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Bone Morphogenetic Proteins (BMPs) are secreted signaling molecules belonging to the TGF-β superfamily. Initially discovered for their ability to induce ectopic bone formation, their signaling pathways are transduced via Type I (BMPR-IA/IB) and Type II serine-threonine kinase receptors.

The BMP signaling pathway plays a pivotal role in metazoan biology, intricately shaping embryonic development, maintaining tissue homeostasis, and influencing disease progression. Dysregulation of BMP signaling has been implicated in a wide range of pathological conditions, including cancer, cardiovascular diseases, and developmental disorders. The BMP signaling pathway encompasses a rich repertoire of ligands, with over 20 members identified to date. These ligands can be classified based on their nucleotide or amino acid sequence similarities. The pathway is activated when a dimeric BMP ligand binds to two cognate Type II receptors, thereby facilitating the formation of a heterotetrameric complex with two Type I receptors. The Type II receptor kinases remain constitutively active, phosphorylating specific serine residues within the Type I receptors and thereby activating them. The BMP signaling pathway comprises four Type I receptors—ALK1 (ACVRL1), ALK2 (ACVR1), ALK3 (BMPRIA), and ALK6 (BMPRIB)—as well as three Type II receptors: BMP Receptor Type II (BMPR2), Activin Receptor Type IIA (ACVR2A), and Activin Receptor Type IIB (ACVR2B). Receptor activation leads to the phosphorylation of Receptor-regulated SMAD (R-SMAD) transcription factors, specifically SMAD1, SMAD5, and SMAD8. These R-SMADs form heterocomplexes with the Common SMAD (Co-SMAD), SMAD4, and translocate into the cell nucleus, where they regulate the expression of target genes at the transcriptional level.

The BMP Effector Reporter Cell model accurately recapitulates the in vivo signal transduction processes of BMPs; the underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the HEK293 Human BMP Effector Reporter Cell Cell Model

Figure 2. Dose Response of Ligands in BMP Effector Reporter Cell(C38).

Cell Resuscitation
1)Rapidly thaw the frozen cells in a 37 °C water bath for approximately 60 seconds. Once thawed (which may take slightly less or more than 60 seconds), immediately transfer the cell suspension from the cryovial into a 15 mL centrifuge tube containing 10 mL of pre-warmed HEK293 Human BMP Effector Reporter Cell complete culture medium.
2)Centrifuge cells at 1000 rpm for 5 min to remove medium, then resuspend cells in 5 mL of pre-warmed complete medium.
3)Transfer the cell suspension into a T25 culture flask and incubate at 37 °C with 5% CO₂.
4)After approximately 24–36 hours, replace the medium or passage the cells to remove non-adherent dead cells.

Subculturing procedure
1)When the cell density reaches the appropriate confluency for passaging, wash the cells with PBS, then add 1 mL trypsin to detach the cells. When more than 80% of the cells detach upon gently tapping the culture flask, add complete culture medium to terminate digestion. Gently pipette to obtain a single-cell suspension, transfer to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.

2)Discard supernatant after centrifugation. Resuspend cells in fresh medium to a single-cell suspension and transfer to a new culture flask for continued growth.

Cell Freezing
After trypsinization and centrifugation of cells from each T75 flask or 10 cm culture dish, discard the supernatant. Add 2 mL of cryopreservation medium (90% FBS + 10% DMSO), gently resuspend thoroughly, and aliquot into two cryovials. Immediately place the cryovials into a controlled-rate freezing container (e.g., Nalgene 5100-0001), fill with isopropanol to the indicated level, and store at −80 °C. After 24 hours, transfer the cryovials to liquid nitrogen for long-term storage.