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. 2019 Jun 27;9:9361. doi: 10.1038/s41598-019-45731-w

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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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NFATc3 is required for glioma proliferation and migration. U251 cells were transduced with control (KD-Sc), NFATc1 (KD-c1) and NFATc3 (KD-c3) shRNA lentiviral vectors (LV) as before. (A) KD-Sc and KD-c3 cells were labelled with Violet proliferation Dye 450 and left in 2% FBS media. Cells were analysed by flow cytometry 1 and 3 days after treatment. Panel I cytometry histograms showing dilution of fluorescent compound in each division. Panel ii Division index: as the average number of cell divisions that a cell in the original population has undergone, and Proliferation index: the total number of divisions divided by the number of cells that went into at least one division. A representative experiment is shown in panel i, and indexes are measures of 4 independent experiments. ***P < 0.001 with respect to index in KD-Sc cells. (B) Wound-healing assay from KD-Sc, KD-c1 and KD-c3 U251. Panel i, photomicrograph of representative fields at initial time (0 h) and after 24 hours (1d) 2% FBS stimulation. Dotted lines mark the limits of the unpopulated area. Panel ii shows quantification of migrated area (the proportion of the denuded area repopulated by migrating cells) at 24 hours (mean ± SD; n = 3). *P < 0.05 with respect to KD-Sc cells. (C) Transmigration assay was carried out using Boyden chamber assays (scheme on panel i). KD-Sc and KD-c3 cells were platted on the Transwell inserts and 2% FBS was added to the lower chamber. After 4 hours, non-migrated cells were washed and fixed as in material and methods. Panel i show microphotographs of randomly chosen fields of paraformaldehyde fixed cells stained with Hoechst 33342 to visualize nuclei. Panel ii shows quantification of migrating cells per chamber. Data are shown as the mean ± SD of three independent experiments. **P < 0.01 with respect to KD-Sc cells without FBS in the lower chamber.