Antibiotic antimycotic mixture

Perfusion was done with M199 medium (Gibco). Paired adult worms were collected using fine tweezers and washed with M199 medium (2x). Subsequently, they were maintained in culture in M199 supplemented with FCS (Gibco; 10%), HEPES (Sigma; 1 M, 1%), and antibiotic/antimycotic mixture (Sigma; 1%) at 37°C and 5% CO₂[36] (link), [38] (link). Inhibitor treatment was performed for 24 h or 48 h with 50 µM Imatinib (Imatinib mesylate, C₂₉H₃₁N₇O·CH₃SO₃H, dissolved in water; Enzo Life Sciences) as previously described [38] (link). Control couples were kept in culture for 24 h or 48 h without inhibitor addition but otherwise treated using the same conditions. During the treatment periods, pairing stability and vitality were checked regularly. We defined pairing stability of couples when males kept their female partners within the gynecophoral canal while being sucked with their ventral suckers to the Petri dish. When couples separated, or when males stopped sucking to the Petri dish and/or lay on the side (a sign of decreasing vitality), the appropriate worms were not used for experiments and removed. After completion of treatment, the couples (inhibitor-treated and control) were carefully separated using featherweight tweezers, immediately shock-frozen in liquid nitrogen, and stored at −80°C.

A detailed description of the method used for the gill cell line culturing is provided by Mardones et al. [28 (link)]. The RTgill-W1 cell line (CRL-2523) was acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cell line was maintained in the dark at 19 °C in 25 cm² culture-treated flasks containing Leibovitz’s L-15 medium (Sigma, St. Louis, MO, USA, L1518) supplemented with an antibiotic–antimycotic mixture (Sigma, A5955, St. Louis, MO, USA), comprising penicillin (10,000 units mL⁻¹), amphotericin B (25 mg mL⁻¹) and streptomycin (10 mg mL⁻¹), and 10% (v/v) fetal bovine serum (FBS) (Sigma, 12003C, St. Louis, MO, USA). For sub-culturing twice per week, TrypLE™ Express (Gibco™, Billings, MT, USA) solution was used to detach the cells from the bottom of the flasks, and L-15 medium was added at a ratio of 1:2.

Hank’s balanced salt solution (HBSS) and 0.05% trypsin were purchased from Mediatech (Washington, DC). Fetal bovine serum (FBS) was obtained from Atlas Biologicals (Fort Collins, CO). Antibiotic-antimycotic mixture was obtained from Sigma-Aldrich (St. Louis, MO).

Gastric epithelial cultures were generated as previously described (Duckworth et al., 2015a (link)) and were maintained in 12-well tissue culture plates on glass cover slips (Appleton Woods, Selly Oak, UK) that contained 1.0 mL/well DMEM-Ham’s F-12 mix (Sigma–Aldrich), 10% fetal calf serum (Invitrogen, Paisley, UK), 1.25% L-glutamine (Sigma–Aldrich), and 1% antibiotic/antimycotic mixture (Sigma–Aldrich). Following digestion and plating, glands were maintained at 37°C in a humidified environment containing 5% CO₂ for 24 h. Media was changed to fresh complete media after a further 24 h. Forty-eight hours after initial plating, cells were treated with HECb, brasiliensic and isobrasiliensic acids (12.5 to 100 μg/mL) for 24 h, 2 h before fixation EdU was added to the culture media. Cells were fixed in 2% formaldehyde for 30 min followed by three washes in PBS. Treatments were repeated a minimum of four times using glands extracted from a different mouse on each occasion.