Sv40 renilla luciferase construct

The predicted miR-34a and miR-200 target sites in the 3′UTR of Snail and Zeb1 were identified as described previously [11 (link),14 (link)]. A luciferase expression construct with multiple cloning sites for UTRs was used as described previously [3 (link),14 (link)]. The 3′UTRs of Snail and Zeb1 were amplified from the genomic DNA of MCF-7 cells and sub-cloned into the BamH1 and NotI sites downstream of luciferase. For targeting of endogenous miR-34a and miR-200, cells were transiently transfected with 3′ UTR reporter constructs (1–5 ng) and miR vectors that induce expression or suppression (500 ng) using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA). The activity of 3′ UTR reporter constructs was normalized to the activity of the cotransfected SV40-renilla luciferase construct (2 ng) (Promega, Madison, WI, USA). Cells were lysed 48 h after transfection and the relative ratio of renilla luciferase to firefly luciferase activity was measured in a dual luciferase assay (Promega, Madison, WI, USA).

Cells were transiently transfected with an NF-κB-responsive7 (link) promoter firefly and a SV-40-Renilla luciferase construct (Promega) for normalization of transfection efficiency, grown for 48 h under sphere conditions and assayed for luciferase activity with the Dual-Luciferase Reporter-Assay System (Promega).

Cells were transiently transfected with constructs expressing firefly luciferase fused to HIF1α-ODD (Addgene plasmid #18965) together with an SV40-Renilla luciferase construct (Promega) used for normalization of transfection efficiency. Cells were cultured for 18 h under hypoxia and assayed for luciferase activity with the Dual-Luciferase Reporter-Assay System (Promega).