Pgl 3 firefly reporter vector

The putative FXR binding regions in the promoter of IL-6 were firstly predicted with online databases PROMO and NUBIscan. Then the regions were amplified by PCR from genomic DNA extracted from splenocytes in C57BL/6 WT mice and cloned using Kpn I and Sma I into the pGL-3 firefly reporter vector (Promega). All the constructs were verified by sequencing. Then respective luciferase reporter constructed or basic pGL-3 vector, as with Renilla plasmid and the FXR overexpression vector (Genecopoeia, Guangzhou, China) were co-transfected into 293T cells using lipofectamine 2000 (Thermo). Transfected cells were then lysed and luciferase activity was quantified with the Dual-Luciferase Reporter Assay (Promega) following the manufacturer’s instructions and normalized to the activity of the co-transfected Renilla reporter gene.

HRE regions (I–V) of Il10 promoter (Supplementary table 2) were amplified by PCR from genomic DNA extracted from splenocytes in C57BL/6 WT mice and cloned into the pGL3 firefly reporter vector (Promega). 293T cells were co-transfected with luciferase reporter construct and β-gal plasmid using Lipofectamine 2000 (invitrogen). Transfected cells were cultured under normoxic (21% O₂) or hypoxic (1% O₂) conditions for 24 h. Cells were then lysed and luciferase activity was quantified and normalized to the activity of the co-transfected β-gal reporter gene.