Renilla protein activity

HeLa cells were dual-infected with STAT3 lentiviral luciferase and Renilla lentiviral luciferase (#CCS-9028L, Qiagen, Hilden, Germany), selected by puromycin (stable cell line-Hela STAT3 Luc/Ren). Cells were grown to 70–80% confluence in 6-well plates and transiently transfected with encoding emerin and incubated for 24 h at 37 °C in a CO₂ incubator. Then, cells were treated with IL-6 for 6 h, lysed in 5× passive lysis buffer (Promega, Madison, WI, USA), and analyzed for dual luciferase activity with a GloMax^® 96-well microplate luminometer system (Promega). The luciferase reporter activity in each sample was normalized to Renilla protein activity (Promega).

Our lab previously made an inducible NICD-expressing stable cell line (HeLa RBP-JK (Luc/Ren), through infecting HeLa cells through Lentivirus [50 (link)]. Cells were grown to about an 80% cell concentration in 96-well plates and treated with several compounds for 24 h. Then, the cells were lysed in 5× Passive Lysis Buffer (Promega, Madison, WI, USA) and dual-analyzed for Luciferase and Renilla activity with a GloMax^® 96-well microplate luminometer system (Promega). The Luciferase reporter activity in each sample was normalized with Renilla protein activity (Promega).