Antibiotic antimycotic

Cytokine production by immune cells from lamina propria was checked by using gut explant immersion cultures which were done with small fragments from jejunum and ileum. This approach was adapted from Bareiss et al.^{85 (link)} and Randall et al.^{86 (link)}. Briefly, the entire jejunum and ileum were aseptically removed and placed in sterile Petri dishes with cold HBSS containing 1% of antibiotic/antimycotic (Sigma-Aldrich). The cleaned sections (4 -5 mm), devoid of faeces and Peyer's patches, were sectioned longitudinally to expose the mucosal surface and placed in a 48-well plate. Each well contained two explants immersed in 0.5 ml of RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (Gibco, United States), 4 mM of l-glutamine (Sigma-Aldrich), 1% of sodium pyruvate (Sigma-Aldrich), 1% of nonessential amino acids (Sigma-Aldrich) and 2% antibiotic/antimycotic (Sigma-Aldrich). The cultures were incubated in a humidified incubator at 37 °C and 5% CO₂ for up to 6 h after which the supernatants were stored at − 20 °C for further cytokine quantification.

Canine inflammatory mammary carcinoma cell line, IPC-366, was cultured in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 Ham (DMEM/F12) containing 10% fetal bovine serum, 1% L-glutamine, and 1% antibiotic-antimycotic (Sigma Aldrich). Its human counterpart, SUM149 cell line, was obtained from Asterand plc (Detroit, MI) and was cultured in Ham's F12 (Fisher Scientific) supplemented with 10% fetal bovine serum, 5 μg/mL insulin, 1 μg/mL hydrocortisone, and antibiotic-antimycotic (Sigma Aldrich).
All cell lines were cultured in 25 cm² culture flasks and were maintained in a humidified atmosphere of 5% carbon dioxide at 37°C. Cell culture was observed daily by a phase-contrast microscopy.

Madin-Darby canine kidney (MDCK) cells were maintained in Dulbecco’s Modified Eagles Medium (DMEM, Sigma-Aldrich, St Louis, MO) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, St Louis, MO), 2mM L-glutamine (Sigma-Aldrich, St Louis, MO), and 1% antibiotic/antimycotic (Sigma-Aldrich, St Louis, MO). 3D4/21 cells were maintained in Roswell Park Memorial Institute media (RPMI-1640, Sigma-Aldrich, St Louis, MO) supplemented with 10% FBS, 2mM L-glutamine, 1% antibiotic/antimycotic, 1 mM non-essential amino acids (Sigma-Aldrich, St Louis, MO), and 1 mM sodium pyruvate (ThermoFisher Scientific, Waltham, MA). All cell lines were cultured at 37°C under 5% CO₂. Viruses used in this study were: a reassortant carrying seven genes from A/turkey/Ohio/313053/2004 and the pandemic H1N1pdm09 (A/California/04/09) matrix gene (sOH/04); a reassortant carrying the HA and NA genes from A/Victoria/361/2011 and internal genes from sOH/04 (hVIC/11); and hVIC/11^A138S which only differs from hVIC/11 by an A to S amino acid substitution at position 138 in the HA gene (H3 nomenclature). Notably, the PB2, PB1, PA, NP, M, and NS genes were the same for all viruses. These viruses have been previously reported by our group [35 (link)]. Viral titers were determined by TCID₅₀ using the Reed and Muench method [78 (link)].

hBM‐MSCs (ATCC, USA) and MG‐63 cell line (ECACC, Sigma‐Aldrich, USA) were cultured in minimum essential medium alpha (α‐MEM) (Thermo Fisher Scientific, USA) supplemented with sodium bicarbonate (2.2 g L⁻¹, Sigma‐Aldrich, USA), 10% heat‐inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, USA), and 1% antibiotic/antimycotic (Thermo Fisher Scientific, USA). SaOS‐2 cell line (ECACC, Sigma‐Aldrich, USA) was cultured in Dulbecco's modified Eagle's medium (DMEM) low glucose (Sigma‐Aldrich, USA) supplemented with sodium bicarbonate (3.7 g L⁻¹), 10% heat‐inactivated FBS, and 1% antibiotic/antimycotic. A549 cell line (ATCC, USA) was cultured with Nutrient Mixture F‐12 Ham (Sigma‐Aldrich, USA) supplemented with sodium bicarbonate (2.5 g L⁻¹), 10% heat‐inactivated FBS, and 1% antibiotic/antimycotic. All cells were cultured in T‐flasks, maintained under 5% CO₂ atmosphere at 37 °C (standard culture conditions) and passaged at about 80% confluence. The medium was replaced every 2 to 3 d.

Human HeLa and mouse C2C12 cells were grown in high-glucose DMEM medium (Lonza) supplemented with 10% fetal bovine serum (Sigma) and 1x antibiotic/antimycotic (Sigma). Human skeletal myoblast (HSkM) cells were grown in HAM F-10 medium (Sigma) supplemented with 20% FBS, 1x antibiotic/antimycotic, 0.39 µg/ml dexamethasone (Sigma) and 10 ng/ml epidermal growth (Sigma). All cells were grown at 37 °C and in a 5% atmosphere of CO₂. HeLa, C2C12 or HSkM cells plated in 12-well plates were transfected at 50–60% confluence with Lipofectamine® 2000 (Invitrogen™) according to the manufacturer’s protocol. Single transfection was conducted with an siRNA mix against MBNL1 and MBNL2 (25 nM each) (FUTURE synthesis and RiboTask™, respectively^{67 (link),68 (link)}), 50 nM AllStars negative control siRNA (Qiagen), or a specific AON at 125 nM, in which AON-Ctrl was 2′OMe-PS unspecific to a tested transcript. Co-transfection was conducted with 200 ng of the minigene and 500 ng (or as indicated in the figures) of the MBNL1, SRSF1 or GFP expressing vector. For verification of the MBNL-binding regions and inhibition of the MBNL/(CUG)^exp interaction, the co-transfection was followed by a 4-hour incubation and transfection with selected AONs. Erythromycin was added directly to the medium. The cells were harvested 48 hours after transfection.