Fig 1: Diurnal rhythms in microsomal proteins in mouse liver indicate temporal compartmentalization of the early secretory pathway steps.a, Experimental design (created with BioRender.com). WT mice were maintained under a 12 h light–dark cycle and exposed to NR feeding. Liver tissue was collected every 3 h over two consecutive days (n = 16 (8 timepoints × 2 biological replicates)). b, Heatmap representation of the rhythmic microsomal proteome (adjusted P < 0.1) analysed across two independent days (day 1 and day 2). Data are row-standardized. c, Number of rhythmic microsomal liver proteins as a function of minimal amplitude. The colour code represents different false discovery rate (FDR) values. d, Phase distribution of rhythmic microsomal liver proteins. The colour code represents different FDR values. e, Scatterplot showing the relationship between acrophase and amplitude of rhythmic microsomal proteins. f, Examples of two rhythmic microsomal proteins confirmed by western blot: STX4 and ARFGAP1 (top). Quantification of western blot analysis (bottom). LC, loading control. MS: n = 16, 8 timepoints × 2 biological replicates; WB: n = 23, 8 timepoints × 3 biological replicates, except ZT21 (two biological replicates). g, Gene set enrichment analysis of rhythmic proteins in the secretory pathway. Adjusted P = 0.05, indicated by a dashed line. h, Phase distribution of microsomal rhythmic proteins associated with the ER and GA (statistical difference in phase evaluated by Kolmogorov–Smirnov test, P = 2.2 × 10⁻¹⁶). i, Scatterplot showing the relationship between acrophase and amplitude of rhythmic ER-associated (left) and GA-associated (right) proteins. j, Representative images (left) and quantitative analysis (right) of electron microscopy imaging of mouse livers at one timepoint during the day (ZT4) and night (ZT16) under NR feeding (n = 4 biological replicates with GA ZT16 (n = 3). ER and GA are highlighted in orange and blue, respectively. N, nucleus; mt, mitochondria. Each colour in the graphs represents an independent biological replicate. Several independent liver areas per biological replicate were analysed. k, Heatmap representation of rhythmic proteins involved in protein glycosylation, categorized by function. Data are row-standardized. l, Scatterplot showing the relationship between acrophase and amplitude of N-glycans on the indicated protein. Non-complex (oligomannose) and complex glycosylation modifications. m, Phase difference between rhythmic oligomannose (top) and complex (bottom) N-glycans in mouse liver (two-tailed Kolmogorov–Smirnov test for the difference in phase, P = 0.011). n, Temporal profiles of the microsomal proteins and N-glycans at indicated sites of RAB2 and H2-K1. Temporal profiles of microsomal proteins, oligomannose and complex N-glycans at indicated positions (n = 16 (8 timepoints × 2 biological replicates)). Data are displayed as means; error bars, s.e.m. Tukey boxplots show the median and interquartile range, whiskers extend to the most extreme values within 1.5× the interquartile range and outliers are shown as individual points. A detailed description of the statistical analysis is available in Source Data Fig. 3. See also Extended Data Fig. 3 and Supplementary Tables 3 and 4 for related data.Source data