Figure 3.

c-MYC regulates the transcription of zonula occludens-1 (ZO-1), and c-MYC depletion reduces ZO-1 expression in human brain microvascular endothelial cells (HBMECs). (a) Results of dual luciferase reporter experiments. HBMECs were transfected with different pGL3 vectors containing different truncations of the ZO-1 promoter and pRL-TK, and luciferase activity was detected after 24 h. One-way ANOVA was used to compare the luciferase activity. Data are shown as the mean ± SD, n = 3. **p < 0.01 vs. the luciferase activity of the −2787 truncation. (b) Possible transcription factor binding to the −43 to −1 region. c-MYC binding to this region was predicted using the Matinspector online software. (c) Effect of c-MYC interference on ZO-1 expression. sic-MYC-587 reduced c-MYC and (d) ZO-1 mRNA levels significantly. (e) sic-MYC-587 decreased c-MYC and (f) ZO-1 protein levels significantly. One-way ANOVA was used for repeated measurements. Data are shown as the mean ± SD, n = 3. *p < 0.05 and **p < 0.01 vs. NC. (g) Immunofluorescent staining of ZO-1 expression. HBMECs from the NC and c-MYC-587 groups were stained with ZO-1 (green) and 4,6-diamidino-2-phenyl-indole (DAPI) (blue), and the down-regulation and disassembly of ZO-1 (not continuous) were indicated by the white arrows. Scale bar = 20 µm.