(A) Cleaved Caspase 3 staining was absent in wild-type and atp6v1f−/− mutant neuromasts at 4 dpf, but was detected in the retina of atp6v1f−/− embryos, and in wild-type hair cells treated with neomycin (Neo) that induces Caspase 3 activation (yellow arrowheads). Nuclei were detected using DAPI. White arrowheads indicate pyknotic nuclei. (B) DAPI staining of neuromast nuclei at 4 dpf in wild-type and atp6v1f−/− embryos treated with either DMSO (control) or 300 µM ZVAD-FMK from 2 dpf to 4 dpf. White arrowheads indicate pyknotic nuclei. Dashed line circles indicate the neuromast boundary. Scale bars: 5 µm. (C) Quantification of pyknotic nuclei per neuromast in control and 300 µM ZVAD-FMK-treated embryos. n=number of embryos. ns, not significant by unpaired Student's t-test with Welch's correction. (D) 3D rendering of time-lapse snapshots of Tg(myo6b:tdtomato); atp6v1f−/− hair cells undergoing necrosis-like morphological changes. The white arrowhead follows one hair cell swelling and then bursting over time. Scale bars: 3 µm.
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