KC-4305

293T-SMAD-Luc2-GARP-LTGFβ-Cell-Line

44358

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Background of 293T-SMAD-Luc2-GARP-LTGFβ-Cell-Line

Transforming growth factor-β (TGF-β) is a potent effector in the liver, which is involved in a plethora of processes initiated upon liver injury. TGF-β affects parenchymal, non-parenchymal, and inflammatory cells in a highly context-dependent manner. Its bioavailability is critical for a fast response to various insults. In the liver - and probably in other organs - this is made possible by the deposition of a large portion of TGF-β in the extracellular matrix as an inactivated precursor form termed latent TGF-β (L-TGF-β). The intracellular effectors of TGF-beta signalling, the Smad proteins, are activated by receptors and translocate into the nucleus, where they regulate transcription. Although this pathway is inherently simple, combinatorial interactions in the heteromeric receptor and Smad complexes, receptor-interacting and Smad-interacting proteins, and cooperation with sequence-specific transcription factors allow substantial versatility and diversification of TGF-beta family responses.

Specifications

Catalog Number	KC-4305
Cell Line Name	293T-SMAD-Luc2-GARP-LTGFβ-Cell-Line
Host Cell Line	293T-SMAD-Luc2
Description	Stable 293T cell line expressing exogenous luciferase gene under the control of TGFβ signaling pathway.
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS +150μg/ml Hygromycin B+1μg/ml puromycin
Selection Marker	Hygromycin and Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T-SMAD-Luc2-GARP-LTGFβ cell line was generated using lentivirus expressing GARP-LTGFβ sequence.

Characterization

Figure 1. 293T-SMAD-Luc2-GARP-LTGFβ cell line was seeded into the 96-well plate, co-cultured with SK-BR-3 cell line at a maximum density of 80000cells/well for 16 hours, then readout with Bright-Glo system.

Figure 2. 293T-SMAD-Luc2-GARP-LTGFβ cell line was seeded into the 96-well plate, and treated with ABBV-151 at a maximum concentration 20μg/mL for 1 hours, then co-cultured with SK-BR-3 cell line (30000cells/well) for 16 hours and readout with Bright-Glo system.

Figure 3. 293T-SMAD-Luc2-GARP-LTGFβ cell line was seeded into the 96-well plate, and treated with hGA17-hIgG1 at a maximum concentration 20μg/mL for 1 hours, then co-cultured with SK-BR-3 cell line (30000cells/well) for 16 hours and readout with Bright-Glo system.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 150μg/ml Hygromycin and 1μg/ml Puromycin)in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage.

References

Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature. 2003 Oct 9;425(6958):577-84. doi: 10.1038/nature02006. PMID: 14534577.
Li Y, Fan W, Link F, Wang S, Dooley S. Transforming growth factor β latency: A mechanism of cytokine storage and signalling regulation in liver homeostasis and disease. JHEP Rep. 2021 Nov 18;4(2):100397. doi: 10.1016/j.jhepr.2021.100397. PMID: 35059619; PMCID: PMC8760520.