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. 2019 Jul 29;10:3362. doi: 10.1038/s41467-019-11325-3

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© The Author(s) 2019

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. 7 — Reduced actomyosin contractility following Par3 loss drives ectopic differentiation and suprabasal fate. a Immunoblot analysis for Keratin1 expression in keratinocyte lysates following 48 h CalA (1 nM) treatment. GAPDH served as loading control. b Quantification of a. Keratin1 levels were first normalized to GAPDH and then expressed as relative values. n = 5 biological independent samples; ***p = 0.001 (Ctrl vs. Par3KO, DMSO-treated); ***p = 0.0003 (Par3KO, DMSO-treated vs. CalA-treated), ***p < 0.0001 (Par3KO/DMSO-treated vs. Ctrl/CalA-treated), mean ± SD, two-way ANOVA/Tukey’s multiple comparisons test. c Immunoblot analysis for Keratin1 expression in keratinocyte lysates following siCtrl or sip53 transfection. GAPDH served as loading control. Relative p53 protein levels are shown below the immunoblot. d Quantification of c. Keratin1 levels were first normalized to GAPDH and then expressed as relative values. n = 3; ***p = 0.0009 (Ctrl vs. Par3KO, siCtrl-treated); ***p = 0.0001 (Par3KO siCtrl-treated vs. Ctrl sip53-treated); **p = 0.008 (Par3KO siCtrl-treated vs. sip53-treated); **p = 0.0049 (Ctrl sip53-treated vs. Par3KO sip53-treated); mean ± SD; two-way ANOVA/Tukey’s multiple comparisons test. e Schematic representation of the Transwell filter culture system and experimental setup. Par3KO keratinocytes were positive for H2B-GFP, control keratinocytes isolated from littermates were negative for H2B-GFP. f Immunofluorescence micrographs of stratified cultures incubated for 10 days either in DMSO or in 1 nM CalA. Par3KO keratinocytes were H2B-GFP (green) labeled, DAPI is shown in magenta. Bottom panels show zx-projection. Scale bar: 50 µm. g Pie graphs showing cell distribution according to basal or suprabasal position following a 10-day culture period of mixed control and Par3KO keratinocyte cultures on Transwell filters. DMSO-treated samples: n = 180 cells (basal/DMSO-treated: n = 171 cells; suprabasal/DMSO-treated: n = 9 cells); CalA-treated samples: n = 170 cells (basal/CalA-treated: n = 143 cells; suprabasal/CalA-treated: n = 27 cells). An experimental representaton of three independent experiments is shown. Cropped immunoblot data are shown. Image intensity was enhanced for better visualization. Ctrl control, KO Par3KO, CalA Calyculin A, ns non-significant, SE short exposure, LE long exposure, siCtrl small interfering non-targeting RNAs, sip53 small interfering RNAs targeting p53