Pgl3 basic system

The pGL3-Basic system (Promega, Madison, USA) was used for the promoter activity analysis of PCK1 as previously described [50 (link)]. The putative upstream promoter regions were cloned from the genomic DNA of cattle and buffalo (Fig. 7a and Additional file 8: Fig. S2). Fragments were inserted into the KpnI and XhoI restriction sites of the pGL3-Basic vector to obtain recombinant plasmids (pGL3-cattle-PCK1 and pGL3-buffalo-PCK1). The pLR-TK vector was used as an internal control.

ChIP assays were conducted with ChIP Kit (Thermo, MA, USA) using HIF1A and IgG antibodies. The primers used for amplifying potential binding site of HIF1A in the Abhd16a and RhoA were shown in Supplementary Table S4. The promoter of Abhd16a or RhoA with HIF1A binding motif (WT) or without HIF1A binding motif (MUT) was constructed into the pGL3 basic system (Promega). The pGL3-ABHD16A-promoter-WT or the pGL3-ABHD16A-promoter-Mut and the pGL3-RhoA-promoter-WT or the pGL3-RhoA-promoter-Mut were transfected into the MGC-803 cells together with the pCMV-Renilla control. Following the transfection for 30 h, the luciferase activity was detected. Transient transfection and luciferase assays were performed as described previously [12 (link)]. HEK-293, MGC-803 or SGC-7901 cells were co-transfected with miR-4646-5p mimics (GenePharma, Shanghai) and pMIR-PHD3 3′-UTR (wild-type or mutant) and pRL-TK control vector using lipofectamine 2000 (Invitrogen, Carlsbad). Renilla and firefly luciferase activities were measured with a Dual-Luciferase Reporter System (Promega, USA) after culture for 30 h.