Stable 3T3-L1 PPARγ and STAT3 reporter cell lines were generated as follows. Briefly, consensus binding sites of PPARγ and STAT3 (AGGACAAAGGTCA for PPARγ and TTTCCGGGAA for STAT3) were identified using the TRANSFAC public database. The response element (RE) oligonucleotides, containing three consensus binding sequences, were cloned into the GLuc-DRE2-viral vector, wherein GLuc is regulated by a minimal promoter [14 (link)]. The expression of GLuc is induced when a target transcription factor binds to its consensus binding site. Thereafter, stable 3T3-L1 PPARγ and STAT3 reporter cell lines were generated through lentiviral transduction.
To profile the activation of PPARγ and STAT3, the reporter cells were differentiated in 12-well plates as described above. The supernatant was harvested on day 1, day 3, and day 5 post-differentiation induction and used to measure GLuc activity. GLuc activity (Relative Light Units; RLU) was assessed using the Gaussia Luciferase Flash Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and TriStar2 LB 942 Modular Multimode Microplate Reader (Berthold technologies, Bad Wildbad, Germany) in accordance with the manufacturers’ instructions. After normalization with cell density, relative fold changes were determined at each time point.
To profile the activation of PPARγ and STAT3, the reporter cells were differentiated in 12-well plates as described above. The supernatant was harvested on day 1, day 3, and day 5 post-differentiation induction and used to measure GLuc activity. GLuc activity (Relative Light Units; RLU) was assessed using the Gaussia Luciferase Flash Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and TriStar2 LB 942 Modular Multimode Microplate Reader (Berthold technologies, Bad Wildbad, Germany) in accordance with the manufacturers’ instructions. After normalization with cell density, relative fold changes were determined at each time point.
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