Fig. 4

In vitro autophagy stimulation and anti-atherosclerosis effects. (A) Western blot analysis of p62, LC3-I, LC3-II, and GAPDH protein expressions from foam cells treated with or without free trehalose and nanogel in comparison to the control); i = control, ii = foam cells, iii = foam cells + trehalose, and iv = foam cells + TNG. (B, C) Quantification of LC3-II/LC3-I and p62/GAPDH ratios from the Western Blot images. (D) Lipid profile in foam cells treated with or without free trehalose and nanogel in comparison to the control upon oil Red O (ORO) staining, following quantification of (E) lipid droplet area (% of total) and (F) Abs at 524 nm; i = control, ii = foam cells, iii = foam cells + trehalose, and iv = foam cells + TNG. (G, I) Fluorescence imaging and (H, J) quantification of p62 and LC3 expressions from foam cells treated with or without free trehalose and TNG in comparison to the control. (K) Fluorescence images and (L, M) quantification by flow cytometry showing intracellular ROS generation after treatment with different formulations and stimulation with H₂O₂. RAW264.7 cells were incubated with medium alone or trehalose or TNG for 4 h, followed by stimulation with H₂O₂ for 0.5 h. Cells unstimulated with H₂O₂ served as the negative control. Then fluorescence microscope and flow cytometric analyses were performed. Scale bar: 50 μm. Data are presented as mean ± SD, n = 3, ns: no significance, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001