Cell culture flask

A highly virulent S. aurantiacum WM 06.482 (CBS 136046) strain⁴⁰ was obtained from the culture collection of the Medical Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology, Westmead Hospital, Sydney, Australia. Conidia were prepared and stored as described previously^{40 ,41} .
The A549 human epithelial cell line derived from a lung carcinoma was obtained from the American Type Culture Collection (ATCC^® CCL-185^™). The A549 cells were maintained in RPMI 1640 medium (Life technologies, Australia) supplemented with 10% (v/v) FBS (fetal bovine serum, Life technologies, Australia), 1 mM glutamine (Life technologies, Australia), 100 U/ml penicillin and 100 µg/ml streptomycin in cell culture flasks (Sigma-Aldrich, Australia) at 37 °C in a humidified 5% CO₂ atmosphere. Cell count and viability were calculated with TC20^TM Automated Cell Counter (Biorad, Australia) according to the manufacturer’s protocol. Cells were then seeded in RPMI 1640 medium in 8-chamber tissue culture slides (BD Falcon™ CultureSlides) approximately 1 × 10⁵ cells/well at 37 °C.

The KGN cells were thawed on day 1, placed in a cell culture flask (75 cm³, Sigma-Aldrich), grown up to 80% confluence, and sub-cultured on day 4. On day 7, viable cells were seeded in a six-well plate at a density of 3×10⁵ per well in 3 ml of medium and grown for 72 h to obtain full confluence. For all experimental conditions, the medium was then replaced with DMEM/F12 containing antibiotics, 100 nmol L^-1 of 4-androstene-3,17-dione (product A9630, Sigma-Aldrich, St. Louis, MO) and 1% of low fatty acid BSA. Plates were incubated for 24 h in a humidified incubator at 37 °C in 5% CO2:95% air after adding either ethanol (less than 0.1% of the culture volume) as a control or the fatty acid combination described above with or without the addition of human insulin. Control wells received the same volume of ethanol as the test groups.

The KGN cells were cultured as previously described [43 (link)]. Briefly, the cells were cultured in DMEM/F12 medium (Life technologies) supplemented with 10% fetal bovine serum (Corning, NY, USA) and ×100 Penicillin-Streptomycin (10,000 U/mL) (Life technologies, Waltham, MA, USA) in an atmosphere of 5% CO₂/95% O₂ at 37 °C. The cells were thawed on day 1, placed in a cell culture flask (75 cm³, Sigma-Aldrich), and sub-cultured on day 3. Confluence was never over 80%. On day 6, cells were placed in a 6-well dish at a density of 1.510⁵ cells per well in 1.5 mL of medium and grown for 72 h, at which point they were reaching almost full confluency. For all experimental conditions, the medium was replaced with charcoal-stripped (SVFA) DMEM/F12 containing antibiotics and 100 nmol L⁻¹ of 4-androstene-3,17-dione. The 24 h treatments consisted of adding either ethanol as a control (less than 0.05% of the final culture volume) or additional stimulators in ethanol to obtain either 1 nmol of melatonin L⁻¹ or 1 μmol of FSK L⁻¹ as final concentrations. The experiment was repeated four times (four different weeks) to generate four biological replicates for RNAseq analysis.