Fig. 3: Delayed YAP-mediated control of the cyclin D1/p27 ratio and cell cycle arrest.

a Schematic for RNA-sequencing of doxycycline-induced CA-YAP expression in RPE1 cells. Cells were seeded densely (38,000 cells/well) to induce low endogenous YAP activity and pre-conditioned with starvation or serum media and palbociclib-arrested (CDK4/6i, 1 μM), prior to doxycycline-induction over 36 h (0.5 μg/mL DOX). See also “Methods”. b Up-regulated genes (q < 0.05, Log2 fold-change >1) for 12, 24, and 36 h YAP induction (- serum) showed the Cordenonsi YAP signature (using ToppGene)³³. Log2 fold-change (Log2FC) induction of c CYR61 (canonical YAP target gene, gray) compared with CCND1 (cyclin D1, red) and d regulation of WWTR1 (suppressed YAP target gene TAZ, gray), CDKN1A (p21, dark blue), and CDKN1B (p27, blue) in starved and serum conditions (dashed or solid line, respectively). Adjusted p-values reported in Supplementary Table S3. e Mean YAP ratio in starved WT cells following LATSi treatment (0.5 µM TDI-011536) or co-treatment with TEADi (1 µM GNE-7883). N = 3 independent experiments, not significant (NS) by ordinary one-way ANOVA (p > 0.10 for all time points). f Cyclin D1/p27 single-cell scatterplots (G1-gated), colored by p-Rb state (purple, p-Rb-negative; orange, p-Rb-positive) for cells treated as in (e) with LATSi for 0 h (DMSO), 6 h, 12 h. g Mean cyclin D1/p27 ratio (left) and percent p-Rb (right) for cells treated as in (e). N = 3 independent experiments. Ordinary one-way ANOVA with Dunnett’s multiple comparisons: cyclin D1/p27 ratio, p = 0.14 (3 h), 0.0017 (6 h), 7 × 10⁻⁴ (12 h), 0.0084 (24 h), 0.92 (TEADi); p-Rb, p = 0.31 (3 h), 0.0044 (6 h), 7 × 10⁻⁴ (12 h), 0.017 (24 h), >0.99 (TEADi). h Mean YAP ratio in starved CA-YAP cells following TEADi treatment (1 µM GNE-7883) for 0–36 h. Ordinary one-way ANOVA with Dunnett’s multiple comparisons: p < 1 × 10⁻⁴, all time points. i Cyclin D1/p27 single-cell scatterplots (G1-gated), colored by p-Rb state for cells treated as in (h) with TEADi for 0 h (DMSO), 6 h, and 24 h. j Mean cyclin D1/p27 ratio (left) and percent p-Rb (right) for G1-gated cells treated as in (h). N = 3 independent experiments. Ordinary one-way ANOVA with Dunnett’s multiple comparisons: cyclin D1/p27 ratio, p > 0.99 (3 h), 0.75 (6 h), 0.36 (12 h), 0.0074 (24 h), < 1 × 10⁻⁴ (36 h); p-Rb, p = 0.68 (3 h), 0.17 (6 h), 0.043 (12 h), 0.0030 (24 h), 1 × 10⁻⁴ (36 h). k Schematic for computational alignment of asynchronously cycling cells based on mitosis time (M), followed by analysis of CDK2 activity reporter and its bifurcation into CDK_high and CDK_low fractions for cells born after variable time in DMSO, LATSi, or TEADi (see also “Methods”). l Mean CDK2 activity in cells aligned by mitosis and gated to have received drug in the first 3 h of G1 (left), or in the preceding G2/M of the mother cell (3–6 h before mitosis, middle; 6–9 h before mitosis, right). Shaded error are 2 × SEM (n > 500 cells/condition). The dashed line at 0.8 indicates the CDK_high threshold for G1/S progression. m Violin plot distribution of G1 lengths in WT cells treated with DMSO, LATSi, or TEADi during G1 (G1), or in G2/M of the previous cell cycle (M, in the mother cell). G1 cells were gated for birth prior to drug treatment and S-phase entry following drug addition. M cells were gated for birth 6–9 h after drug treatment (during G2/M in the mother cell). Solid lines are median and dotted lines are interquartile range (IQR), n = 1000 random cells/condition. Mood’s median test: p = 0.19 (DMSO), 1.13 × 10⁻⁸⁴ (LATSi), 7.11 × 10⁻²⁷ (TEADi). n Proportion of cells entering S-phase (by FUCCI reporters), binned by window of time in drug as in (m). o Fold-change in the fraction of cells activating CDK2 within 3 h of mitosis (short G1), binned by time in LATSi or TEADi (bin size = 3 h). N = 3 independent experiments, n > 5000 cells/condition. All single-cell (n = 5000 cells/scatterplot) and live-cell data are representative of 3 independent experiments.