was
tested in a range of concentrations (1, 5, 10, 17, 25, and 35 μM).

Monocyte/macrophage cultures (n = 4) were pre-treated for 24 h with IFNγ at a concentration of 1000 ng/ml or left un-treated (Ctr). Cells were infected with F. noatunensis (NCIMB 14265) diluted in L-15+ with 5% FBS Gold (PAA) at a multiplicity of infection (MOI) of 50–100. Uninfected cells were incubated with cell media/FBS Gold alone and treated otherwise similarly as infected cells. The cell culture plates were centrifuged (500 × g, 5 min) to enhance the initial contact with the cells. Two hours after infection, the cells were washed three times in L-15++ and pulse-treated for 1 h with 50 μg/ml gentamicin (Sigma-Aldrich) to kill extracellular bacteria. The cells were washed three times to remove gentamicin and fresh media supplemented with 5 μg/ml gentamicin (Sigma-Aldrich) were added. Cell cultures were further incubated before harvesting in 1x lysis buffer (Applied Biosystems) at sequential time points (6, 10, 24, 48 and 72 h). All samples were stored at −80 °C for further gene expression analysis as described below.