Figure 1: TGF-β1, TGF-β2 or TGF-β3 induced concentration-dependent stimulation of intracellular luciferase accumulation in HEK293/SBE-Luc cells.
HEK293/SBE-Luc cells were plated and incubated at 37 ℃ for 16-18 hours prior to the addition of different concentrations of TGF-β1 (Cat. No. Z03411, GenScript), TGF-β2 (Cat. No. Z03429, GenScript) and TGF-β3. (Cat. No. Z03430, GenScript). After 6 hours of incubation, Fire-Lumi™ Reagent (Cat. No. L00877C, GenScript) was added and the luminescence signal was measured by Thermo Scientific Varioskan LUX. The relative light unit (RLU) were recorded and normalized to plot against the log of the cumulative doses of TGF-β (mean ± SD, n = 2). The EC50 of TGF-β1, TGF-β2, TGF-β3 on HEK293/SBE-Luc cells were 0.095 ng/ml, 0.096 ng/ml, 0.135 ng/ml, respectively.
Notes:
EC50 value is calculated with four parameter logistic equation:
Y=Bottom + (Top-Bottom) / (1+10^ ((LogEC50-X)*Hill Slope))
X is the logarithm of concentration. Y is the response.
Y is RFU and starts at Bottom and goes to Top along a sigmoid curve.
Figure 2: M7824 induced blockade of intracellular luciferase accumulation triggered by TGF-β1, TGF-β2 or TGF-β3 in HEK293/SBE-Luc cells.
HEK293/SBE-Luc cells were plated and incubated at 37 ℃ for 16-18 hours. Mix TGF-β1 (0.3 ng/ml), TGF-β2 (0.3 ng/ml) or TGF-β3 (0.3 ng/ml) with different concentrations of M7824 for 30 min. After 30 min of incubation, the mixture was added into the 384-well plate and incubated at 37℃ for 6 hours. After incubation, Fire-Lumi™ Reagent (Cat. No. L00877C, GenScript) was added and luminescence measured by Thermo Scientific Varioskan LUX. The relative light unit (RLU) were recorded and normalized to plot against the log of the cumulative doses of TGF-β (mean ± SD, n = 2). The IC50 of M7824 on HEK293/SBE-Luc cells were 0.0048 μg/ml (TGF-β1 activation), 1.221 μg/ml (TGF-β2 activation), 0.0005 μg/ml (TGF-β3 activation).
Notes:
EC50 value is calculated with four parameter logistic equation:
Y=Bottom + (Top-Bottom) / (1+10^ ((LogEC50-X)*Hill Slope))
X is the logarithm of concentration. Y is the response.
Y is RFU and starts at Bottom and goes to Top along a sigmoid curve.
TGF-β/SMAD Signaling Pathway SBE Reporter-HEK293 Cell Line
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