Fig. 4

Melatonin protected JEV-mediated neurotoxicity via inhibition of CaN (A) The protein levels of phospho-bcl10 were examined via western blot. (B) CaN activity was measured using a CaN activity assay. (C) SK-N-SH cells were previously treated with melatonin (100 µM) for 6 h, and then infected with JEV at an MOI of 5.0 for 1 h. Cells were collected after incubation with fresh media for 24 h. The protein levels of phospho-bcl10 were examined via western blot using the indicated primary antibodies. (D) CaN activity was measured by CaN activity assay. (E) Cells were observed under light microscopy (100x). (F) Viable cells were measured by crystal violet. SK-N-SH cells were pretreated with FK506 (10 µM) for 1 h, and then infected with JEV at an MOI of 5.0 for 1 h. Cells were collected after incubation with fresh media for 96 h. (G) Graphical data showing the OD550 nm from 1% SDS elution with crystal violet stain. (H) SK-N-SH cells were subjected to infection and treatment with FK506. Cells were pretreated with FK506 (10 µM) for 1 h, and then subjected to JEV infection at an MOI of 5.0 for 1 h. Cells were collected after incubation with fresh media for 24 h. The mRNA and protein levels of TNF-α and IL-6 were measured via quantitative real-time PCR (H, I) and ELISA (J, K), respectively. (H, I) The expression levels of TNF-α and IL-6 were calculated by normalizing to those of GAPDH; the graph indicates the relative quantification of gene expression normalized to control samples. (J, K) The data are expressed as the mean concentration (pg/ml) of TNF- α and IL-6, secreted in the supernatant. Values represent the mean ± SEM (n = 5 (B, D), 10 (G, H, I, J, K)). *p < 0.05, **p < 0.01, ***p < 0.001 vs. control (B) or JEV (D, G, H, I, J, K)