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. 2021 Apr 6;12(4):348. doi: 10.1038/s41419-021-03631-w

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© The Author(s) 2021

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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Fig. 1 — A C3G (RAPGEF1) mRNA levels in GBM patients and normal brain (171 patients) obtained from TCGA database normalized with GUSB. B Western-blot analysis of C3G and Vimentin protein levels in the indicated GBM cell lines and human astrocytes (HAs) normalized to β-actin. Densitometric quantification of C3G/β-actin and Vimentin/β-actin ratios are shown. C, G Western-blot analysis of C3G normalized to β-actin to confirm silencing in U87 and 12Ф12D cells as compared to parental cells and/or cells with control shRNA (NTC). Densitometric quantification of C3G/β-actin ratio is shown. D Phase-contrast microscopy images of parental U87, U87shC3G and NTC cells maintained either in the presence (10% FBS) or absence (0% FBS) of serum for 24 h. Scale bars: 50 µm. E Representative images of β-catenin (red) and DAPI (blue) staining analyzed by confocal microscopy. Scale bars: 25 µm. F and H Left panels, immuno-fluorescence microscopy images of phalloidin staining (red) in parental and C3G-silenced U87 and 12Ф12D cells, maintained as indicated. Cell nuclei were stained with DAPI (blue). Scale bars: 50 µm. An amplification of cells inside the square is also shown. Right panels, histograms showing the quantification of the number of cells presenting migratory structures (filopodia, blebs, stress fibers and/or lamellipodia) expressed as fold increase.