Human white preadipocytes

Human white preadipocytes derived from subcutaneous adipose tissue of a female Caucasian subject (BMI 21; age 44 years) were obtained from PromoCell (Germany). The preadipocytes were cultured in T25 flasks then sub-cultured into 12-well plates (seeding density: 5000 cells per cm²) in preadipocyte growth medium (PromoCell, Germany) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B, and incubated at 37 °C in 95% air and 5% CO₂.

Human white preadipocytes derived from subcutaneous adipose tissue of a female Caucasian subject (BMI 21 kg/m²; age 44 yr) were obtained from PromoCell (Heidelberg, Germany). Cells were cultured in preadipocyte growth medium supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Lonza, Tewkesbury, UK) at 37°C in a humidified atmosphere of 5% CO₂-95% air. Preadipocytes were seeded at 40,000/cm² and grown in 6- or 24-well plates until confluence. At confluence, cells were induced to differentiate (day 0) by incubation for 3 days in Dulbecco's modified Eagle's medium (DMEM)-Ham's F-12 (1:1) medium containing 32 μM biotin, 1 μM dexamethasone, 200 μM 3-isobutyl-1-methylxanthine, 100 nM insulin, 11 nM l-thyroxine (all from Sigma, Poole, Dorset, UK), 8 μM rosiglitazone (GlaxoSmithKline, Uxbridge, UK), and 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B. After induction, cells were cultured in maintenance medium containing 3% fetal calf serum (FCS, Sigma), 100 nM insulin, 32 μM biotin, and 1 μM dexamethasone until full differentiation into adipocytes.

Human white preadipocytes and their corresponding incubation media were obtained from PromoCell (Heidelberg, Germany) and negative tested for mycoplasma. Differentiation was conducted according to the manufacturer’s instructions (Fig. S1). Mature adipocytes were infected with IAV/PR8 at a multiplicity of infection (MOI) of 1. Cells were incubated with viral dilutions prepared in Dulbecco’s phosphate-buffered saline (DPBS; Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 0.2% (v/v) bovine serum albumin (BSA; Carl Roth GmbH, Karlsruhe, Germany), 1 mM MgCl₂, and 0.9 mM CaCl₂ at 37 °C and 5% CO₂ for 30 followed by a wash step with DPBS. Afterwards, cells were incubated in adipocyte nutrition medium supplemented with 1 mM MgCl₂, 0.9 mM CaCl₂, and 30 ng L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Thermo Fisher Scientific) for 1 day and 3 days. Infection with the SARS-CoV-2 alpha, delta, and omicron variants was performed at MOI1. Cells were infected with virus diluted in adipocyte nutrition medium supplemented with 10% (v/v) FBS for 60 min. After one washing step cells were cultured in nutrition medium for 1 day and 3 days.