Pa5 109888

The cells were plated onto 12-well plates at a density of 60,000 per well, maintained until 80% confluence, and treated with either miR-16-5p, miR-27b-3p, or a negative control mimic, for 24 h (n = 3 per group). The cells were then cultured in ameloblast differentiation medium for another 48 h. The treated cells were lysed with RIPA buffer (Thermo Fisher Scientific) containing a protease inhibitor cocktail (Roche) and centrifuged at 21,130 × g for 20 min at 4°C. The protein concentration of the supernatants was measured with the BCA protein kit (Pierce). Protein samples (30 μg) were applied to Mini-PROTEAN TGX Gels (Bio-Rad) and transferred to a polyvinylidene difluoride (PVDF) membrane. Anti-AMELX rabbit polyclonal antibody (ab153915, Abcam, 1:1,000), anti-KLK4 rabbit polyclonal antibody (PA5-109888, Thermo Fisher Scientific, 1:750), anti-MMP20 rabbit polyclonal antibody (55467-1-AP, Proteintech, 1:750), and anti-GAPDH mouse monoclonal antibody (MAB374, Millipore, 1:6,000) were used for immunoblotting. Peroxidase-conjugated anti-rabbit IgG (7074, Cell Signaling Technology, 1:100,000) and anti-mouse IgG (7076, Cell Signaling Technology, 1:100,000) were used as secondary antibodies. All immunoblotting experiments were performed three times to validate the results.

The cells were plated onto ibiTreat 8-well μ-slides (ibidi GmbH, Munich district, Germany) at a density of 5000/chamber and cultured until 80% confluency. Then, cells were treated with mimic for miR-3195, miR-1306-5p, miR-3914, or control using Lipofectamine RNAiMAX transfection reagent (4.8 pmol of mimic with 0.48 µL of transfection reagent in 200 µL of keratinocyte-SFM medium). After 24 h, the cells were cultured with differentiation medium for 72 h. Immunofluorescence analysis was performed, as previously described [55 (link)], using rabbit polyclonal antibodies against KLK4 (PA5-109888, Thermo Fisher Scientific, 1:200) and MMP20 (55467-1-AP, Proteintech, 1:250). Images were taken with a confocal microscope (Ti-E, Nikon USA, Melville, NY, USA).