Figure 3.
Rev-erbα circadian power fraction anticorrelates with nuclear YAP. (A) Representative maps of traction force modulus on micropatterns of 1,600 and 900 µm2. The dashed line indicates the contour of the cell. Inset: brightfield (BF) image of corresponding cell and fibronectin (FN) micropattern (orange). Scale bar is 50 µm. (B) Box plot representing the mean traction force modulus of cells on micropatterns of 1,600 and 900 µm2. Two sample t test; ** indicates P value <0.01. (C) Violin plots representing the distribution of the single-cell RevVNP circadian power fractions of cells cultured in fibronectin stadium-shaped micropatterns of 1,600 and 900 µm2; n = 154 and 161, respectively, from four 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; full P values are reported in Table S1. (D) Confocal microscopy images of cells under different conditions (high-density control, low-density control, micropatterned cells of 1,600 and 900 µm2, low-density treated for 24 h with jasplakinolide 1 µM, latrunculin A 200 nM, cytochalasin D 1 µM, para-nitro-blebbistatin 10 µM, and low-density cells grown on polyacrylamide gels with a stiffness of 300 Pa). The cells were stained with an anti-YAP antibody (green, top) and Hoechst (blue, bottom); the cell perimeter is represented with a dashed white line. Scale bar, 20 µm. (E) Violin plots representing the distribution of the single-cell YAP nuclear to cytosolic ratios for the conditions depicted in C; n = 554, 589, 208, 331, 203, 171, 238, 153, and 315 cells, respectively, from three to seven experiments depending on the condition. Medians and interquartile ranges are depicted as white circles and black bars, respectively. The P values of the comparisons of every condition against the low-density, obtained with a two-sided Wilcoxon rank sum test, are represented in Table S1. (F) Correlation between circadian power fraction and YAP nuclear to cytosolic (n/c) ratio of all the aforementioned conditions. The values represented are the means of the medians of each independent experiment for every condition. The error bars refer to the corresponding SD. Pearson’s correlation coefficient is r = −0.93 with a P value of 0.0003.

Rev-erbα circadian power fraction anticorrelates with nuclear YAP. (A) Representative maps of traction force modulus on micropatterns of 1,600 and 900 µm2. The dashed line indicates the contour of the cell. Inset: brightfield (BF) image of corresponding cell and fibronectin (FN) micropattern (orange). Scale bar is 50 µm. (B) Box plot representing the mean traction force modulus of cells on micropatterns of 1,600 and 900 µm2. Two sample t test; ** indicates P value <0.01. (C) Violin plots representing the distribution of the single-cell RevVNP circadian power fractions of cells cultured in fibronectin stadium-shaped micropatterns of 1,600 and 900 µm2; n = 154 and 161, respectively, from four 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; full P values are reported in Table S1. (D) Confocal microscopy images of cells under different conditions (high-density control, low-density control, micropatterned cells of 1,600 and 900 µm2, low-density treated for 24 h with jasplakinolide 1 µM, latrunculin A 200 nM, cytochalasin D 1 µM, para-nitro-blebbistatin 10 µM, and low-density cells grown on polyacrylamide gels with a stiffness of 300 Pa). The cells were stained with an anti-YAP antibody (green, top) and Hoechst (blue, bottom); the cell perimeter is represented with a dashed white line. Scale bar, 20 µm. (E) Violin plots representing the distribution of the single-cell YAP nuclear to cytosolic ratios for the conditions depicted in C; n = 554, 589, 208, 331, 203, 171, 238, 153, and 315 cells, respectively, from three to seven experiments depending on the condition. Medians and interquartile ranges are depicted as white circles and black bars, respectively. The P values of the comparisons of every condition against the low-density, obtained with a two-sided Wilcoxon rank sum test, are represented in Table S1. (F) Correlation between circadian power fraction and YAP nuclear to cytosolic (n/c) ratio of all the aforementioned conditions. The values represented are the means of the medians of each independent experiment for every condition. The error bars refer to the corresponding SD. Pearson’s correlation coefficient is r = −0.93 with a P value of 0.0003.

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