Figure 3.

Analysis of Circadian Rhythmicity in SPC Proliferation and Migration

(A) Two examples of BrdU staining showing BrdU incorporation 14–20 hr (47%) and 22–28 hr (31%) after serum shock.

(B) Circadian biphasic rhythm in BrdU incorporation of SPCs after serum shock (period 12 hr; p < 0.001; n = 4 independent experiments) but not in unsynchronized cells (p = 0.776; n = 4 independent experiments).

(C) Example of scratch-wound migration assay picture taken directly and 6 hr after scratch.

(D) No rhythmicity was present in migration of SPCs into the scratch (p = 0.442, n = 4).

(E) Combination of proliferation and migration experiments showing that the majority (83.1% ± 2.0%) of cells present in the scratch after 6 hr did not proliferate. White line indicates border of the scratch.

(F) Percentage of growth back into scratch in conditions with lowest and highest cell density and in scratches perpendicular and parallel to cell orientation. No significant differences were found between groups (low density 36.1 ± 6.0 versus high density 31.0 ± 6.5, p = 0.59; perpendicular orientation 32.4 ± 4.9 versus parallel orientation 37.9 ± 5.2; p = 0.11; n = 12 locations for all).

Data are presented as mean ± SEM; ^∗p < 0.05 for significant circadian oscillation. Scale bars, 100 μm.