Fig 1: Hepatocyte clock proteins regulate Col18a1 transcription and endostatin secretion.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 Bmal1hep-/- ZT12 n = 3, all other groups n = 4 mouse livers. Gray shading indicates lights off. Rhythmicity analyses – Cosinor: WT p = 0.00993, Bmal1hep-/- p = 0.8212; RAIN: WT p = 0.083, Bmal1hep-/- 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. 7a. 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 Bmal1hep-/- ZT4, 12, 20 n = 3, all other groups n = 4 mouse livers. Rhythmicity analyses – Cosinor: WT p = 0.6118, Bmal1hep-/- p = 0.2977; RAIN: WT p = 0.99, Bmal1hep-/- 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 Bmal1hep-/- fast male n = 3, WT refeed female, WT fast female, Bmal1hep-/- refeed female, and Bmal1hep-/- fast female n = 4, WT fast male and Bmal1hep-/- 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 Bmal1hep-/- 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 Source data file. ZT – Zeitgeber time.
Fig 2: Hepatocyte Bmal1 regulates hepatic protein secretion.Proteomic analyses of liver ex vivo secreted fractions of WT and hepatocyte-specific Bmal1 knockout (Bmal1hep-/-) male and female mice aged 12–16 weeks at two diurnal time points, ZT8 (inactive, fasting, day) and ZT20 (active, feeding, night), n = 4 per sex, per group. a Comparison of time-dependent secreted proteins in each genotype, Student’s t test, two-sided, FDR < 0.1, > ± 50% change. b Example WT time-dependent secreted proteins that lost time-dependence in Bmal1hep-/-, two-way ANOVA, Fisher’s LSD, COL18A1 *p = 0.0313, LUM *p = 0.0369, IGHG2C **p = 0.0069, IGH-3 **p = 0.0089, IGKV6-15 *p = 0.0236, SOD3 *p = 0.0119, and IGFBP2 *p = 0.0342. Data are presented as mean ± SEM. Outliers removed by Grubb’s test (p < 0.05). COL18A1 – collagen type XVIII alpha 1 chain; LUM – lumican; EFEMP1 – EGF-containing fibulin extracellular matrix protein 1; IGHG2C – immunoglobulin heavy constant gamma 2C; IGH-3 – immunoglobulin heavy constant gamma 2B/heavy chain 3; IGKV6-15 – immunoglobulin kappa variable 6–15; SOD3 – superoxide dismutase 3; IGFBP2 – insulin-like growth factor binding protein 2. c–e Comparison of Bmal1-dependent secreted proteins at each time point, FDR < 0.1, > ± 50% change. Proteins without identifiers were removed from analysis. d Directionality of Bmal1-driven secretory effects. e Outcyte classification of secretory mechanism. f Same color coding as in (e). Plot of logFC (Bmal1hep-/-/WT) in the liver and secreted fraction for all proteins quantified in both compartments. Pearson correlation (r), simple linear regression line shown. g Volcano plots showing individual Bmal1-dependent secreted proteins. Dark red data points = Student’s t test, two-sided, FDR < 0.1, > ± 50% change. The top 5 proteins by logFC are labeled. h Gene Ontology enrichment analysis (biological process) of Bmal1-dependent secreted proteins that contain signal peptides or are unconventionally secreted. The arrows indicate the direction of the change. ZT – Zeitgeber time.
Supplier Page from Abcam for Mouse Endostatin ELISA Kit