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. 2018 Jul 9;9:2372. doi: 10.1038/s41467-018-04590-1

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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. 4 — YAP1 is required for HNSCC survival and proliferation in vitro and in vivo. a Analysis of FAT1 expression in a panel of epithelial cells, including HNSCC. b Expression of CD4ext and CD4-FAT1-TM/ICD chimera in the HNSCC cell line CAL33 by CD4 FACS analysis. c TEAD-Luciferase reporter assay in CAL33 cells transiently transfected with the CD4-FAT1 chimeric constructs. Luciferase expression was evaluated in exponentially growing cultures 36 h after transfection. Bars represent mean Renilla-normalized luciferase expression ± SEM (N = 4). d Quantitative PCR depicting gene expression levels of the YAP1 transcriptional targets CTFG and CYR61 in CAL33 stably expressing the CD4-FAT1 ICD constructs. Bars represent the GAPDH-normalized mean ± SEM (N = 3). e In vivo xenograft assay. One million CAL33 cells expressing indicated constructs were injected in nu/nu mice. Data points represent mean volume (N = 10 tumors per group) ± SEM. f YAP-rescue experiments. In vivo flank xenograft assay as in (e) using CAL33 expressing CD4-FAT1-TM/ICD and control or the indicated YAP expression vector. Data points represent mean volume (N = 10 tumors per group) ± SEM. g Spheroid formation assay of stable CAL33 shRNA control and YAP1 shRNA cell lines. Representative pictures are shown on top and diameter quantifications (>200 colonies per group) are shown below. Black lines represent mean ± SEM. h CAL33 stably expressing control and YAP1 shRNAs were stimulated with doxycycline for five days (1 µg/ml) and then transfected with a 8xTEAD-luciferase reporter. Renilla-normalized reporter activity is expressed as % of control. Bars represent mean ± SEM (N = 4). i Apoptosis assay by propidium iodide staining of CAL33 cell lines expressing control or YAP1 shRNA after 5d of Doxycycline stimulation. Bars represent mean ± SEM (N = 4). j In vivo xenograft assay. One million cells were injected in nu/nu mice. Animals were fed Doxycycline food (6 g/Kg) ad libitum 24 h h after tumor cell injection for the duration of the experiment. Data points represent mean volume per group (N = 10 tumors) ± SEM. k Representative immunohistochemical stainings of CAL33 tumors from panel (j). In the Cytokeratin 10 (CK10) panels the dotted red line delimits the proliferating front of the tumor. Scale bar, 100 µm. l Automated histological quantification of stainings in (k), bars represent mean ± SEM (N = 3). **P < 0.01, ***P < 0.001 (One-way ANOVA)