Serum free medium

The GFP reporter vector (pEGFP-N1, Invitrogen, CA, USA) was used for fast detection of positive clones in subsequent FACS analysis. Transfection was performed with the lipofectamine 2000 reagent (Invitrogen, CA, USA) combined with 5 μg of the pEGFP-N1 plasmid for first and second transfection time. Briefly, the cells were seed in 3 × 6-well plates and cultured until 80% confluence. The day before transfection, the medium was removed and 2 ml of fresh complete medium was added to cells prior to transfection. Experimental mixture was prepared by adding the following reagents: pEGFP-N1 plasmid, lipofectamine 2000 reagent and serum free medium (Life Technologies, USA) as recommended by the manufacturer. The mixture was incubated at room temperature for 5 min then added to the cells dropwise. In continuation, the cells were incubated in a CO₂ incubator at 37°C. Then, three plates per treatment were collected for GFP expression assay as a following procedure: days 1 and 2 of first transfection continued with 3 days post-first transfection (day 5). Next 9 plates from treated cells were prepared for second transfection. Second transfection was done at day 5 of transfection for 48 hrs (day 7) as described above. Finally, three plates of cells were collected after 7 days and the rest were incubated 3 days post transfection for single cell cloning.

GUMC-395 cells (p3) were cultured in a collagen type I-coated flask using F medium supplemented with 10% fetal bovine serum, 0.125 ng/mL epidermal growth factor (Invitrogen, Carlsbad, CA, USA), 25 ng/mL hydrocortisone (Sigma–Aldrich, Burlington, MA, USA), 5 μg/mL insulin (Sigma-Aldrich), 0.1 nM cholera toxin (Sigma-Aldrich), in addition to 10 μM Y-27632 (Sigma-Aldrich). HFK cells (p4) were grown in serum-free medium (Life Technologies, Carlsbad, CA, USA). All cells were maintained in a humidified incubator with 5% CO₂ at 37 °C [15 (link),16 (link)]. Dneasy Blood & Tissue Kit (Qiagen, Hilden, Germany) was used to isolate DNA from cells. HFK and GUMC-395 DNA preparation were subjected, using HPV16 primers, to polymerase chain reaction (PCR) analysis according to the protocol previously described [15 (link)]. Primer sequences: HPV16, 5-TTATGAGCAATTAAATGACAGCTCAG-3, and 5′-TGAGAACAGATGGGGCACACAAT-3′. EVs were isolated from conditioned media (25 mL collected from 6 million cells) after being centrifuged at 1000× g for 10 min then filtered using 0.22 nm filter. The resulting supernatant was used to isolate EVs using ExoQuick reagent (SBI, product #EXOQ5A, lot #140624-001) following the manufacturer’s protocol and resuspended in 50 µL of PBS.

Antibody titers against Anc80L65 in CSF and serum were determined through neutralization assays. Using a 96-well format, heat-inactivated CSF or serum samples (collected as described above) were serially diluted in serum-free medium (Life Technologies), and then treated with Anc80L65-luciferase (10⁷ GC/well) for 1 h at 37 °C. The sample/Anc80L65-luciferase mix was then transferred onto HEK293 cells (10,000 cells/well), which had been treated with adenovirus (MOI 100) the day before. After 1 h at 37 °C, diluted serum medium (1 part serum-free, 2 parts serum) was added to each well. Two days later, the cells were treated with lysis buffer (Promega) and frozen at −80 °C for 30 min. The cells were then thawed at 37 °C for 15 min before being treated with substrate buffer (Tris-HCl, MgCl₂, ATP) (Life Technologies) and D-luciferin (Caliper Life Sciences). Luminescence output was read using the Synergy BioTek Plate Reader (BioTek). Data Analysis was performed using Microsoft Excel.

Three T125 cm² plates of Sf9 cells adapted for growth in serum-free medium (Invitrogen) were each co-transfected with 9 μg of pIEx10-LRIM1^HIS and 21 μg of pIEx10-APL1C^HIS using Escort IV transfection reagent (Sigma-Aldrich). Two batches of conditioned medium (180 mL total) were collected over 6 days, 0.45 μm filtered and supplemented with 0.1% Triton X-100. Conditioned medium was affinity purified in batch purified using Ni-NTA resin (Qiagen) producing LRIM1^HIS/APL1C^HIS heterodimer as well as some LRIM1^HIS and APL1C^HIS monomers and homodimers, which these cells also produce [47 (link)]. The purified protein was used to generate a guinea pig polyclonal antibody (Eurogentec).