Fig. 3: Impact of CLU on OPC differentiation and apoptosis in vitro.

A Schematic diagram illustrating OPC differentiation, proliferation, and apoptosis assays treated with CLU. B, C CLU exhibited a dose-dependent decrease in the number of MBP⁺ cells in the OPC differentiation assay (n = 3 technical replicates/group, 50 ng/mL vs. 0 ng/mL p = 0.0141; 100 ng/mL vs. 0 ng/mL p = 0.0032; 200 ng/mL vs. 0 ng/mL p = 0.0013; 400 ng/mL vs. 0 ng/mL p = 0.0001). D, E In the OPC proliferation assay, CLU dose-dependently reduced the number of EdU⁺Olig2⁺ cells (n = 3 technical replicates/group, 50 ng/mL vs. 0 ng/mL p = 0.0035; 100 ng/mL vs. 0 ng/mL p = 0.0025; 200 ng/mL vs. 0 ng/mL p = 0.0008; 400 ng/mL vs. 0 ng/mL p = 0.0005). F, G CLU dose-dependently increased OPC apoptosis, as detected by cleaved caspase-3 immunohistochemical staining (n = 3 technical replicates/group, 50 ng/mL vs. 0 ng/mL p = 0.0005; 100 ng/mL vs. 0 ng/mL p = 0.0005; 200 ng/mL vs. 0 ng/mL p < 0.0001; 400 ng/mL vs. 0 ng/mL p < 0.0001). H, I CLU dose-dependently increased OPC apoptosis, confirmed by CC3 expression via western blot (n = 3 technical replicates/group, 1 h vs. 0 h p = 0.9447; 3 h vs. 0 h p = 0.4328; 6 h vs. 0 h p = 0.2383; 12 h vs. 0 h p = 0.0017; 24 h vs. 0 h p = 0.0014). J, K CLU inhibited the ability of OPCs to form a myelin sheath in cerebellar organic slice culture (n ≥ 3 technical replicates/group, p = 0.0033). L–N Numbers of Olig2⁺ OPCs and CC1⁺ OLs in the cerebellar slices were reduced after CLU treatment (n = 5 technical replicates/group, p < 0.0001; p = 0.0003). For immunofluorescence pictures: Scale bar: 50 μm. Statistical analysis was performed using unpaired two-tailed Student’s t-tests, mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001. ns. = p not significant. CC3 cleaved capase-3.