Fig 1: The transcription factor c-Jun element regulates the activity of the CH24H promoter. A. N2a cells were transfected with CH24H promoter-reporter plasmids (TSS-500, TSS-1000, TSS-1500, TSS-2000, and TSS-2500) for 24 h. Then the cells were either infected with VSV at an MOI of 0.01 or uninfected, and the luciferase activities were measured after another 18 h of infection (n = 3). B. N2a cells were transfected with the CH24H promoter-reporter plasmid: TSS-500. At 24 h post-transfection, cells were infected with VSV at an MOI of 0.01. At different time points after VSV infection, the culture medium was replaced in the presence of different signaling pathway-specific inhibitors (p38 inhibitor, 1 μM, 24 h; NF-κB inhibitor, 5 μM, 18 h; JNK inhibitor, 0.5 μM, 12 h; ERK inhibitor, 5 μM, 18 h) or DMSO. After 18 h of infection, the luciferase activity of the CH24H promoter was measured (n = 3). C. Schematic representation of point mutations in the wild-type promoter-reporter (TSS-500). N2a cells were transfected with the CH24H promoter reporter (TSS-500 and its mutant: TSS-500-mut). At 24 h post-transfection, cells were either mock-infected or infected with VSV at MOI 0.01, and luciferase activity was measured after 18 h of infection (n = 3). D. N2a cells were co-transfected with CH24H promoter-reporter (TSS-500) plasmid and pCAGSS-HA-c-Jun expressing plasmid (named HA-c-Fos) or pCAGGS vector for 24 h, and then CH24H promoter luciferase activity was measured (n = 3). E. N2a cells were mock-infected (top) or infected with VSV-GFP (bottom) at an MOI of 0.01. At 18 h post-infection, the cells were fixed, and c-Jun proteins (left), GFP (middle), and cell nuclei (right) were observed by confocal microscopy. Scale bar = 10 μm. F. N2a cells were transfected with HA-c-Jun for 24 h and then infected with VSV at an MOI of 0.01 for 18 h. Fixed chromatin from N2a cells was prepared and immunoprecipitated with anti-HA antibodies. ChIP primers were designed to amplify the region containing the c-Jun binding site in the CH24H promoter. PCR products were separated by acrylamide gel electrophoresis. Statistical analysis of comparisons between groups was performed by Student's t-test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Bar graph shows mean ± SD.