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. 2019 Oct 10;9:14527. doi: 10.1038/s41598-019-50742-8

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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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In PI3K-dysregulated cells, PDK1 homodimerisation and localisation is independent of PtdIns(3,4,5)P₃ levels. All experiments were performed under resting conditions (−), stimulation of the PI3K pathway with EGF (+) or treated with the PI3K inhibitor (LY294002) prior to stimulation. SKBR3 cells were cotransfected with GFP-PDK1 and PDK1-mCherry. (A) Representative intensity and FRET-FLIM f_D images. (B) Relative distribution of full-length PDK1 at the PM and the cytoplasm of SKBR3 cells. The dashed line indicates the reference level for a control cytoplasmic protein (Table 1). (C) Quantification of the homodimerisation efficiency of full-length WT PDK1 and its mutants. E_D calculation is based on FRET-FLIM measurements on a single-cell level, as illustrated in (A). (D) Localisation of the PDK1 homodimers in SKBR3 cells. The graph shows the ratio of PDK1 dimers at the PM and the cytoplasm based on the segmentation of the f_D images shown in (A). Scale is 20 µm. N > 30. Error bars: SEM. (C,D) N > 30. Box: 2xSEM; Whiskers: 80% population. Mann-Whitney test *p < 0.05. Three independent experiments.