Fig. 1. Circadian variation in cytosolic protein content in mouse fibroblasts.

a Cell-autonomous circadian mTORC1 activity detected by immunoblots of phospho-S6 kinase and S6 kinase abundance in fibroblasts sampled every 3 h over 3 days in constant conditions (n = 3) (right). Blot quantification and parallel PER2::LUC bioluminescent clock reporter (n = 3) (left). b Total and soluble protein quantification of cell lysates sampled ever 4 h (n = 3) and parallel PER2::LUC bioluminescent clock reporter in fibroblasts (n = 3). Protein abundance values were normalized for the maximal value in each timeseries. c ³⁵S-met/cys incorporation assay at peak (36 h/60 h) and trough (24 h/48 h) of S6K phosphorylation rhythms (n = 4) and parallel PER2::LUC activity (n = 3). d Protein quantification of total cell lysates, nuclear/organellar, and cytosolic fractions at peak (36 h/60 h) and trough (24 h/48 h) of protein rhythms (n = 3). e Effective diffusion rate of QDs in fibroblasts (n = 81, 83, 51, 63, respectively) and representative tracking images. Color key represents diffusion rate. Scale bar: 1 µm. Mean ± SEM shown throughout. p-values in (b) indicate comparison of damped cosine wave with straight-line fit (null hypothesis = no rhythm). Significance was calculated using one-way ANOVA and Tukey’s multi comparisons test (MCT) (c), one-way ANOVA and Sidak’s MCT (d), and Kruskal-Wallis with Dunn’s MCT (e). Cell type used was lung fibroblasts for (a–d) and cardiac fibroblasts for (e).