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. 2017 Aug 22;7:9031. doi: 10.1038/s41598-017-09539-w

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© The Author(s) 2017

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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Cell function assays. Knockdown TMSB4X in SAS cells may inhibit their cell proliferation and invasion ability in vitro, as well as suppress cervical LN metastasis in vivo. (A) The panel of TMSB4X expression level in 6 HNSCC cell lines by RT-PCR. The relative aggressive SAS cells have abundant TMSB4X. (B) In TMSB4X knockdown clones of SAS, the expression was revealed by RT-PCR. (C) The growth curves of TMSB4X knockdown clones of SAS. (D) The invasion ability of TMSB4X knockdown clones of SAS was performed by Boyden chamber assay. Cell photography and number were taken and counted at 200x magnification (high power field, HPF). (E) Bioluminescence images were taken from the buccal orthotopic xenograft mouse model. The total flux of bioluminescence was measured separately at mouse buccal (Tumor) and neck lymph node (LN metastasis). (F) The box-plot of LN metastasis was drawn according to the total flux value in region of interest (ROI). Asteroid *denotes P < 0.05. Statistics was calculated by student t-test. (shTb4-1: clone1 with shRNA-TMSB4X).