Dharmafect duo transfect reagent

Mesangial renal cells or endothelial renal cells were co-transfected with the vector pEZX-MT01-HIF1A 3′UTR and mimic miR-107, miR-31, or miR-135b-5p using DharmaFECT Duo transfect reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer protocols. After 48 h, luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA, detailed information in Supplementary Materials).

Human kidney cells were plated in 96-well plates for 24 h and then were cotransfected using DharmaFECT Duo transfect reagent (ThermoFisher Scientific, Waltham, MA, USA) with 40 ng of pEZX-MT06-SP1 3′UTR plus 10 nM of miR-21, miR-150, or miR-29c analog or miR negative control (5 duplicates per group). The pEZX-MT06-SP1 3′UTR plasmid contains firefly luciferase and miR binding site from SP1 3′UTR, which is inserted between firefly luciferase cDNA and PolyA (more binding site details in Figure S1). This plasmid also includes Renilla luciferase as an internal control (GeneCopoeia, Rockville, MD, USA). Transfected cells were lysed by reporter lysis buffer (Promega, Madison, WI, USA). Dual luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) by FLUOstar Omega (BMG Labtech, Oternberg, Germany).

Primary HUVECs or primary monocytes were co-transfected with the vector pEZX-MT01-PTEN 3′UTR or pEZX-MT01-NOTCH1 3′UTR and miR-483-3p or miR-326 mimics (10 μM) (mirVanaTM, Life technologies, Carlsbad, CA, USA) using DharmaFECT Duo transfect reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer protocols. After 24 h, the luciferase activity of firefly and Renilla was measured by Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Renilla luciferase was used as internal control.