Fig. 3: Accumulation of unsaturated lipids and cholesteryl esters in lipid droplets (LDs) of de-differentiated M381 cells.

a A representative SRS image of M381 cells imaged in the CH₂ (2845 cm⁻¹) channel. LDs are indicated. A zoomed-in image at right highlights a single LD. b The hSRS spectrum of the zoomed-in LD in a at the C–H stretch region (2800–3050 cm⁻¹). c Heatmap for scores of the top two constraints (λ₀–λ₁) by surprisal analysis of hSRS spectra on LDs across five cell lines (n = 30 LDs per cell line examined over three independent experiments). Each column represents an individual LD and each row represents the constraint scores. d The average score of λ₁ across five cell lines (n = 30 LDs). e Raman peak assignments for constraint 1 (λ₁). The pink shadowed range from 2957 to 2997 cm⁻¹ is assigned to cholesteryl esters (CE), and the 3022 cm⁻¹ peak (violet arrow) is assigned to unsaturated lipids (=C–H, UL). f hSRS spectra (normalized at 2908 cm⁻¹, the zero point revealed in e) of LDs across each cell line (n = 30). g Quantification of relative CE (2974 cm⁻¹/2908 cm⁻¹, top panel) and UL (3022 cm⁻¹/2908 cm⁻¹, bottom panel) enrichment in LDs across cell lines from f (n = 30). h GC-MS measurement of fatty acids extracted from bulk melanoma cells. The percentages of 16:0, 16:1, 18:0, 18:1, 18:2, and 20:4 are normalized to all extracted fatty acids (n = 4 independent experiments for M262, M229, M397; n = 5 independent experiments for M409 and M381). i Average ratio of unsaturated fatty acids (UFA) to saturated fatty acids (SFA) in each lipid class from lipidomics of M381 cells (n = 3 independent experiments). j Percentage of major lipid classes from lipidomics of M381 cells (n = 3). ***p < 0.001 from two-tailed unpaired t-tests. Scale bars, 20 μm. Data shown as mean ± SEM. Lipidomics data are provided as Supplementary Data 1. Source data are provided as a Source data file.