Jetpei huvec dna transfection reagent

The functional m6A binding sites were accessed from the website https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE118093. Potential HIF1α binding sites were identified on the human lncRNA MIAT promoter by using the Binding Site Scanner (http://nrmotif.ucr.edu) based on the Support Vector Machine (SVM). The 5′-promoter fragment of the human lncRNA MIAT locus (−1940 to +166 bp) was amplified by PCR and cloned into the pGl3-basic plasmid (Promega, USA) at KpnI and BamHI sites^{30 (link),31 (link)}. Serial deletion of MIAT constructs as well as a construct with HIF1α motif deletion were generated by using the Q5 Site-Directed Mutagenesis Kit (NEB) as per the manufacturer’s protocol. All constructs were confirmed by sequencing. Co-transfections were performed with the jetPEI-HUVEC DNA transfection reagent (Polyplus, USA) containing Renilla luciferase (Promega, E2231) bearing MIAT-pGl3-basic plasmids. Luciferase activity was assessed at 24 h after transfection by using the Dual-Glo Luciferase Assay System (Promega, E1980). The sequences of primers for serial constructs are in Table S1.

Human umbilical vein endothelial cells were grown in 12-well plates and transfected using the jetPEI-HUVEC DNA transfection reagent (PolyPlus) using a total amount of 2 μg of DNA with 2 μg jetPEI in 100 μl 150 mM NaCl, which was added to 400 μl serum-free medium for 4 h. The following reporter and expression plasmids were used: pNL3.2.NF-κB-RE (Promega), SELE-luc [1.3 kb/MAM(ex)neo-luc], pmaxGFP (Amaxa). Expression levels of NanoLuc luciferase were analyzed using the NanoGlo Luciferase Assay (Promega) according to the manufacturer’s protocol. Firefly luciferase-based reporter gene assays were performed as described (de Wet et al., 1987 (link)). Experiments were performed in triplicates, and luciferase values normalized to co-transfected EGFP levels, and are depicted as mean fold induction.
For the transactivation assay, HUVECs were grown as above and transfected by electroporation using a BioRad Gene Pulser with the settings 200 V/960 μF; 5 × 10⁶ cells were electroporated in 400 μl RPMI medium in 0.4 cm cuvettes with a total of 10 μg plasmids. The reporter plasmid gal4-luc and the vectors for expression of the p65 transactivation domain (TAD) fused to the gal4 DNA binding domain (DBD; gal4/p65-TAD) and empty gal4 control (gal4/-), were kindly provided by M. L. Schmitz (Schmitz and Baeuerle, 1991 (link)).