REV-ERBα levels decrease with cell density in a cell division–independent manner. (A) Confocal microscopy images of low- and high-density cell populations immunostained against REV-ERBα. All images are displayed under the same brightness and contrast settings. Scale bar, 20 µm. (B) Violin plots representing the single-cell levels of nuclear REV-ERBα. n = 104 for low density and 142 for high density, from one representative of five independent experiments. Two-sided Wilcoxon rank sum test; **** indicates P value <0.0001. (C) Violin plots representing the distribution of the single-cell RevVNP circadian power fraction of low- and high-density populations for three alternative clonal RevVNP cell lines; from left to right: n = 73, 197, 2,342, 3,857, 1,424, and 4,297, from three independent experiments; medians and interquartile ranges are depicted as white circles and black bars, respectively. Two-sided Wilcoxon rank sum test; **** indicates a P value <0.001. (D) Kymograph of Fig. 2 B with a modified color map dynamic range so the oscillations of the bulk population after the gap closure are more conspicuous. (E–G) RevVNP-expressing cells were cultured in two adjacent compartments separated by a barrier until they reached high density. Next, thymidine 2 mM was added, the barrier was released, and time-lapse confocal microscopy was performed during and after gap closure every 15 min for 4 d. A selection of time-lapse images showing the closure of the gap is depicted in E while F is a kymograph representing the average RevVNP intensity of all the cells along the x axis per time point. The average RevVNP intensity over time of the cells at the edge in comparison to those residing in the confluent zone is shown in G. The prior location of the barrier is depicted in the left image as a white dashed line. Notice that inhibition of proliferation causes a delay in gap closure and a decrease of overall cell density, which has a potential effect on RevVNP expression and rhythmicity. Scale bar, 100 µm.