Journal: Nature Communications

Article Title: Timed secreted proteomes reveal regulation of hepatokines by the liver circadian clock

doi: 10.1038/s41467-026-73840-4

Figure Lengend Snippet: a – h Data are presented as mean ± SEM. a Expression of Col18a1 by qPCR in the liver at 6 diurnal timepoints. Two-way ANOVA, Fisher’s LSD post hoc test, ZT0 * p = 0.0399, ZT12 ** p < 0.0033, ZT16 * p = 0.0112, and ZT20 * p = 0.0335. WT ZT0 and Bmal1 hep-/- ZT12 n = 3, all other groups n = 4 mouse livers. Gray shading indicates lights off. Rhythmicity analyses – Cosinor: WT p = 0.00993, Bmal1 hep-/- p = 0.8212; RAIN: WT p = 0.083, Bmal1 hep-/- p = 0.985. b Transient transfection assay in AML12 hepatocytes. Luciferase driven by Col18a1 promoter region (−2 kb to +1 kb relative to TSS). Scramble-luc = randomized DNA sequence control. One-way ANOVA, Tukey’s post hoc test, * p = 0.0132, *** p = 0.0002, and **** p = < 0.0001, n = 5. One representative experiment is shown. See also Supplementary Fig. . c , d Western blot of whole liver lysates. c Two-way ANOVA, Fisher’s LSD post hoc test, * p < 0.05. WT ZT4, 8, 16, and Bmal1 hep-/- ZT4, 12, 20 n = 3, all other groups n = 4 mouse livers. Rhythmicity analyses – Cosinor: WT p = 0.6118, Bmal1 hep-/- p = 0.2977; RAIN: WT p = 0.99, Bmal1 hep-/- p = 0.38. d One-way ANOVA, Fisher’s LSD post hoc test, * p = 0.0438, n = 4 mouse livers. e Left – cathepsin L protein abundance in liver from proteomics dataset, n = 8. Right – cathepsin L enzymatic activity from liver, n = 4. Left and right – two-way ANOVA, Fisher’s LSD post hoc test, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. f Acute ex vivo secretion assay on livers of fasted or fasted-refed mice. Secreted endostatin measured by ELISA. One-way ANOVA, Fisher’s LSD post hoc test, WT refeed male and Bmal1 hep-/- fast male n = 3, WT refeed female, WT fast female, Bmal1 hep-/- refeed female, and Bmal1 hep-/- fast female n = 4, WT fast male and Bmal1 hep-/- refeed male n = 5. g 4-h conditioned media generated from unsynchronized primary hepatocytes. Secreted endostatin measured by ELISA. Student’s t test, two-sided, ** p = 0.0056, n = 3. One representative experiment is shown. h Left – schematic of primary hepatocyte synchronization with dexamethasone (1 h, 100 nM) and subsequent collection of conditioned media from separate wells at 4 h intervals. Created in BioRender. Koronowski, K. (2026) https://BioRender.com/zg2obsf . Middle – qPCR confirming synchronization. Two-way ANOVA, Sidak’s post hoc test, Bmal1 ** p = 0.0096, **** p = <0.0001, Per2 * p = 0.0353, **** p = < 0.0001, and Col18a1 ** p = 0.0055. All groups for Per2 and Col18a1 n = 3 except for WT Hr 24 n = 2. All groups for Bmal1 n = 3 except for WT Hr 24 and Bmal1 hep-/- Hr 20 n = 2. Right – quantification of secreted endostatin by ELISA. Two-way ANOVA, Sidak’s post hoc test, * p = 0.0251 (Hr 28), * p = 0.0459 (Hr 32), ** p = 0.022, and **** p = < 0.0001, n = 3. One representative experiment is shown. Source data are provided in the file. ZT – Zeitgeber time.

Article Snippet: Primary hepatocytes were isolated from male WT or Bmal1 hep-/- mice, ages 8–16 weeks, using the Liver Perfusion Kit, mouse and rat (Miltenyi Biotec, 130-128-030) and the gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec, 130-096-427) according to the manufacturer’s instructions.

Techniques: Expressing, Transient Transfection Assay, Luciferase, Sequencing, Control, Western Blot, Quantitative Proteomics, Activity Assay, Ex Vivo, Enzyme-linked Immunosorbent Assay, Generated