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. 2017 Aug 21;7:8944. doi: 10.1038/s41598-017-09387-8

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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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mSREBP1c inversely regulate signalling output driven by oncogenic H-ras or K-ras. (A,B) Western blot analysis of mSREBP1c in HEK cells transiently expressing (A) mGFP-H-rasG12V or (B) mGFP-K-rasG12V. Cells were incubated for 24 h with increasing concentrations of amino acids (1x, 4x or 8x) in the absence of serum. Numbers indicate β-actin normalized mSREBP1 levels (n = 3). (C,D) Nanoclustering-FRET analysis in HEK cells transfected with mature Flag-tagged mSREBP1c or empty vector, co-expressing mGFP- and mCherry-tagged (C) H-rasG12V or (D) K-rasG12V. The numbers in the bars indicate the number of analyzed cells (mean ± SEM, n = 4). (E,F) Nanoclustering-FRET analysis in HEK cells transfected with scrambled siRNA or siRNA directed to SREBP1, and co-expressing mGFP- and mCherry-tagged (E) H-rasG12V or (F) K-rasG12V. The numbers in the bars indicate the number of analyzed cells (mean ± SEM, n = 4). Note that the variations in FRET efficiency (%) of the control levels in C, E and D, F are due to the different experimental procedures. (G,H) Western blot analysis of Ras- and mTORC1-signalling in HEK cells transfected with siRNA SREBP1 or Flag-tagged mSREBP1c vector, transiently expressing (G) mGFP-H-rasG12V or (H) mGFP-K-rasG12V. Numbers indicate the ratio of phosphorylated to respective total protein levels (n = 3). Statistical significance levels are annotated as *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.