Gaussia luciferase flash assay kit

The exosome fraction was prepared as described earlier in the Exosome Isolation section. The Gaussia luciferase activity in the exosome was measured by using the Gaussia Luciferase Flash Assay Kit (Thermo Fisher Scientific) according to the protocol supplied by the manufacturer.

For short-term treatments, 293T cells were transfected with pCMV-Gaussia Luc Vector (ThermoFisher Scientific, 16147) as described above. After 18 h, medium was removed and replaced with fresh medium containing 6-TG, BFA, or DMSO. After 6 h, medium was removed, debris was cleared by centrifuging samples at 5,000 × g for 5 min, then samples were stored at -80°C until measuring Gaussia luciferase activity using the Gaussia Luciferase Flash Assay Kit (ThermoFisher Scientific, 16158) as per the manufacturer’s directions on a FLUOstar Omega microplate reader (BMG Labtech). For long-term treatments, 293T cells were co-transfected with pCMV-Gaussia Luc and pLJM1-Luc2 and treated 6 h post transfection. After 24 h, supernatants were harvested as described above. Cell monolayers were lysed in Reporter Lysis Buffer (Promega) as described above. To measure both Luciferase and Gaussia luciferase activity in samples, we used the Dual-Luciferase Reporter Assay System (Promega, E1910), which although designed for firefly and Renilla luciferase, can be readily adapted for firefly and Gaussia luciferase as both Gaussia and Renilla luciferase both use coelenterazine as a substrate.

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.

All compounds were acoustically transferred to the assay plates using ECHO555 (Beckman Inc. Sykesville, MD, USA) at a final desired concentration. All plates included DMSO and cytosine arabinoside (AraC) controls. Cells were trypsinized, resuspended in a fresh medium, and counted before mixing with diluted reporter viruses to reach the desired multiplicity of infection (MOI). The cell–virus mixture was then plated in 96-well plates pre-coated with library drugs along with positive (AraC) and negative (DMSO) controls. The plating procedure was carried out using an automated microplate dispenser, and 50 µL of the virus–cell mixture (~5000 cells) were dispensed into each well. The plates were then incubated at 37 °C with 5% CO₂. After 24 h, luciferase activities were measured using a Glomax luminometer with a Gaussia luciferase flash assay kit (Thermo Fisher 16158).