Fig. 4
ApoHPC-MPs are cytotoxic to HPCs. A The same amount (20 µg) of apoLTC-MPs, apoHep-MPs or apoHPC-MPs was incubated with cultured WB-F344 cells (5 × 103) for 24 h. The killing of WB-F344 cells was observed by microscopy. B WB-F344 cells were treated with 20 µg of apoHPC-MPs, apoHep-MPs or apoLTC-MPs for 1 h. The cells were washed and cultured in fresh medium for 24 h. Cell proliferation was detected by CCK8 assay. Data were collected from three independent experiments. Data are presented as mean ± SD. ***p < 0.001, ****p < 0.0001. C 40 µg of apoHPC-MPs, apoHep-MPs or apoLTC-MPs were intrasplenically injected into rats, which were treated with DEN for the previous 6 weeks. 24 h after injection, liver slices were acquired for fluorescence detection. HPCs were stained with antibodies against SOX9 (red) and cleaved caspase 3 (green) for fluorescence microscopy analysis. Nuclei were stained with DAPI (blue). Representative pictures of staining are shown. White asterisks show the SOX9-positive HPCs which are also positive for cleaved caspase 3. D Cells positive for both SOX9 and cleaved caspase 3 were counted in each field of view in C; the combined data from three experiments are shown. **p < 0.01, ****p < 0.0001 E 40 µg of apoHPC-MPs, apoHep-MPs or apoLTC-MPs were intrasplenically injected into rats, which were treated with DEN for the previous 6 weeks. Injections were administered twice every week for 4 weeks. Rats were then sacrificed, and liver tissue sections were acquired for IHC assay. The HPCs were stained with an antibody against SOX9. Representative photographs are shown. In the apoHPC-MP-treated liver, SOX9 staining is faint and very scarce. The apoHep-MP-treated liver shows a moderate level of SOX9 staining. The other livers show strong and diffuse staining. F Computer-assisted IOD evaluation of SOX9 staining in E. Data were collected from three independent experiments. Data are presented as mean ± SD. *p < 0.05, ***p < 0.001