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. 2017 Aug 18;7:8710. doi: 10.1038/s41598-017-09504-7

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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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MSI1/TNS3-regulated GBM cell migration and actin remodeling is potentially governed by RhoA-GTP activation. (A) MSI1 and TNS3 protein levels were assessed in 05MG cells transiently transfected with siMSI1 (clone #1 and #2) and/or siTNS3 (clone #1 and #2) using Western blotting. (B) MSI1- and TNS3-depleted 05MG cells were subjected to TransWell migration assay. The number of migratory cells were counted and presented as percentages relative to the controls. The percentage of cell numbers were indicated by alamar blue assay shown in bar chart. (C) MSI1-mediated actin remodeling with TNS3 inhibition were shown with the immune-stained F-actin distribution in MSI1- and TNS3-depleted 05MG cells (left); the formation and the angular distribution of F-actin orientation were analyzed by assessing and quantifying the immunofluorescence intensities using Matlab software. Mean of Kurtosis was shown on the right (n = 45). (D) GTP-bound RhoA was pulled down (IP) with Rhotekin-RBD agarose beads from cell lysates and blotted with anti-RhoA antibodies. The intensities of RhoA-GTP were normalized to that of total RhoA and presented as relative fold change in the graph. (E) MSI1- and TNS3-depleted 05MG cells were subjected to Western blot for MSI1, TNS3 and RhoA protein levels. The intensity of RhoA-GTP blot at each group be calculating and presented as a relative ratio in comparison to the control group (siCON.). (F) TNS3-overexpressed (mycTNS3) and MSI1-overexpressed (FlagMSI1) U251 cells were subjected to Western blot for Flag-tag, myc-tag and RhoA protein levels. The intensity of RhoA-GTP blot was calculated and presented as a relative ratio compared to control (Flag). (G) Transwell migration assay was performed in MSI1-overexpressed and TNS3-overexpressed U251 cells. The number of migratory cells were counted and presented as percentage relative to the control group (Flag), while the cell numbers were indicated by alamur blue assay shown in bar chart. The uncropped blots were demonstrated in Supplementary Figure 4A. Each result is shown as the mean ± SEM. The experiments were repeated at least three times. *p < 0.05.