Zebrafish were handled according to the vertebrate animal handling protocol. AB (wild-type) zebrafish were used for all experiments. For morpholino experiments, 2-cell-stage zebrafish were injected with 1–2 nl 500 µM p53-morpholino (5′-GCGCCATTGCTTTGCAAGAATTG-3′, GeneTools PCO-ZebrafishP53-100). Non-injected embryos were used as control.
At 6 hpf, zebrafish were treated with TG003 or 2-deoxy-D-glucose (2DG, Sigma-Aldrich, # D8375-1G). Water quality conditions were: dissolved oxygen = 6.50±0.25 mg/L, salinity = 0.0 practical salinity unit (PSU) pH 7.2, conductivity = 520µS/cm), temperature = 28°C. Drug was washed out at 24 hpf (i.e.18 hours after treatment) and no additional drug was added. Water was changed every subsequent day, and fish were collected at 5 days post fertilization (dpf) for cartilage staining.
Fish were fixed, bleached and stained with Alcian Blue as described previously (Dingerkus and Uhler, 1977 (link); Sakata-Haga et al., 2018 (link)). Fish were imaged using a Leica M205 FCA stereo microscope, using the same settings for all samples (bright-field, exposure = 1 ms). Lengths of craniofacial structures were analyzed using Fiji, by an investigator to whom the treatment groups had been anonymized.
At 6 hpf, zebrafish were treated with TG003 or 2-deoxy-D-glucose (2DG, Sigma-Aldrich, # D8375-1G). Water quality conditions were: dissolved oxygen = 6.50±0.25 mg/L, salinity = 0.0 practical salinity unit (PSU) pH 7.2, conductivity = 520µS/cm), temperature = 28°C. Drug was washed out at 24 hpf (i.e.18 hours after treatment) and no additional drug was added. Water was changed every subsequent day, and fish were collected at 5 days post fertilization (dpf) for cartilage staining.
Fish were fixed, bleached and stained with Alcian Blue as described previously (Dingerkus and Uhler, 1977 (link); Sakata-Haga et al., 2018 (link)). Fish were imaged using a Leica M205 FCA stereo microscope, using the same settings for all samples (bright-field, exposure = 1 ms). Lengths of craniofacial structures were analyzed using Fiji, by an investigator to whom the treatment groups had been anonymized.
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