Nano glo dual luciferase reporter assay system

The selected promoters and enhancer sequences analyzed are listed in Table 1. Each fragment was PCR amplified and cloned into the basic pNL-vector (Promega) upstream of the NanoLuc luciferase reporter gene. For promoter assays, the fragments were cloned into KpnI/HindIII sites of the promoterless pNL plasmid. For enhancer assays, the fragments were first inserted upstream of the minimal promoter element into the KpnI/HindIII sites of the pNL-minP NanoLuc luciferase reporter plasmid (Promega). Subsequently, the fragment (Xist bp 2000-1380) that displayed the highest enhancer activity was cloned upstream of the putative XistAR promoter sequence (Xist bp 3000-2750). The plasmids were transfected into female XEN cells plated in a 96-well format. Each well was transfected with 100ng of pNL-NanoLuc reporter construct and 6ng of transfection control plasmid encoding the firefly luciferase gene (pGL3-firefly; Promega), with Lipofectamine 2000 (Invitrogen) transfection reagent, following manufacturer's instructions. Each construct was tested in triplicate in each of 3 separate transfections. Seventy two hours after transfection, the activities of firefly and NanoLuc luciferase were measured using the Nano-Glo Dual-Luciferase reporter assay system (Promega, #N1610). The NanoLuc luciferase activity was normalized with firefly luciferase activity for each transfection.

HeLa cells and PLAT-E cells were cultured in 10% fetal bovine serum–Dulbecco’s modified Eagle’s medium containing 1% penicillin–streptomycin (all from Sigma-Aldrich). PLAT-E cells were transfected with the pMIGR construct (as a control) or the pMIGR-3×FLAG-BHLHA9 construct with use of FugeneHD (Promega). Forty-eight hours later, the media were collected, filtered, and transferred to the HeLa stable cell line in a medium containing puromycin (1μg/ml). Control or 3×FLAG-BHLHA9-expressing HeLa stable cells were cotransfected with each TP63 promoter–nanoluciferase reporter gene construct and the pGL4.13 luciferase construct (for normalization; Promega) with use of Fugene HD (Promega). Cell extracts were prepared 48 h after transfection, and luciferase activity was measured with a Nano-Glo dual-luciferase reporter assay system (Promega).