RRD-251 promoted apoptosis during the early development of otic vesicles.
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After treatment with DMSO (A1–A4), RRD-251 20 µM (B1–B4), or RRD-251 40 µM (C1–C4), the balance between proliferation, apoptosis, and quiescence during otic development was shifted. In the DMSO control group, nearly half of the cells incorporated EdU (48.78%), only 7.32% of the cells were Caspase-3–positive, and 43.9% of the cells in the otic vesicles were double negative (A4). After treatment with RRD-251, the portion of EdU-positive cells was reduced to 29.85% and 20.84% for doses of 20 µM (B4) and 40 µM (C4), respectively, and the portion of cleaved Caspase-3–positive cells was increased to 7.46% and 47.22%, respectively. Furthermore, compared with controls, the portion of double-negative cells was increased at the lower dose of RRD-251 (B4) to 62.69% and was reduced with the higher dose (C4) to 31.94%. Green arrows indicate the EdU-positive cells, red arrows indicate the cleaved Caspase-3–positive cells, and the white arrows indicate the double-negative cells. The differences between the three separate cell populations within each group were estimated using the χ2 test (P<0.001). Scale bar = 10 µm.