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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 regulates immune/inflammatory responses and 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. TGF-β ligands 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 heterotrimeric 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 regulating target genes by recruiting other cofactors (such as coactivators or corepressors).

SBE-Luc CHO-K1 Reporter Cells are a line of CHO-K1 cells engineered to express the 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 CHO-K1 Principle

Figure 2. Dose Response of Human TGFβ1 in SBE-Luc CHO-K1 (C17).

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 CHO-K1 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.