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Figure 4. Lipids exposed at the outer leaflet of the plasma membrane of drug-naïve, DTP and PDR melanoma cells. (A) Pie charts show the mole percentage (mol%) of the four major GPL classes obtained on the outer leaflet of the plasma membrane (O, above histogram) and the lipids of the inner cytoplasmic leaflet and intracellular organelles (I, below histogram). Histograms show the proportion of each lipid class exposed on the outer leaflet (O, PC: blue, PE: yellow, PI: Purple and PS: red) and found inside the cells (I, light blue, yellow, purple and red), which include lipids of the inner leaflet combined with intracellular organelles, as quantified via mass spectrometry with error bars as SD (n = 3-4 biological replicates). Statistical analyses using 2-way ANOVA with Tukey’s multiple comparison test show no significant difference in the phospholipid proportions across the three different states (see also Supplementary File 2). Differences between total, outer leaflet and inner compartments within individual cell states are also not statistically significant (see also Supplementary Figure 4). Average surface charge (mV ± SD) of cells treated with dabrafenib for 0, 30 and 105 days, as measured using zeta potential (250,000 cells/sample, n = 3 biological replicates); differences are not statistically significant; (B) Histograms represent the normalised abundance of the most common PC, PE, PI, and PS species quantified at the sum composition level of lipids found inside cells including the inner leaflet (I) and lipids found on the outer leaflet (O). WM164 and HT144 drug-naïve, DTP and PDR states which correspond to 0, 30 and 105 days of treatment with dabrafenib. Data represent mean values from three to four separate experiments. See Supplementary File 2 for absolute values and statistical analyses using 2-way ANOVA with Sidak’s multiple comparison test. DTP: Drug-tolerant persister; PDR: permanent drug-resistant; GPL: glycerophospholipid; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; PS: phosphatidylserine; SD: standard deviation; ANOVA: analysis of variance; FA: fatty acid; PDRCs: permanent drug-resistant cells.