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. 2023 Jan 28;14:465. doi: 10.1038/s41467-023-36128-5

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© The Author(s) 2023

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 Diagram of GSL synthetic pathways. UGCG, UDP-glucose ceramide glucosyltransferase. b Basal glycolysis rate of WT and KRASG12V (KG12V) expressing MDCK cells measured in a Seahorse XF analyzer (±SEM, n = 3 independent biological relicates, **p < 0.01). Time course of changes in 2DG (10 mM)-treated MDCK cells of: GFP-KRASG12V (KG12V) PM binding quantified by EM as mean gold labeling density (c), and nanoclustering quantified as L_max (d) (n = 12 PM sheets for each group). For the DMSO control time course see Fig. S1J–L. Time course of changes in relative ATP levels for control (t = 0) and glucose-depleted (GS) MDCK-KRASG12V cells (±SD, n = 3 wells, *p < 0.05, **p < 0.01) (e). PM localization (f) and nanoclustering (g) of KRASG12V-expressing MDCK (n = 14 for Control, n = 12 for GS) or Caco-2 cells, evaluated by EM, after 4 h of glucose depletion (n = 14 for Control, n = 13 for GS). Time course of GFP-KRASG12V mislocalization in glucose-depleted MDCK cells co-expressing mCherry-CAAX (an endomembrane marker) evaluated using Manders coefficients (±SD, n = 3 independent replicates, ***p < 0.001) (h). Time course of changes in PM binding (i) and nanoclustering (j) of GFP-KRASG12V in DL-PDMP-treated MDCK cells. EM evaluation of PM binding (k) and nanoclustering (l) of GFP-KRASG12V in UGCG knockdown (sgUGCG) and non-targeting control (sgCtrl) Caco-2 cells (n = 41 PM sheets). In (c, d), (f, g), and (i–l) data are mean ± SEM. Differences in gold labeling densities and Lmax were evaluated in two-tailed Student’s t-tests and bootstrap tests respectively (*p < 0.05, **p < 0.01, ***p < 0.001). Exact p-values and source data are provided as a Source data file.