Luciferase reporter assay kit

GCs were isolated and cultured using previously described methods [12 (link)]. Briefly, fresh ovaries from adult sows were washed alternately with saline and 75% alcohol, and follicular fluid from healthy follicles was drawn, collected into centrifuge tubes, and centrifuged to collect cells. After washing with phosphate-buffered saline, the cells were resuspended using DMEM/F12 medium (containing 15% fetal bovine serum and 1% penicillin), seeded into cell culture plates, and incubated at 37 °C in a 5% CO₂ incubator. When both GC density and status reached the transfection requirement, plasmids (1000 ng per well) were transfected into GCs by using HighGene (ABclonal, Wuhan, China) according to instructions. After 24 h of transfection, GCs (5 × 10⁵) were collected for luciferase reporter assay. Luciferase activity was measured using a luciferase reporter assay kit (Vazyme, Nanjing, China) according to the instructions.

HEK-293T cells expressing human ACE2 (293T/ACE2) were plated in 96-well plates (Corning, 3599) at a density of 20,000 cells per well. On the following day, monoclonal antibodies (mAbs) were serially diluted in complete media, combined with wild-type (WT) pseudoviruses (Vazyme) or Omicron pseudoviruses (Vazyme), and incubated for 1 h at 37°C. Subsequently, the culture media of 293T/ACE2 cells were replaced with the pre-incubated mixture of mAbs and pseudoviruses, and the cells were cultured for an additional 16 h. Luciferase activity in 293T/ACE2 cells was measured using a luciferase reporter assay kit (Vazyme, DD1201). The IC₅₀ (50% inhibitory concentration) values were determined by fitting a non-linear four-parameter dose-response curve using GraphPad Prism 8.0.

Mammalian two‐hybrid assays were performed as previously reported. Briefly, HMGB1 and HDAC1 cDNA were cloned into the pBIND and pACT vectors, then co‐transfected with the pG5luc vector (Promega). After 48 h, reporter activity was measured using the Luciferase Reporter Assay Kit (Vazyme), and Renilla activity was used to normalize firefly luciferase activity.