Fig. 6.

Modulation of Afdn expression by hypermethylation of its gene promoter contributes to the increased BBB permeability. (A) Detection of methylation levels of Afdn using JEV supernatant treated bEnd.3 cell by BSP. (B) qPCR was performed to detect the expression of Afdn after JEV-infected supernatant treated in bEnd.3 cell. (C) Western blot was performed to measure Afdn protein expression in bEnd.3 cells treated with JEV-infected supernatant. The right histogram shows a densitometric analysis of the Western blot results. (D) Schematic diagram showing the transwell assay to assess the permeability of bEnd.3 cells after mock or JEV supernatant treatment for 72 h. (E) In vitro, transwell experiment was performed to measure bEnd.3 cells permeability after JEV infected mice brain supernatant treatment. (F) qPCR analysis of Afdn expression changes after Afdn siRNA transfection in the bEnd.3 cell. (G) Western bolt was detected the expression of AFDN after siRNA transfection in bEnd.3 cells. The right histogram shows a densitometric analysis of the Western blot results. (H) Schematic diagram showing the detection of permeability of bEnd.3 cells after transfection with Afdn siRNA. (I) Transwell experiment to measure the permeability of bEnd.3 cell after Afdn siRNA transfection for 72 h. The schematic diagrams were made with the free version of BioRender. Data are represented as the mean values ± standard deviations (SD) of at least three biological replicates using Student’s t-test (*p < 0.05, **p < 0.01, and ***p < 0.001) between the indicated groups