Mem vitamin

HBMECs were cultured in RPMI 1640 medium (Thermo Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, Marlborough, MA, USA), 10% Nu-serum (BD Biosciences, Franklin Lakes, NJ, USA), 2 mM glutamine, 1% MEM nonessential amino acids (Wako, Osaka, Japan), 1 × MEM vitamin (Sigma, Irvine, UK), 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 mM sodium pyruvate (Thermo Scientific). E. coli K1 strains were resuscitated from −80 °C, cultured overnight, and then subcultured in fresh BHI medium at a dilution of 1:100 until strains were grown to the exponential phase at an OD₆₀₀ of 0.6. Bacteria were collected by centrifugation and resuspended in RPMI 1640 medium containing 10% FBS. HBMECs infected at a multiplicity of infection (MOI) of 100 were incubated for 1.5 h in a cell incubator. The HBMECs were then washed with phosphate-buffered saline (PBS) to remove unbound bacteria and incubated with new medium containing gentamicin (100 mg/mL) for 1 h to kill extracellular E. coli. HBMECs were washed, lysed with 0.5% triton X-100, and plated on Luria broth (LB) agar plates for the determination of CFUs. A binding assay was performed similarly to the invasion assay except that the gentamicin treatment step was omitted. All experiments were conducted in duplicate and performed at least in triplicates.

Hearts were dissected from E8.5–9.5 mouse embryos, washed in PBS without Ca²⁺, and digested using 0.04% Trypsin and 0.05% Collagenase IV. After incubation at 37 °C for 20 min (E8.5 embryonic hearts) or 30 min (E9.5 embryonic hearts), cells were gently pipetted and transferred to DMEM containing 10% fetal bovine serum for termination. Cells were plated on matrigel (1:60, BD) coated plates in the in vitro CMs culture medium (IVCC), with 4 ng/mL zbFGF. The in vitro CMs culture medium (IVCC) is composed of DMEM/F12 (Gibco) medium with 2% B27 (Gibco), 1% N2 (Gibco), 1% Glutamax (Gibco), 1% non-essential amino acids (NEAA; Gibco), 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco). 50 µg/mL 2-phospho-l-ascorbic acid (Vitamin C, Sigma), 0.001% Progesterone (Sigma), 1% MEM Vitamin (Sigma, M6875), 0.1% trace element B (Corning), and 0.1% trace element C (Corning).
After 24 h culture, CMs were transfected with 50 nmol/L miRNA mimics (miRNA-1-3p, miR-541-5p and NC-miRNA (GenePharma) using Lipofectamine^® 2000 (Thermo Fisher) following the manufacturer’s instructions. The same transfection protocol was used for SEVC4–10, mouse Yolk sac dissociated cells, human iPSC derived ECs and mESCs.

At the time of recruitment (baseline visit), peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood with Ficoll using density centrifugation. After isolation, PBMC numbers were adjusted to a concentration of 10⁶ viable cells/mL in complete culture medium. For cell culture, Roswell Park Memorial Institute 1640 medium supplemented with 25 mmol/L HEPES (Gibco, Invitrogen, Darmstadt, Germany) was used. Furthermore, 100 IU/mL penicillin, 100 µg/mL streptomycin, 50 µmol/L β‐mercaptoethanol, 1% l‐glutamine (200 mmol/L), 1% MEM Vitamin, 1% nonessential amino acids, 1% sodium pyruvate, and 10% fetal bovine serum were added (complete culture medium); these reagents were purchased from Sigma‐Aldrich (Steinheim, Germany). The PBMCs were cultured in complete culture medium for 24 hours at 37°C and 5% CO₂, whereby parts of them were challenged in vitro with 10 µg/mL PHA (Sigma‐Aldrich) or with RV (RV1b). The growth of RV1b and the description of the RV1b infection itself have been published previously in detail elsewhere.¹Human IFNβ and interleukin 10 (IL‐10) was detected in the cell‐culture supernatants by using IFNβ ELISA kit from PeproTech (Hamburg, Germany) and IL‐10 OptEIA™ sandwich ELISA kit from BD Bioscience (Heidelberg, Germany), respectively, according to the manufacturer's protocol.