Mirna mimic control

The pcDNA-EZH2 plasmid was purched from Addgene (Cambridge, MA). The let-7c mimic and the miRNA mimic control were acquired from Dharmacon (Lafayette, CO). The miRNA mimic is a double-stranded oligonucleotide devised to mimic the function of an endogenous mature miRNA.

The miRNA-664a-5p mimic and miRNA mimic control were purchased from Dharmacon (Lafayette, CO, USA). The cells were transfected with 10 nM miRNA-664a-5p mimic or 10 nM pre-miR-664a using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) for 24 h at 37 °C and 5% CO₂. For control experiments, the cells were also transfected with miRNA mimic control or pre-miRNA-U1A control. The next day, the medium was replaced with fresh growth medium or differentiation medium containing 30 µM retinoic acid (Wako Pure Chemical Industries, Osaka, Japan). The cells were again transfected with miRNA mimics, pre-miR-664a, or pre-miRNA-U1A control in growth medium or differentiation medium after 2 days. After 24 h, the medium was replaced with fresh growth medium or differentiation medium, and the cells were cultured for an additional 2 days.
To induce apoptosis, the cells were transfected with 10 nM pre-miR-664a or pre-miR-664-U1A using Lipofectamine 3000 (Invitrogen) for 24 h at 37 °C and 5% CO₂. For control experiments, the cells were also transfected with pre-miRNA-U1A control.