Dy293b

Cells were cultured identically to previous, except they were plated in 96 well cell culture treated plates and exposed to gamma-irradiated Mtb for a total of 24 h to facilitate VEGF production and secretion. Supernatants were collected and spun down and then the upper layer was collected for further analysis. ELISA was performed according to the manufacturer’s instructions (R&D Systems #DY293B). Absorbance was read on an Agilent Synergy LX plate reader.

The levels of VEGFA and hCG‐β secreted by the established placenta‐like tissue were detected using human VEGFA (DY293B; R&D) and hCGβ ELISA kits (ab100533; Abcam) according to the manufacturer's instructions. After culturing for 48 h, the collected culture medium was centrifuged to remove debris. Then the supernatant was stored at −80°C before use. The absorbances were measured using TECAN Infinite M Nano.

HGSOC cells were plated in 12-well plates at the same cell densities as the MMP2 expression assay and allowed to attach overnight. Cells were then rinsed with PBS and serum-free media was added. After serum starving for 24 h, cells were washed with PBS and vehicle (0.1 % BSA in PBS) or 10 ng/mL HB-EGF, NRG1β, IGF1, or HGF was added in serum-free media. After 24 h of growth factor treatment, conditioned media was collected, and VEGF levels were determined by ELISA (#DY293B, R&D Systems) according to the manufacturer’s instructions.

Blood-derived secretomes—fresh plasma and fresh serum, hypoxia preconditioned plasma (HPP) and serum (HPS), and hypoxia preconditioned media (HPP/HPS -NaCl/-PBS/-G-5%/-AIM) were sampled and analyzed by ELISA for vascular endothelial growth factor (VEGF-A), epidermal growth factor (EGF), thrombospondin-1 (TSP-1), and platelet-factor-4 (PF-4) (DY293B for VEGF-A, DY236 for EGF, DY3074 for TSP-1, DY795 for PF-4, Duoset, R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer’s instructions. Factor concentrations in blood-derived secretomes/media were measured immediately after the predefined hypoxic incubation period (2, 4, or 8 days). All conditions were tested in triplicate per blood donor, and a total of four donors was taken for final evaluation.

Male and female aVICs and qVICs were seeded in 10 cm dishes at a density of 50,000 cells/cm² and cultured in standard growth medium (low-glucose DMEM with 10% FBS and 1% penicillin-streptomycin solution) for 48 h. The aVICs were cultured on 10 cm tissue culture coated plates while qVICs were cultured on 10 cm collagen-coated plates in order to retain their qVIC phenotype for the duration of the experiment. Seven ELISAs were run on the conditioned culture media and culture lysate (collected in RIPA buffer, 89900; ThermoFisher): endothelin-1 (DY1160; R&D Systems, Minneapolis, MN), platelet derived growth factor-A (PDGF-A, DY221; R&D), vascular endothelial growth factor-A (VEGF-A, DY293B; R&D), basic fibroblast growth factor (bFGF, DY233; R&D), epidermal growth factor (EGF, DY236; R&D), insulin-derived growth factor-1 (IGF-1, DY291; R&D), and thrombospondin-2 (TSP2, DTSB20; R&D), all per manufacturer's instructions. The media and lysate were differentially diluted in the sample dilution buffer specific to each ELISA kit such that all sample absorbance readings fell within the dynamic range of each ELISA. A Quant-iT™ PicoGreen™ dsDNA Assay (P11496; Thermo Fisher Scientific) was performed on the lysate to normalize to cell number across all samples.