Figure 4.
YAP perturbs the circadian clock via TEAD. (A) Violin plots representing the distribution of the single-cell RevVNP circadian power fractions of YAP-overexpressing cells carrying different mutations and the control; n = 6,466, 440, 5,320, and 4,571 for control, 5SA-YAP, 5SA-CCDEL-YAP, and 5SA-S94A-YAP, respectively, from five independent experiments; medians (0.21, 0.09, 0.13, and 0.19) and interquartile ranges are depicted as white circles and black bars, respectively. Two-sided Wilcoxon rank sum test; all comparisons show statistically significant differences; full P values are reported in Table S1. (B) Raw data of the RevVNP intensities over time, represented in kymograph style, of 440 cells grown and tracked under low- or high-density conditions, from a representative experiment of three. The single tracks are ordered from less (top) to more (bottom) circadian power fraction and aligned along the time axis according to maximum cross-correlation with the median track. (C) Confocal microscopy images of control, 5SA-YAP, 5SA-CCDEL-YAP, and 5SA-S94A-YAP cells immunostained for YAP (left) and a REV-ERBα (right). All images are displayed under the same brightness and contrast settings. Scale bar, 20 µm. (D and E) Violin plots representing the single-cell levels of nuclear YAP (D) and nuclear REV-ERBα (E) for all the conditions depicted in C. n = 294, 400, 383, and 393 for control, 5SA-YAP, 5SA-CCDEL-YAP, and 5SA-S94A-YAP, respectively, from three independent experiments. Medians (21.99, 36.35, 50.13, and 50.77 for YAP; 30.75, 61.89, 45.58, and 36.51 for REV-ERBα) and interquartile ranges are depicted as white circles and black bars, respectively. Two-sided Wilcoxon rank sum test; all comparisons show statistically significant differences; full P values are reported in Table S1. (F) mRNA expression of Rev-erbα, Bmal1, Cry1, and Per2 in high-density cells subjected to constitutive overexpression of an empty vector (control), 5SA-YAP, and 5SA-S94A-YAP, measured by RT-qPCR. The values refer to the logarithms of the fold change relative to the first time point in the control condition. Cells were synchronized with a 2-h horse serum shock. The first time point corresponds to cells collected 24 h after the shock. The graphs represent the mean of n = 3 biological repeats, and the error bars represent the SD. The depicted sinusoidal fits and the statistical analysis were performed using single cosinor analysis. The reported P values (P) refer to an F test against a null hypothesis of zero amplitude. Exact values are listed in Table S1.

YAP perturbs the circadian clock via TEAD. (A) Violin plots representing the distribution of the single-cell RevVNP circadian power fractions of YAP-overexpressing cells carrying different mutations and the control; n = 6,466, 440, 5,320, and 4,571 for control, 5SA-YAP, 5SA-CCDEL-YAP, and 5SA-S94A-YAP, respectively, from five independent experiments; medians (0.21, 0.09, 0.13, and 0.19) and interquartile ranges are depicted as white circles and black bars, respectively. Two-sided Wilcoxon rank sum test; all comparisons show statistically significant differences; full P values are reported in Table S1. (B) Raw data of the RevVNP intensities over time, represented in kymograph style, of 440 cells grown and tracked under low- or high-density conditions, from a representative experiment of three. The single tracks are ordered from less (top) to more (bottom) circadian power fraction and aligned along the time axis according to maximum cross-correlation with the median track. (C) Confocal microscopy images of control, 5SA-YAP, 5SA-CCDEL-YAP, and 5SA-S94A-YAP cells immunostained for YAP (left) and a REV-ERBα (right). All images are displayed under the same brightness and contrast settings. Scale bar, 20 µm. (D and E) Violin plots representing the single-cell levels of nuclear YAP (D) and nuclear REV-ERBα (E) for all the conditions depicted in C. n = 294, 400, 383, and 393 for control, 5SA-YAP, 5SA-CCDEL-YAP, and 5SA-S94A-YAP, respectively, from three independent experiments. Medians (21.99, 36.35, 50.13, and 50.77 for YAP; 30.75, 61.89, 45.58, and 36.51 for REV-ERBα) and interquartile ranges are depicted as white circles and black bars, respectively. Two-sided Wilcoxon rank sum test; all comparisons show statistically significant differences; full P values are reported in Table S1. (F) mRNA expression of Rev-erbα, Bmal1, Cry1, and Per2 in high-density cells subjected to constitutive overexpression of an empty vector (control), 5SA-YAP, and 5SA-S94A-YAP, measured by RT-qPCR. The values refer to the logarithms of the fold change relative to the first time point in the control condition. Cells were synchronized with a 2-h horse serum shock. The first time point corresponds to cells collected 24 h after the shock. The graphs represent the mean of n = 3 biological repeats, and the error bars represent the SD. The depicted sinusoidal fits and the statistical analysis were performed using single cosinor analysis. The reported P values (P) refer to an F test against a null hypothesis of zero amplitude. Exact values are listed in Table S1.

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