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. 2018 Jul 27;9:2961. doi: 10.1038/s41467-018-05388-x

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© The Author(s) 2018

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. 2 — High confluent cells show dramatic reduction of stress fibers formation and have low expression of actomyosin proteins via YAP/TAZ inhibition. a mRNA levels of the indicated proteins relative to GAPDH (control) in MCF10A cells plated at different confluencies: LC and HC. b mRNA levels of the indicated proteins relative to GAPDH (control) in MCF10A cells treated with YAP/TAZ siRNAs. c Densitometry of the indicated protein levels relative to GAPDH (control) in MCF10A cells plated at different confluencies: LC and HC. d Densitometry of the indicated protein levels relative to GAPDH (control) in MCF10A cells treated with YAP/TAZ siRNAs. e Relative promoter enrichment in YAP of the indicated genes. MCF10A cells were seeded at LC and HC and subjected to ChIP:YAP analysis. Bars represent the mean ± s.d. of one representative experiment performed in triplicates (***P < 0.001, **P < 0.01, *P < 0.05; two-tailed t-test). The experiment was repeated at least another 2 times with similar results. f Representative images of F-actin (Phalloidin) immunostaining in MCF10A cells plated at low and high confluencies and exposed to either Ctrl or YAP/TAZ double knockdown. Scale bar is 10 µm. g Representative images of F-actin (Phalloidin) immunostaining in MCF10A cells plated at high confluency and treated with LATS1/2 siRNAs. The number of cells with stress fibers is rescued by LATS1/2 KD (see right panel). h Representative western-blots for the indicated proteins in MCF10A cells plated at high confluency and treated with LATS1/2 siRNAs. The levels of the indicated proteins are rescued by LATS1/2 KD. i Relative mRNA levels of the indicated genes to GAPDH in MCF10A cells exposed to LATS1/2 and/or YAP/TAZ siRNAs. The treated cells were seeded at low and high confluencies. Bars represent the mean ± s.d. of one representative experiment performed in triplicates (***P < 0.001; two-tailed t-test). j MCL2 levels assessed by immunoblotting in HeLa cells plated at different confluencies and overexpressing control (Ctrl), YAP(5SA) or YAP(5SA/S94A). GAPDH was used as loading control. MLC2/GAPDH densitometry is displayed on the right panel. The graphs show the mean ± s.e.m. (n = 4; ***P < 0.001, **P < 0.01, *P < 0.05; two-tailed one sample t-test). Unless otherwise stated, bars—mean ± s.d. (n = 3;***P < 0.001, **P < 0.01, *P < 0.05, NS, not significant; two-tailed one sample t-test). n = number of independent biological replicates unless otherwise stated