Experimental animals and animal care

The care and experimental use of chickens were approved by the Institute of Laboratory Animal Resources, Seoul National University, and conducted in accordance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Chickens were maintained following standard procedures at the University Animal Farm. Methods were approved by the Institutional Animal Care and Use Committee (IACUC, SNU-220311-1) of Seoul National University.

Western blotting analysis

To analyze hADPN present in egg white (EW) from OVAADPN knock-in (KI) chickens, western blotting procedures were performed as previously described [29]. Briefly, for non-reducing conditions, samples were mixed with an equal volume of native sample buffer (BioRad, Hercules, CA, USA). For reducing conditions, samples were denatured by adding an equal volume of beta-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) in 2× Laemmli sample buffer (BioRad) and boiled at 95 °C for 5 min. In the western blotting analysis, EW samples were diluted to 500 times their volume and an equal volume of each sample was loaded. A total of 100 ng of commercial recombinant hADPN derived from human embryonic kidney (HEK) 293 cells (RD172023100; Biovendor R&D, Asheville, NC, USA), High-Five (Hi-5) cell-derived recombinant hADPN (450 − 24; PeproTech, Cranbury, NJ, USA), serum-derived commercial hADPN (CYT-024; ProSpec, East Brunswick, NJ, USA), or purified recombinant hADPN from OVA ADPN KI chicken EW was used for each analysis. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membranes, and blocked with 5% skim milk (Becton Dickinson, East Rutherford, NJ, USA) for 1 h at room temperature. Subsequently, membranes were incubated with primary antibodies at 4 °C overnight, followed by appropriate horseradish peroxidase-conjugated secondary antibodies. Primary antibodies used in this study included anti-ADPN antibody (Abcam, Cambridge, UK) and anti-mouse IgG HRP-linked antibody (Santa Cruz Biotechnology, Dallas, TX, USA). Immunoreactive proteins were visualized using ECL Select Western Blotting Detection Reagent (Amersham, Buckinghamshire, UK), and signals were detected using a ChemiDoc XRS imaging system (BioRad).

Quantification of ADPN protein

The quantities of total ADPN and HMW ADPN from OVA ADPN KI EW were measured using human total ADPN quantikine ELISA kit (DRP300; R&D systems, Minneapolis, MN, USA) and human HMW ADPN quantikine ELISA kit (DHWAD0; R&D systems) respectively. These assays were conducted following the manufacturer’s instructions and as described previously [29]. Briefly, these kits utilize the double-antibody sandwich method, where the optical density is proportional to the amount of anti-total ADPN or anti-HMW ADPN monoclonal antibody present. We determined the concentration of EW-derived hADPN by comparing with the optical density of the standard protein.

Purification of hADPN from genome-edited hen EW

Purification of hADPN from OVA ADPN KI chicken EW was conducted according to previously established protocols [29]. Briefly, EW was combined with five times its volume of 40% ammonium sulfate (Thermo Fisher Scientific, Waltham, MA, USA) and stirred for 4 h at 4 °C. Subsequently, the mixture was centrifuged at 3000 g for 1 h at 4 °C, and the resulting pellet was resuspended in four volumes of 20 mM Tris-HCL and 50 mM NaCl (pH 8.0) to reach the original EW volume. The sample was filtered through a 0.2 μm bottle filter and loaded onto a 5 mL HiTrap Q column (Cytiva, Marlborough, MA, USA). The protein was eluted with 20 mM Tris-HCl and 1 M NaCl (pH 8.0), and further purified via size-exclusion chromatography using a Superdex 200 Increase 10/300 GL column (Cytiva) pre-equilibrated with 20 mM Tris-HCl and 50 mM NaCl (pH 8.0).

