Figure 5. Conserved influence of the cell cycle on the circadian clock in human U2OS osteosarcoma cell.

(a) Mean luminescence intensities (±SD, n=3) from non-dividing NIH3T3 and U2OS cells grown at 37°C expressing a Bmal1-Luc reporter. Values in the legend correspond to the mean periods ±SD. (b) Semi-automated segmentation and tracking of U2OS cell lines expressing the Rev-erbα-YFP circadian fluorescent reporter. Red vertical lines represent cell divisions (cytokinesis) and blue vertical lines show Rev-erbα-YFP signal peaks. (c) Top: stack of divisions (red) and Rev-erbα-YFP peaks (blue) for single U2OS traces centered on divisions. Bottom: distribution of the time of division relative to the next Rev-erbα-YFP peaks (in read mean±SD, n=1298). (d) Divisions and Rev-erbα-YFP peaks from single non-synchronized (top), and dexamethasone (dex)-synchronized (bottom) U2OS traces ordered on the first division. (e) Synchronization index (SI) from non-synchronized (black) and dex-synchronized (red) traces for the circadian phase (top) and cell-cycle phase (bottom) estimated as in Bieler et al. ⁵. The circadian SI from non-synchronized cells is relatively high due to plating. Dashed gray lines show 95^th percentiles of the SI for randomly shuffled traces. (f) Cell-cycle and circadian periods for U2OS cells grown at 34°C and 37°C (n > 90 for all distributions). (g) Mean luminescence intensities (±SD, n=3) for non-dividing U2OS cells grown at 34°C expressing a Bmal1-Luc reporter. Values in the legend correspond to the mean periods ±SD. (h) Mean and standard deviation of the circadian period for non-dividing U2OS cells grown at 34°C and 37°C (n=8 at 34 and n=9 at 37, two-sided Wilcoxon’s test). (i) Coupling function F(θ, ϕ) optimized on n=551 dividing U2OS traces grown at 37°C, superimposed with the attractor (T_ϕ = 22h) obtained from deterministic simulations (green line).