Luciferase and renilla

For the luciferase reporter assays, 3 × 10⁴ iPS cells were seeded on collagen IV‐coated well of a 12‐well plate in DM containing VEGF. Seventy‐two hours later, cells were transfected with the luciferase plasmids under the control of the promoter of the VEGF receptor (Addgene [plasmid 21307] generated by Mammoto et al.) 23, the JAG1 3′UTR Lenti‐reporter‐Luc Vector (ABM), and the Pre‐199b, LNA‐199b and controls. Briefly, 0.33 µg/well of the reporter plasmids was cotransfected with the Pre‐199b, or LNA‐199b and controls (2 µl/well) using jetPRIME (Polyplus‐transfection SA) according to the protocol provided. pGL3‐Luc Renilla (0.1 µg/well) was included in all transfection assay as internal control. Luciferase and Renilla (Promega) activity assays were detected 48 hours after transfection using a standard protocol 24. The relative luciferase unit was defined as the ratio of luciferase activity to Renilla activity with that of control set as 1.0.

For the luciferase reporter assays, 3 × 10⁴ ES cells were seeded on collagen-coated well of a 12-well plate in DM. 72 h later, cells were transfected with the QKI 3′UTR Lenti-reporter-Luc Vector (ABM, QKI-UTR-wt) or QKI 3′UTR Lenti-reporter-Luc vector with mutated miRNA 214 binding site (QKI-UTR-mt), and the indicated miRNAs or controls. Briefly, 0.33 μg/well of the reporter plasmids were co-transfected with the miR-214 mimics, miR-214 inhibitor or respective controls (2 μl/well) using jetPRIME® (Polyplus-transfection SA) according to the protocol provided. pRenilla (0.1μg/well) was included in all transfection assay as internal control. Luciferase and Renilla (Promega) activity assays were detected 48hr after transfection using a standard protocol [7 (link)]. Relative luciferase unit (RLU) was defined as the ratio of luciferase activity to Renilla activity with that of control set as 1.0.