Fig. 2
- ID
- ZDB-FIG-250814-48
- Publication
- Kelu et al., 2025 - Muscle peripheral circadian clock drives nocturnal protein degradation via raised Ror/Rev-erb balance and prevents premature sarcopenia
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MuRF accumulation at night limits nocturnal muscle growth. (A) The UPS pathway highlighting relevant proteins and inhibitors (red). (B) Circadian mRNA profiles of muscle-specific E3 ligase transcripts murf1a/trim63a and murf2b/trim55b in control (blue, solid line) and mΔCLK siblings (dark blue, dotted line). RNA collected from 3xLD entrained fish under free-run was analyzed by RT-qPCR (SI Appendix, Fig. S1C). Statistics from JTK_Cycle. (C) Circadian accumulation of MuRF and Bmal proteins under free-run between 3 and 4 dpf analyzed by western blotting. Note that MuRF antibody may detect the products of more than one zebrafish MuRF gene and that protein quantifications are relative to total protein in the sample. Statistics are two-way ANOVA (Lay/Time) with Bonferroni’s post hoc test. (D) Circadian variations in proteasomal degradation of ubiquitinated proteins. Larvae under free-run between 3 and 4 dpf were treated with either DMSO, bortezomib (BTZ) alone, or BTZ and Myomed during either CT0-12 or CT12-24, and protein analyzed by western blotting. Statistics are two-way ANOVA (Lay/Treatment) with Bonferroni’s post hoc test. (E) Circadian muscle growth after MuRF inhibition in larvae under free-run between 3 and 4 dpf that were either active or inactive and treated with DMSO or Myomed. Statistics are two-way ANOVA (Time/Treatment) with Bonferroni’s post hoc test. (F) Nocturnal protein degradation in muscle measured by OPP decorporation. Newly synthesized proteins in DL-entrained larvae at 3 dpf were labeled with OPP between CT12 and 14 and allowed to degrade overnight under free-run. Fish fixed at 0, 2, 6, and 10 h post OPP-washout were imaged in single confocal XY parasagittal slices and OPP signal in S17 visualized with fluorescent Click-iT chemistry and quantified after background subtraction (SI Appendix, Fig. S5 D and E). MG132 (green symbols) or bafilomycin A1 (orange symbols) treatment after OPP-washout reduced decorporation. Log2 fold change in fluorescence signal relative to control samples at 0 h. Statistics are two-way ANOVA (Lay/Treatment) with Bonferroni’s post hoc test. |