Fig. 3: Cellular uptake of GO/LDL complexes, intracellular localization, and influence on macrophage lipid droplet formation.

a Cellular uptake of GO/LDL complexes assessed by Raman mapping of GOs, showing D-band (red color) and G-band (green color) signal intensity. Scale bar represents 10 μm. b Confocal images depicting the uptake of GO/LDL complexes using laser reflectivity microscopy. Fluorescent signals in red, green, and blue correspond to cytoplasmic membrane, GOs, and the nucleus, respectively. Scale bars represent 7.5 μm. c Subcellular localization of GOs and lipid droplets observed through TEM images. Red arrows indicate lipid droplets, while blue arrows indicate GOs. Scale bar represents 1 μm. Cells are treated with 50 μg/mL LDL and a mixture of 50 μg/mL LDL with 20 μg/mL GOs for 24 h. d Accumulation of lipid droplets in macrophages when treated with LDL and GO/LDL complexes, visualized by confocal imaging. Red color indicates lipid droplets, while blue indicates nucleus. Scale bar represents 10 μm. e Quantitative analysis of total lipid droplet mean fluorescent intensity (MFI) per cell using high-content analysis. Data are presented as mean ± standard deviation of n = 3 biological replicates. Statistical significance is calculated by one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s., not significant (p > 0.05). f, g Soft X-ray cryo-tomographic images of lipid droplets in macrophages before and after exposure to LDL and GO/LDL, obtained through soft X-ray transmission microscopic imaging and 3D reconstruction (nano-CT). Cells are treated with 50 μg/mL LDL or a mixture of 50 μg/mL LDL with 20 μg/mL GOs for 24 h, and wet samples are frozen in liquid nitrogen for nano-CT imaging. f Soft X-ray transmission cryo-microscopic and 3D tomographic imaging of frozen cells and lipid droplets at 520 eV and −80 °C. Red stars indicate lipid droplets, while purple stars represent organelles. g Spatial distribution of lipid droplets (in yellow) and organelles (in red) based on 3D reconstruction of the macrophages. Scale bar represents 5 μm. Source data are provided as a Source Data file.