fig5

Figure 5. Comparing biophysical properties of melanoma drug-naïve, DTPs and PDR cell membranes using Laurdan, Di-4-ANEPPDHQ and FLIPPER-TR dye. (A) GP measurements of drug-naïve, DTP and PDR WM164 and HT144 cells labelled with Laurdan using 2-photon microscopy at 37 °C. Each point represents mean GP calculated from several cells captured in 5-7 fields of view taken per condition. The horizontal line represents the mean value and SD. One-way ANOVA with Tukey’s multiple comparison test was performed for statistical analyses. Differences are not statistically significant (ns); (B) GP measurements of WM164 and HT144 drug-naïve, DTP, and PDR melanoma cells (cells), as well as LUVs generated from melanoma cell lipid extracts or defined synthetic lipid mixtures (model membranes). Cells were plated at a density of 15,000 cells and labelled with Di-4-ANEPPDHQ for 15 min at 37 °C. LUVs prepared from cell lipid extracts or from synthetic POPC, POPC/POPS (90:10), POPC/PS/Chol/SM (17:10:33:40) and POPC/Chol/SM (27:33:40) were labelled with 2.5 µM Di-4-ANEPPDHQ at 37 °C and used as model membranes. Arrows indicate the direction of increasing membrane order, corresponding to liquid-ordered (L_o) and liquid-disordered (L_d) phases. Fluorescence emission spectra were measured with excitation at 480 nm. Data represent mean GP ± SD (n = 3-5); (C) Representative FLIM measurements of membrane tension on drug-naïve, DTP and PDR melanoma cells labelled with FLIPPER-TR probe. DTPs: Drug-tolerant persisters; PDR: permanent drug-resistant; GP: generalised polarisation; SD: standard deviation; ANOVA: analysis of variance; LUVs: large unilamellar vesicles; POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPS: 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine; PS: phosphatidylserine; Chol: cholesterol; SM: sphingomyelin; FLIM: fluorescence lifetime imaging.