Firefly luciferase reporter gene assay kit

A total of 1 × 10³ tachyzoites of RH-Luc strains were inoculated into HFFs grown in 96-well plates using DMEM containing gradient diluted iron chelating agents DFO (Topscience, Shanghai, China) or iron supplements of ammonium iron(II) sulfate (Aladdin, Shanghai, China). Dimethyl sulfoxide (DMSO, M&C Gene, Beijing, China) was used as a control. After 72 h of culture, the relative luminescence units (RLU) were measured using a fluorescence microplate reader (Tecan, Spark 10M, Männedorf, Switzerland) and Firefly Luciferase Reporter Gene Assay Kit (Beyotime Biotech, Shanghai, China). The inhibition rate was calculated using the formula: inhibition rate = [(RLUDMSO − RLUexperimental group)/RLUDMSO] × 100%. Nonlinear regression curve fitting was employed to determine the median lethal concentration (IC₅₀) and 95% confidence interval (CI). Three independent experiments were performed.

microRNA.org prediction website was used for the analysis of the target gene of miR-203. The bioinformatics was applied to predict the target binding site of miR-203 and PRC1 gene. Synthesized primers were obtained from Shanghai GeneChem (Shanghai, China). The WT vector was constructed as follows: amplified 3′ UTR sequence of PRC1 was cloned and inserted in pmiR-RB-REPORT vector digested by restrictive endonuclease XhoI/NotI. The MUT vector was constructed with WT vector used as a template. MUT primers were designed and two mutation subsections were obtained by PCR, and lastly the products of PCR were purified and cleaved by restrictive endonucleases XhoI/NotI, which were harvested and inserted into pmiR-RB-REPORT vector. The generated PRC1-MT or PRC1-MUT plasmid was co-transfected with miR-203 mimic into SCL-1 cells (Guangzhou Jennio Biotech, Guangdong, China), followed by luciferase activity detection at 560 nm with the use of a Firefly Luciferase Reporter Gene Assay Kit (RG005, Beyotime Biotechnology, Shanghai, China) and a microplate reader (MK3, Thermo Fisher Scientific, Waltham, MA, USA). The miScript target protectors were modified RNA oligonucleotides complementary to specific target sites that could not bind to other sequences.³⁹ (link)

1×10⁴ BV2 cells were seeded in 24-well plates, and transfected with lentivirus NF-κB and AP-1 reporters (QIAGEN, Hilden, Japan) for 24 h and then screened with puromycin (2.5 μg/ml) to obtain BV2 cell lines stably expressing NF-κB and AP-1 reporter elements. Subsequently, BV2 cells stably expressing NF-κB and AP-1 reporter elements were treated with LPS (100 ng/ml) with or without RRx-001 (5 μM). After treatments, the activities of NF-κB and AP-1 promoters were detected with Firefly Luciferase Reporter Gene Assay Kit (Beyotime Biotechnology, Shanghai, China) at 560 nm by using Luminoskan™ Ascent (Thermo Fisher Scientific, Waltham, United States).