Opti mem 1 1x

PRNT50 assays were performed as described previously. Briefly, monoclonal antibodies were 3-fold serially diluted in 60 μL Opti-MEM I (1X) + GlutaMAX (Gibco). Four hundred PFU (based on 8 h titrations) in 60 μL were added per well to a final volume of 120 μL and a serum dilution of 1:20 in the first well. Plates were incubated for 1 h at 37 °C. Next 100 μL of virus and serum mix was added to confluent monolayers of Calu-3. SARS-CoV-2 infected plates were incubated for 8 h at 37 °C before fixing in formalin and permeabilizing in ethanol. Plates were then washed in PBS and stained as described for virus titrations. Nuclei were stained with Hoechst for 30 min. Cells were imaged using the Opera Phenix spinning disk confocal HCS system and the number of GFP-positive/Alexa Fluor 488-positive infected cells were quantified using the Harmony software (version 4.9, PerkinElmer). The PRNT50 was calculated based on non-linear regression. All analyses were performed using GraphPad Prism 9 software.

Cells were transfected with synthetic miRNAs once or several times at various time points, between 0 and 90 h after culture start. We used mirVana miRNA mimics (Ambion, LifeTechnologies) of Hpo-miR-71-5p (mature sequence: UGA​AAG​ACA​UGG​GUA​GUG​AGA​C), Hpo-miR-100-5p (mature sequence: AAC​CCG​UAG​AUC​CGA​ACU​UGU​GU), and Hpo-miR-10021-5p (mature sequence: UGA​GAU​CAU​CAC​CAU​AAG​CAC​A), as they are among the most abundant freely circulating miRNAs found in H. polygyrus bakeri culture supernatants (Buck et al., 2014 (link); Tritten et al., 2017 (link)). We used additionally Hpo-miR-1-3p (mature sequence: UGG​AAU​GUA​AAG​AAG​UAU​GUA) the mirVana miRNA mimic universal negative control #1 (Ambion, LifeTechnologies), a random sequence miRNA mimic molecule validated to not produce identifiable effects on known miRNA function in human cells. miRNA mimics were transfected into 2 × 10⁵—1 × 10⁶ cells using lipofectamine RNAiMax (Invitrogen) and OPTI-MEM I 1x (Gibco) following the manufacturer’s protocol at final concentrations of 10 nM or 50 nM. A combination of miR-71, miR-10021, and miR-100 was also tested at the same total concentration (e.g., 3.33 nM of each individual miRNA). Information on miRNAs used in this work is provided in more detail in Supplementary Table S4.

HEK293T cells were seeded in 24 well plates (Greiner Bio-One) at a concentration of 1 × 10⁵ cells per well on a coverslip for immunofluorescence experiments or at 2 × 10⁵ cells per well for secreted embryonic alkaline phosphatase assays. For immunoprecipitation, HEK293T cells were seeded in 10 cm cell culture dishes (Greiner Bio-One) at 4 × 10⁶ cells per dish. Cells were seeded 1 day before transfection. On the day of transfection, FuGENE 6 Transfection Reagent (Roche) was added into Opti-MEM® I (1x) in GlutaMAX(TM)-I (Gibco, Life Technologies) and incubated for 5 min at room temperature before mixed with relevant plasmids according to manufacturer's instructions. Transfection mixtures were incubated for 20 min before added to seeded cells. Transfected cells were placed in 37°C incubator with 5% CO₂ for 18 h incubation before harvesting for other experiments.

1.5 × 10⁸ HEK293-F cells were seeded in 300 ml FreeStyle™ 293 Expression Medium. After 24 h, cells were transfected using PEI. 300 μg DNA and 1.2 ml PEI (1 mg/ml) were dissolved separately in 10 ml Opti-MEM I (1X) + GlutaMAX™ reduced serum medium (Gibco). The solutions were mixed and incubated for 30 min at RT and then added to the flasks. Cells used for incorporation of UAAs were further supplemented with 20 nM PCK. The cells were harvested 30 h after transfection.