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. 2020 Sep 11;11:4581. doi: 10.1038/s41467-020-18368-x

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a N/C trace of an individual MCF10A cell in monolayer and corresponding fluorescence image (cell of interest is false colored in green). b Example traces of YAP N/C over time with large changes in signal highlighted by gray (increase) and purple (decrease) in MCF10A cells. c Overlay of YAP N/C traces for MCF10A, MCF10A + HRas, and MCF10A + 1 μM PP1. Each plot contains 200 traces randomly selected from the pool of all tracks collected from each condition. The mean N/C value is subtracted from each trace to allow visual comparison. d Mean fraction of cells with at least one detectable fluctuation within a 24 h time-course (24 ROIs for each condition compiled from two independent experiments). Box and whisker lines: black-center line is median value, box-bounds are 25th and 75th percentile, and whiskers are 0.05 and 0.95 percentiles. A one-sided Mann–Whitney U-test was used to test whether the fraction of cells displaying a significant fluctuation was changed after HRas expression (N = 24, P = 9.0 × 10⁻⁷, Ranksum-Stat = 734) or PP1-treatment (N = 24, P = 3.52 × 10⁻⁴, Ranksum-Stat = 290.5). e (Top panel) Time-course of MCF10A^YAP-GFP-KI cells following 20 h serum starvation followed by serum replenishment. (Bottom panel) Quantification of YAP N/C following stimulation by serum or 1 µM LPA stimulation. f Fluorescent images of serum starved cells followed by serum stimulation for the indicated times. g Fluctuation of nuclear YAP in a single representative H1 hESC^YAP-GFP-KI cell. (Top panel) Nuclear outline highlighted in dashed line. (bottom panel) Quantification of nuclear YAP intensity over time. At very high density in hESC^YAP-GFP-KI cells it is not feasible to reliably estimate cytoplasmic intensity. h Overlay of nuclear YAP intensity in many individual hESC^YAP-GFP-KI cells showing dynamic changes in intensity levels. Repeated twice with similar results.