Pre mir145 mirna precursor

Transient transfection was performed using Lipofectamine Reagent 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. C666-1 cells (5x105 (link)) were seeded onto fibronectin-coated 35-mm dishes. For siRNA knockdown, 50 nM ON-TARGETplus SMARTpool Human SOX2 siRNA (a pool of 4 siRNA: GCUCUUGGCUCCAUGGGUU; UCAUGAAGAAGGAUAAGUA; GCUUCUAGACCUACAUGAA; CAGUACAACUCCAUGACCA), or 50 nM ON-TARGETplus siCONTROL Non-Targeting siRNA (Dharmacon, Lafayette, CO) was transfected to C666-1. For miRNA precursor transfection, 50 nM pre-miR145 miRNA precursor or 50 nM pre-miR miRNA precursor control (Ambion, Austin, TX) was transfected to C666-1. After 72 hours of transfection, cells were harvested for expression analysis and tumor sphere formation assay.

C666-1 cells were transiently transfected with 50 ng pMirTarget luciferase reporter vector containing SOX2 3'UTR (Origene, Rockville, MD) along with 200 nM pre-miR145 miRNA precursor or pre-miR miRNA precursor control (Ambion, Austin, TX) using Lipofectamine Reagent 2000 (Invitrogen, Carlsbad, CA) mentioned above. The pMirTarget vector contains a red fluorescent protein (RFP) for monitoring the transfection efficiency and normalization. After 48 hours of transfection, the red fluorescent signal was captured under microscopy and then the cells were lysed for measurement using firefly luciferase reporter assay system (Promega, Valencia, CA) according to the manufacturer's manual. The luciferase activities were calculated and normalized to intensity of RFP expression.