HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum in a 37°C humidified incubator with 5% CO2. Transfection was performed using Lipofectamine 2000 (11668019, Invitrogen) according to the manufacturer’s instructions. Once the HEK293T cells transfected with constructs expressing secreted proteins, such as Pinhead, Chd, and BMP1a, reached 80% confluency, they were washed three times with phosphate-buffered saline (PBS) and cultured in serum-free DMEM for 24 hours. CM was then collected from each sample and centrifuged at 3000g for 5 min, filtered through a 0.22-μm filter, and concentrated to 10% of the original volume using Centriplus concentrators (Amicon).

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