Sodium pyruvate

The epithelial human glioblastoma cell line U87 MG (ATCC HTB-14, MGMT^-) and the human hepatocellular carcinoma cell line HepG2 (ATCC HB-8065) were grown in complete growth medium, consisting of Dulbecco’s Eagle’s Minimum Essential Medium (DMEM) (Sigma-Aldrich, Steinheim Germany) supplemented with 10% v/v heat inactivated fetal calf serum (FCS) (Sigma-Aldrich), 1% antibiotics (10,000 IU/mL penicillin, 10 mg/mL streptomycin, Sigma-Aldrich), l-glutamine (Sigma-Aldrich), MEM non-essential amino acid solution 100× (Sigma-Aldrich) and sodium pyruvate (Sigma-Aldrich).The epithelial human cervix carcinoma cell line HeLa (ATCC CCL-2) was grown in complete growth medium, consisting of RPMI-1640 (Sigma-Aldrich, Germany) supplemented with 10% v/v heat inactivated FCS (Sigma-Aldrich), 1% antibiotics (10,000 IU/mL penicillin, 10 mg/mL streptomycin, Sigma-Aldrich), l-glutamine (Sigma-Aldrich) and sodium pyruvate (Sigma-Aldrich). After reaching confluency, adherent growing tumor cells were trypsinized for 2 min at 37 °C in trypsin ethylene diamine-tetra-acetic acid (EDTA) (Sigma-Aldrich). Single cell suspensions with different cell counts were seeded in 15 mL supplemented medium in T-75 ventilated culture flasks. Tumor cells were routinely checked for mycoplasma contamination.

The casein–casein–yeast (CCY) medium was prepared to contain 30 g/L yeast extract (Oxoid, Basingstoke, UK), 20 g/L casamino acids (VWR Amresco, Cleveland, OH, USA), 2.48 g/L Na₂HPO₄, 0.41 g/L KH₂PO₄, 20 mg/L MgSO₄·7H₂O, 7.5 mg/L MnSO₄·H₂O, 6.4 mg/L citric acids, and 6.4 mg/L FeSO₄·7H₂O (Sigma, Darmstadt, Germany) (pH 7.3 ± 0.2 at 25 °C). Then, a 100 mL sodium pyruvate solution was prepared by dissolving 23.2 g of sodium pyruvate (Sigma) in 100 mL deionised water, sterilised by 0.22 µm filtration, and added to 900 mL autoclaved CCY medium at 121 °C for 15 min [32 ]. S. aureus test strains, M1, YF1B-b, and S. aureus control strain HT480, were grown on a Tryptone Soy Agar (CM131, Oxoid) at 37 °C by overnight incubation. Bacterial suspensions were prepared in 0.9% (w/v) NaCl (Sigma) and adjusted to McFarland 0.5 using a densitometer (Den-1B, Biosan, Riga, Latvia). Each bacterial suspension (500 µL) was inoculated to a 50 mL CCY medium and incubated at 37 °C for 24 h by shaking at 120 rpm to promote PVL production.

The H1299-E3 (H1299-ACE2, clone E3, H1299 originally from ATCC as CRL-5803) cell line was derived from H1299 as described in our previous work (Cele et al. 2021a (link), 2021b (link)). The H1299-E3 cells were propagated in growth medium consisting of complete Roswell Park Memorial Institute (RPMI) 1640 medium with 10 per cent fetal bovine serum (Hyclone) containing 10 mM of hydroxyethylpiperazine ethanesulfonic acid (HEPES), 1 mM sodium pyruvate, 2 mM l-glutamine and 0.1 mM nonessential amino acids (all Sigma-Aldrich). Cells were passaged every second day. For virus isolation, Vero E6 cells (originally ATCC CRL-1586, obtained from Cellonex in South Africa) were propagated in complete growth medium consisting of Dulbecco’s Modified Eagle Medium (DMEM) with 10 per cent fetal bovine serum (Hyclone) containing 10 mM of HEPES, 1 mM sodium pyruvate, 2 mM l-glutamine and 0.1 mM nonessential amino acids (all Sigma-Aldrich). Vero E6 cells were passaged every 3–4 days. The VeroE6 cells expressing TMPRSS2 and ACE2 (VeroE6-TMPRSS2), originally BEI Resources, NR-54,970 were used for virus re-expansion of the virus from the day 6 swab and fusion assay. The Vero-TMPRSS2 cell line was propagated in the same way as VeroE6 cells.

The SAS and OECM-1 human oral cancer cell lines were purchased from ATCC (ATCC: American Type Culture Collection, Manassas, VA, USA). SAS cells were cultured in Dulbecco's modified Eagle's medium-F12 supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1.5 g/L sodium bicarbonate, 25 mM HEPES and 1 mM sodium pyruvate (Sigma, St. Louis, Mo, USA). OECM-1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, 1.5 g/L sodium bicarbonate, 25 mM HEPES and 1 mM sodium pyruvate (Sigma, St. Louis, Mo, USA). The cells culture weas maintained at 37°C in a humidified atmosphere of 5% CO₂.