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. 2018 Jun 22;8:9517. doi: 10.1038/s41598-018-27912-1

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© The Author(s) 2018

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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TGFβ treatment increases invasiveness and cisplatin resistance of squamous lung carcinoma cells SK-MES1. (A) Top, schematics of the 2D migration assay. Bottom, migratory properties of SK-MES1 cells are increased upon TGFβ treatment. Cells were seeded in 24-well plate, pretreated with either SB-431542 or DMSO, stimulated with 2 ng/ml TGFβ1 and imaged for 60 hours. Migration speed from each single cell track was quantified. Center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to 5th and 95th percentiles, N indicates the number of quantified single cell tracks per condition. Additional independent experiments are shown in Supplementary Fig. S2A. Statistical analysis was performed using one-way ANOVA; ***P < 0.001; n.s., not significant. (B) Top, schematics of the collagen 3D invasion assay. Bottom, TGFβ stimulation increases number of invading cells and the average invasion depth. SK-MES1 cells were seeded in 96-well plates with precast collagen gels, allowed to attach overnight, growth factor-depleted for three hours, pretreated with either SB-431542 or DMSO, stimulated with 2 ng/ml TGFβ1, allowed to invade for four days, stained with Hoechst and imaged with a confocal microscope. The number of invaded cells and invasion depth were assessed. One representative experiment is shown. Data are presented as median and SD, every dot corresponds to a biological replicate (n = 15). N indicates the number of invaded cells. Additional independent experiments are shown in Supplementary Fig. S2B. Statistical analysis was performed using one-way ANOVA; ***P < 0.001; n.s., not significant. (C) Top, schematics of the experimental setup. Bottom, pre-treatment with TGFβ1 reduces sensitivity of SK-MES1 cells to cisplatin treatment. Cells were seeded in 96-well plate, stimulated with either 2 ng/ml TGFβ1 or left untreated for 3 days and then exposed to increasing doses of cisplatin for 3 days. Cell viability and caspase 3/7 activity were assessed. Data presented correspond to mean and SD, n is the number of biological replicates. Additional independent experiments are shown in Supplementary Fig. S1C. Statistical analysis was performed using one-way ANOVA; **P < 0.01; ***P < 0.001.