miRNA Regulation of Neural Differentiation

mHAT9d cells were plated onto ibiTreat 8-well μ-slides (ibidi GmbH, Munich district, Germany) at a density of 10,000/chamber and cultured until 80% confluence. Cells were then treated with a mimic for miR-16-5p, miR-27b-3p, or control using Lipofectamine RNAiMAX transfection reagent (4.8 pmol of mimic with 0.48 µL of transfection reagent in 200 µL of proliferation medium). After 24 h of transfection, the cells were cultured under differentiation medium for 48 h. In addition, cells were treated with 100 μg/ml BrdU (Sigma Aldrich) for 1 h at day 2 of differentiation (n = 6 per group) and visualized with a rat monoclonal antibody against BrdU (ab6326; Abcam, 1:1,000), as previously described (Yoshioka et al., 2021a (link)). BrdU-positive cells were quantified using images from six independent experiments.