Fig. 7: Autophagy was necessary for CD24-induced sorafenib resistance progress.

The effects of CD24-dependent sorafenib resistance will soon disappear with the inhibition of autophagy using either pharmacological inhibitors or essential autophagy gene knockdown. We treated Huh7/SR cells with 1 nM bafiIomycinA1 (BafA1) to inhibit autophagy by blocking AV-lysosome fusion. Cells were treated with different concentrations (0, 1.5, 3, 5, 10.0 μM) of sorafenib for 48 h. a The relative expression level of LC3B, p62, and CD24 protein was analyzed by western blot. The relative amounts of LC3-II are indicated. b CCK8 assay was used to detect cell sorafenib sensitivity. c Apoptosis detected by AnnexinV-PI assays. Representative images are shown on the left, and quantitative data are on the right. We knocked-down ATG5 with a small hairpin RNA (shATG5) in Huh7/SR cells. d The relative expression level of ATG5, LC3B, and CD24 protein was analyzed by western blot. The relative amounts of LC3-II are indicated. e Evaluation of the AVs by transmission electron microscopy. Cells were cultured with 1.5 μM sorafenib for 48 h then subjected to transmission electron microscopy analysis. The area noted by red arrows is the autophagic vacuole (AV). Scale bars, 0.5 μm. f CCK8 was used to detect the cell sorafenib sensitivity. Each experiment was performed in triplicate. The relative expression level of LC3B, ATG5, and CD24 protein was analyzed by western blot. The relative amounts of LC3-II are indicated. Each experiment was performed in triplicate. The data are presented as the means ± s.e.m. and analyzed with Student’s t-test (*P < 0.05, **P < 0.01)