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. 2019 Nov 6;9:16317. doi: 10.1038/s41598-019-50694-z

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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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CD109 loss promotes EMT process, motility and invasion of SCCs. (A) Expression analysis by qPCR of fibronectin (FN), vimentin (VIM), a-SMA, Collagen 1A1 (Col1A1) and Collagen 3A1 (Col3A1). Results were first normalized with the housekeeping gene GAPDH, and the expressions in parental A431 cells were set at 1. Results are presented as mean ± SD (n = 3 independent experiments repeated in triplicates). (B) Representative Western blot image and (C) quantification of EMT markers. CD109 loss significantly enhanced the EMT marker expressions. (D) Representative images of Western blot and (E) Immunofluorescence microscopy and (F) quantification for P-Smad2 expression in A431 and CD109 KO A431 cells, showing an enhanced p-Smad2 activity in the CD109-KO cells. (G) Representative images and (H) quantification of wound-healing assays on parental and CD109 KO A431 cells, indicating CD109 loss enhanced the migration of SCC cells. Cell migration is expressed as a percentage of the scratch area filled by migrating cells at 24 h post scratch: migration rate = (T0 hr scratch width − T24 hr scratch width)/T0 hr scratch width) × 100%. (I) Representative images and (J) quantification of the invasive assay, suggesting CD109 loss significantly promoted cancer cell invasion in matrigel invasion assay. 50,000 cells of parental or CD109 KOA431 cells were seeded on a BioCoat™ Matrigel^® Invasion Chamber for 24 hours. Cells that invaded through the matrigel-coated membrane were stained with 1% crystal violet, photographed, and counted. All the results are expressed as the mean ± S.D. of three independent experiments. Significance is calculated using a One-Way ANOVA *P < 0.05. **P < 0.01 and ***P < 0.001. Scale bars: 10 μM, 100 μM and 300 μM, as indicated.