Renilla luciferase

A 2000-bp promoter fragment surrounding the TGF-β1 transcription start site was amplified from genomic DNA and subsequently cloned and inserted into the pGL3-Basic vector. The sequences of primers used were as follows: Sense: 5ʹ-atctgcgatctaagtaagcttCGCAGGGTGTTGAGTGACAGGAG-3ʹ, Anti-sense: 5ʹ-cagtaccggaatgccaagcttGGTGACCTCCTTGGCGTAGTAGTCG-3ʹ. Following construction, these plasmids were transfected into HMrSV5 cells using TransIntro PL Transfection Reagent (TransGen). Concurrently, the phRL plasmid containing Renilla luciferase (Promega) was introduced as an internal control. The culture medium was changed to either normal or 60 mM glucose-containing medium 24 h posttransfection. After a 3 day incubation, luciferase activity, with the Renilla luciferase vector phRL serving as the internal reference for luciferase signals, was assessed using the Dual-Luciferase Reporter Assay System (Beyotime, RG042M).

NF-κB luciferase reporter assay was adopted to evaluate relative NF-κB activity. Briefly, the cells were plated in the 24-well plate and cultured to attain a confluency of 70–80%. After adherence, the cells were cotransfected with 600 ng of NF-κB luciferase reporter gene constructs (Beyotime, China) and 200 ng of Renilla luciferase (Beyotime, China), which was used to normalize data for transfection efficiency. After 24 h of transfection, the cells were treated with exogenous SLPI (40 μg/mL) or the same volume of ddH₂O. The cells were then cultivated for 12 h and cell lysates were analyzed using a dual luciferase reporter assay kit (RG027,Beyotime, China) on Modulus™ (Turner Biosystems, Sunnyvale, CA, USA).