Pmir β gal

For luciferase reporter experiments, four Sema3A 3'-UTR segment of ~120 bp was amplified by foot-print two-step PCR, as described previously [19 (link)], and inserted into the pMIR-REPORT™ luciferase miRNA expression reporter vector (Ambion) at sites of SpeI and HindIII. HEK293 cells were cotransfected in 12-well plates using DharmaFECT Duo Transfection Reagent (Thermo Fisher Scientific Inc) according to the protocol of the manufacturer, with 0.4 µg of the 3’-UTR luciferase reporter vector and 0.08 µg of the control vector pMIR-β-gal (Ambion, Inc.). For each well, 100 nM mimic miR-223* or mimic miR control (Thermo Fisher Scientific Inc) was used. Cell lysates were prepared 48-h later. Luciferase activity was measured, using a Monolight 3010 luminometer (Pharmingen), and expressed as relative light units using a luciferase assay kit (Promega). β-galactosidase activity was measured with a commercially available kit (Promega). 3’UTR activity of each construct was expressed as the ratio of luciferase/β-galactosidase activity. All transfections were performed in triplicate from three independent experiments.

A human, mouse, or rat inhibitor-1 3′-UTR segment of ~100 bp and its respective mutant non-complementary nucleotides were amplified by footprint two-step PCR,^{13 (link)} and then cloned into the pMiR-REPORT™ luciferase microRNA expression reporter vector (Ambion) at the SpeI and HindIII sites. Human embryonic kidney (HEK) 293AD cells were co-transfected in 6-well plates using DharmaFECT Duo Transfection Reagent (Thermo Scientific Inc.), according to the protocol of the manufacturer, with 0.4 μg of the 3′-UTR luciferase reporter vector and 0.16 μg of the control vector pMIR-β-gal (Ambion, Inc.). For each well, 100 nM miR-765 mimic or miR-Ctrl mimic was used. Cell lysates were prepared 48 h later. Luciferase activity was measured using a luminometer and expressed as relative light units using a luciferase assay kit (Promega). β-Galactosidase activity was measured with a commercially available kit (Promega).