Prl renilla luciferase construct

MCF7 cells were grown in 24 well plates in phenol red free DMEM supplemented with 10% (v/v) charcoal treated fetal bovine serum 48 h prior to estrogen (E2) treatment. Cells were transfected with pGL3-ERRβ, pEGFP-ERα [41 (link)], pEYFP C1-ERRβ [37 (link)], pRL-Renilla luciferase construct (Promega) in different combinations using jetPRIME-polyplus-transfection reagent (Polyplus transfection, New York, NY, USA) according to manufacture protocol. Post 24 h transfection cells were treated with 100 nM E2 and vehicle and were allowed to grow for 24 h. Luciferase assay was performed using Dual luciferase assay detection kit (Promega) according to manufacture protocol. Luciferase readings were obtained and were normalized with Renilla luciferase activity. The graph was plotted with normalized readings using GraphPad Prism software version 6.01.

HUVECs were transfected with siRNA specific for MCPIP or a negative control scrambled siRNA (Ambion Inc., Austin, TX, USA) with lipofectamine 2000 as previously described [17 (link)]. Cellular lysates were isolated 24 h post-transfection to analyze expression of adhesive molecules (ICAM-1, VCAM-1, E-selectin, and P-selectin), pro-coagulation factors (PAI-1 and TF), cytokines (TNF-α, IL-1β, IL-6, and MCP-1), and miRs (miR-126, -146a, and -223) by qRT-PCR. Primers used for qRT-PCR are indicated in Table S1. To analyze NF-κΒ activity, HUVECs were first transfected with siRNA specific for MCPIP or a scrambled siRNA, and after 24 h the cells were transfected with 1 μg of a 5× NF-κB element-luciferase reporter (Promega, Madison, WI, USA) and 100 ng of pRL Renilla luciferase construct (Promega) (for normalization of transfection efficiency). Cellular lysates were isolated 24 h post-transfection using a passive lysis buffer and luciferase activity was monitored using the Dual Luciferase Reporter Assay System (Promega).