Fig. 4. Hyperosmolarity and treadmill running induce transcriptome-wide changes in gene expression.

a Bioluminescence recording of primary chondrocytes isolated from PER2::Luc mice. Cells were not synchronized at the beginning of the experiment. After 30 h, media osmolarity was increased by +200 mOsm. Parallel samples were harvested every 4 h for RNA isolation and RNAseq analysis. b Principal component analysis (PCA) showing a correlation between RNAseq replicates and a developing trend over time. c Heatmap showing gene expression patterns of 254 rhythmic genes (BHQ < 0.05) following increase of osmolarity. d Volcano plot showing differentially expressed genes between timepoint 0 (T0) and 4 h (T4) after osmotic stress. 630 were up regulated and 927 downregulated at Adj p < 0.05 and Log₂FC = 1 cut off. e Heatmap of circadian clock genes at T0 and T4 after osmotic stress. *p < 0.05. f–i Volcano plot showing differentially expressed genes between sedentary and treadmill running mice in cartilage (f) and IVD (h) at Adj p < 0.05 and Log₂FC = 0.5 cut off. Heatmaps depicts circadian clock genes in cartilage (g) and IVDs (i) from sedentary and treadmill-running mice. The asterisk denotes significant changes, *p < 0.05. j PCA of treadmill-regulated rhythmic genes at ZT2 vs. circadian time series rhythmic genes in cartilage. k, l Bubble plots showing significant canonical pathways (k) and upstream regulators (l) by Ingenuity Pathway Analysis in RNAseq datasets from osmotic stress (0 h vs. 4 h) and treadmill exercise (running vs. sedentary cartilage and IVD). P values were adjusted for multiple comparisons from DESeq2 (d–i) and IPA analysis (k, l) were used to generate the plots. Source data are provided in Supplementary Data.