Figure 2. Rev-erbα circadian... | Rockefeller University Press

Figure 2.

$Rev-erbα circadian oscillations do not depend on cell–cell adhesions or paracrine signaling. (A) RevVNP-expressing cells were cultured in two adjacent compartments separated by a barrier until they reached high density. Next, the barrier was removed, and time-lapse confocal microscopy was performed during and after gap closure every 15 min for 5 d. A sequence of time-lapse images corresponding to the closure of the gap is shown. The prior location of the barrier is depicted in the left image as a white dashed line. Scale bar, 100 µm. (B) Kymograph representing the average RevVNP intensity per time point of all the cells from A along the x axis. The same kymograph with a dynamic range that enhances the fluorescence of the bulk after the gap closure is shown in Fig. S2 D. (C) Average RevVNP intensity over time of the cells at the edge in comparison to those residing in the confluent zone. The inset shows a magnification of the area shaded in gray. This figure shows an example of n = 6 independent experiments. (D) Violin plots representing the distribution of the single-cell RevVNP circadian power fractions of low-density cells grown in fresh medium and conditioned medium; n = 72 and 116 cells, respectively, from three experiments; medians and interquartile ranges are depicted as white circles and black bars, respectively; two-sided Wilcoxon rank sum test; P value = 0.4168. (E) Schematics of the strategy followed to isolate single cells via micropatterning of fibronectin on glass. On the right, phase contrast (PC) and confocal microscopy images of RevVNP cells in stadium-shaped patterns (top), confluent cells cultured in the same well (middle), and non-confined cells cultured on a homogenous fibronectin-coated surface in a density as low as that of the micropatterned cells (bottom). Scale bar, 50 µm. (F) Violin plots representing the distribution of the single-cell RevVNP circadian power fractions of the conditions depicted in E; n = 121, 174, and 115 cells for the high-density, low-density, and micropatterned cells, respectively, from three experiments; medians and interquartile ranges are depicted as white circles and black bars, respectively. Two-sided Wilcoxon rank sum test; ** indicates P value <0.01; **** indicates P values <0.0001. Full P values are reported in Table S1.$

Rev-erbα circadian oscillations do not depend on cell–cell adhesions or paracrine signaling. (A) RevVNP-expressing cells were cultured in two adjacent compartments separated by a barrier until they reached high density. Next, the barrier was removed, and time-lapse confocal microscopy was performed during and after gap closure every 15 min for 5 d. A sequence of time-lapse images corresponding to the closure of the gap is shown. The prior location of the barrier is depicted in the left image as a white dashed line. Scale bar, 100 µm. (B) Kymograph representing the average RevVNP intensity per time point of all the cells from A along the x axis. The same kymograph with a dynamic range that enhances the fluorescence of the bulk after the gap closure is shown in Fig. S2 D. (C) Average RevVNP intensity over time of the cells at the edge in comparison to those residing in the confluent zone. The inset shows a magnification of the area shaded in gray. This figure shows an example of n = 6 independent experiments. (D) Violin plots representing the distribution of the single-cell RevVNP circadian power fractions of low-density cells grown in fresh medium and conditioned medium; n = 72 and 116 cells, respectively, from three experiments; medians and interquartile ranges are depicted as white circles and black bars, respectively; two-sided Wilcoxon rank sum test; P value = 0.4168. (E) Schematics of the strategy followed to isolate single cells via micropatterning of fibronectin on glass. On the right, phase contrast (PC) and confocal microscopy images of RevVNP cells in stadium-shaped patterns (top), confluent cells cultured in the same well (middle), and non-confined cells cultured on a homogenous fibronectin-coated surface in a density as low as that of the micropatterned cells (bottom). Scale bar, 50 µm. (F) Violin plots representing the distribution of the single-cell RevVNP circadian power fractions of the conditions depicted in E; n = 121, 174, and 115 cells for the high-density, low-density, and micropatterned cells, respectively, from three experiments; medians and interquartile ranges are depicted as white circles and black bars, respectively. Two-sided Wilcoxon rank sum test; ** indicates P value <0.01; **** indicates P values <0.0001. Full P values are reported in Table S1.

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