Regulation of AMELX Expression

The cells were plated onto ibiTreat 8-well μ-slides (ibidi GmbH, Munich district, Germany) at a density of 10,000/chamber and maintained until 80% confluency. The cells were then treated with mimics for miR-16-5p, miR-27b-3p, or a negative control, using Lipofectamine RNAiMAX transfection reagent (4.8 pmol of mimic with 0.48 µL of transfection reagent in 200 µL of differentiation medium) (n = 4 per group). After 24 h of treatment, the medium was replaced with differentiation medium for 2 days. AMELX expression was detected with anti-AMELX rabbit polyclonal antibody (ab153915, Abcam, 1:250), as previously described (Yoshioka et al., 2021b (link)). Immunofluorescent images were captured with a confocal microscope (Ti-E, Nikon United States).