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Transforming growth factor-beta (TGF-β) is a cytokine involved in both physiological and pathological processes. During tumor progression, TGF-β signaling modulates immune and inflammatory responses, as well as the tumor microenvironment. The function of the TGF-β signaling pathway relies on the binding of ligands to cell membrane receptors, the activation and nuclear translocation of cytoplasmic mediators, and the subsequent regulation of target gene expression.

The ligands for TGF-β include TGF-β1, TGF-β2, and TGF-β3. The cell- surface receptors for TGF-β signaling are primarily classified into two subtypes: Type I (TGF-βRI) and Type II (TGF-βRII). Signal transduction from the cytoplasm to the nucleus depends on three specific members of the Smad family: Smad2, Smad3, and Smad4. The binding of a ligand to TGF-βRII triggers the phosphorylation of Smad2 and Smad3 by TGF-βRI; these phosphorylated Smads then form a trimeric complex with Smad4 and translocate into the nucleus. Within the nucleus, the Smad trimeric complex binds to Smad-binding elements (SBEs) located on target genes, thereby directly regulating the expression of TGF-β-responsive genes, or indirectly modulating target genes through the recruitment of other cofactors (such as coactivators or corepressors).

SBE-Luc HEK293 reporter cells are HEK293 cells engineered to express a Luciferase (Luc) reporter gene under the transcriptional control of an SBE. The mechanism by which TGF-β binding to its receptor activates the SBE is illustrated in the figure below.

Figure 1. Schematic Diagram of the SBE-Luc HEK293 Principle

Figure 2. Dose Response of Human TGFβ1 in SBE-Luc HEK293 (C27).

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 SBE-Luc 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.