2.2. Dual‐luciferase reporter (DLR) assay

We constructed mouse STRA8 promoter luciferase reporter plasmid by restriction digest with Xhol‐HindⅢ and SETD8 promoter reporter plasmids containing different fragments by restriction digest with KpnI‐HindⅢ. Respectively, they were named pGL3‐STRA8Pro, pGL3‐SETD8ProF1R (−1980~+1 bp), pGL3‐SETD8ProF2R (−1499~+1 bp), pGL3‐SETD8ProF3R (−1000~+1 bp) and pGL3‐SETD8ProF4R (−495~+1 bp). The primers sequences are provided in Table ​Table1.1. Luciferase activity was quantified using the Dual‐Luciferase Reporter assay system (Promega). Firefly (Photinus pyralis) luciferase was used as an reporter gene and Renilla (Renilla reniformis) luciferase (pRL‐CMV) as the internal reference reporter gene. The specific fragment of the target promoter was inserted in front of the luciferase expression sequence. pGL3‐Basic were null reporter vectors without transcriptional activity and acted as a negative control. pGL4 luciferase vectors were used as positive controls as they contained numerous configurations. These included configurations with the synthetic Firefly Luc2 and Renilla hRluc luciferase genes, which have been codon optimized for more efficient expression and risk of anomalous transcription. Approximately 0.998 μg of reporter gene (pGL3‐Basic, pGL3‐target promoter or pGL4) was co‐transfected with 0.0014 μg pRL‐CMV into 293 cells and GC1 cells by Lipofectamine 2000 (Invitrogen) or 6 hours in a 24‐well plate. After 48 hours of continuous culture with complete media, the protein was collected. 100 μL of passive lysis buffer was added to each well to prepare the lysate of the transfected cells. Culture plates were frozen at −80°C for 10 minutes, and then rocked on a rocking platform at room temperature for 15 minutes. After centrifugation at 12000xg for 10 minutes, the supernatant was transferred to new tube for DLR assay. In DLR assay, the activities of Firefly and Renilla luciferases were measured sequentially from each single sample. The Firefly luciferase activity was measured first by adding 50 μL Luciferase Assay Reagent II to generate a stabilized luminescent signal and recorded as F. Renilla luciferase reaction was simultaneously initiated by adding Stop & GloR Reagent to the same tube and Renilla activity was recorded as F. According to the ratio of F/R, the activation degree of target reporter gene in different samples was compared. Luciferase activity is reported as the mean ± SEM of 3 experiments.

Primer sequences used in this study