Fig. 5
C2S NPs promote osteogenesis through an autophagy effect in vitro. a The total protein levels of mTOR, p-mTOR, ULK1, p-ULK1, Beclin, P62, and LC3 in BMSCs treated with C2S NPs (0, 10, 50, 100 μg/mL) at different time points (7 and 14 days) were detected by western blot analysis, and densitometry data were normalized to GAPDH. The relative optical density was analyzed using ImageJ software (below). b BMSCs were treated with 3-MA, rapamycin, C2S NPs (50 μg/mL), C2S NPs + 3-MA, and C2S NPs + rapamycin for 6 h. FITC-labeled LC3 was observed under a fluorescence microscope. The fluorescence intensity ratio was analyzed using ImageJ software (below). ALP activity (c) and the formation of mineralized nodules (d) were detected by BCIP/NBT and Alizarin red S staining, accompanied by 3-MA, rapamycin, C2S NPs (50 μg/mL), C2S NPs + 3-MA, and C2S NPs + rapamycin at different time points (7, 14 days). f Total protein levels of mTOR, p-mTOR, ULK1, p-ULK1, Beclin, P62, LC3 β-catenin, RUNX2, BMP2, OPN, and Axin1 in BMSCs were detected by western blot analysis. Inhabitor + C2S group compared with C2S group. f Densitometry data were normalized to GAPDH. The relative optical density was analyzed using ImageJ software. Values are expressed as the mean ± SEM. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. compared with the control group