Isolation of chicken adipose tissue and oviduct magnum

Wild-type 30-week-old White Leghorn and OVA ADPN KI hens were sacrificed for the isolation of chicken adipose tissue and oviduct magnum. Isolation of chicken adipose tissue was performed as described previously [30] with some adjustments. Briefly, the adipose tissues were separated from the subcutaneous fat tissues of the abdomen, washed with phosphate-buffered saline (PBS), and chopped into small pieces using a thin blade. Isolation of chicken oviduct magnum was conducted using a previously established method [31] with slight modifications. Magnum segments from the oviduct were trimmed and washed with PBS, the inner cell layer of the magnum was scraped using a thin blade, and collected tissues were dissolved in TRIzol reagent (Thermo Fisher Scientific) for subsequent RNA extraction.

Cell culture and recombinant hADPN treatment

Human umbilical vein endothelial cells (HUVECs) were cultured in 12 well-plates (SPL Life Sciences, Pocheon, Korea) in a vascular cell basal medium (ATCC, Manassas, VA, USA). The culture medium comprised 0.2% bovine brain extract (BBE; ATCC), 5 ng/mL recombinant human epidermal growth factor (rhEGF; ATCC), 10 mM L-glutamine (ATCC), 0.75 Units/mL heparin sulfate (ATCC), 1 µg/mL hydrocortisone (ATCC), 50 µg/mL ascorbic acid (ATCC), 2% fetal bovine serum (ATCC), and 1× antibiotic-antimycotic agents (Thermo Fisher Scientific), and culturing was performed in a 5% CO2 incubator at 37 °C. Confluent cells were detached using 0.05% trypsin EDTA (Thermo Fisher Scientific) in Hank’s balanced salt solution (HBSS; Hyclone, Logan, UT, USA). For recombinant hADPN treatment, HUVECs under 10 passages were grown until 80−90% confluency, then treated with 10 µg/mL of EW-derived recombinant hADPN, HEK293 cell-derived commercial recombinant hADPN (Biovendor R&D), or Hi-5 cell-derived commercial recombinant hADPN (PeproTech) for 7 days. After treatment with recombinant hADPN, cells were subjected to stress induced by 100 µM H2O2 for 1 h.

Oil Red O staining and quantification of Oil Red O

The treated HUVECs were fixed with 10% formalin for 1 h at room temperature. After washing with 60% isopropanol (1.09634.1011; Supelco, Bellefonte, PA, USA), cells were incubated with 0.2 μm filtered Oil Red O working solution (5694 − 4125; Daejung, Seoul, Korea) for up to 2 h with gentle rocking. Subsequently, cells with lipid droplets were imaged using an Eclipse Ti-U optical microscope (Nikon, Tokyo, Japan). To quantify accumulated lipid droplets, cells were washed with 60% isopropanol, Oil Red O stain was extracted with 100% isopropanol, and the absorbance was measured at 492 nm.

Quantitative reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was extracted from HEK293 cells, chicken adipose tissue, chicken oviduct magnum, and recombinant hADPN-treated HUVECs by chopping and dissolving tissues in TRIzol reagent (Thermo Fisher Scientific). RNA was then reverse-transcribed using a SuperScript III Reverse Transcription Kit (Thermo Fisher Scientific), and the resulting complementary DNA (cDNA) was amplified using target gene-specific primer sets. Quantitative RT-PCR was performed with an initial denaturation at 95 °C for 5 min, followed by 40 cycles at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min. Expression levels of the target genes were quantified using EvaGreen dye (Biotium, Hayward, CA, USA) and a StepOnePlus real-time PCR machine (Applied Biosystem, Waltham, MA, USA). Each test sample was analyzed in triplicate, and quantification of relative target gene expression was performed after normalization against the expression of chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control. Details about the primer sets used can be found in Supplementary Table 1. Human primer sets were used for HEK293 cell and HUVEC mRNA target gene amplification, and chicken primer sets were used for mRNA target gene amplification in chicken adipose tissue and the oviduct magnum.

Statistical analyses

Statistical analysis was performed using Prism software (GraphPad, Boston, MA, USA). Significant differences between groups were determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison tests for post-hoc analysis. A p < 0.05 was considered statistically significant.