Fig. 4

CREB5 repressed EV-A71 viral replication and expression. a SF268 cells were mock-infected or infected with EV-A71 (MOI = 1) for different time points as indicated (lanes 2–5. 12–48 h after infection). Cell lysates were then harvested for detection of CREB5 protein levels by Western blotting using the anti-CREB5 specific antibody. b Mice were treated with the miRNA hairpin inhibitor negative control in phosphate buffered saline (lane 1) or anti-miR-876-5p inhibitor for 24 h and infected with EV-A71. The protein extracted from the site of injection in the abdominal muscle was collected for CREB5 and viral protein investigations; VP1–3 analysis was performed with Western blotting using specific antibodies as indicated. Lane 1, the miRNA hairpin negative control and no EV-A71 infection; lane 2, the miRNA negative control of mice infected with EV-A71 or anti-miR876-5p inhibitor-treated (lane 3) mice. c Quantitative polymerase chain reaction (qPCR) analysis of CREB5 mRNA level in si-CREB5-transfected or CREB5-overexpressed cells. d Expression of CREB5 determined by Western blotting in pCMV-CREB5-Flag-transfected cells (lane 2) or siCREB5-transfected cells (lanes 3–4). Lane 1, cells were transfected with pCMV-8-Flag plasmid as the negative control. e Accumulation of viral RNA in pCMV-CREB5-Flag-transfected cells or siCREB5-transfected cells upon EV-A71 infection in SF268 cells. pCMV-8-Flag is the empty plasmid used for transfection control. f Expression level of viral protein and 3D and CREB5 protein in the mock-infected, pCMV-CREB5-transfected, and siCREB5-transfected cells upon EV-A71 infection. Lane 1, Mock-infected cells used as negative control; lane 2, cells were treated with si-scramble RNA and infected with EV-A71 for 24 h; lanes 3–4, cells were treated with siCREB5 RNA (5 ng and 10 ng) and infected with EV-A71 for 24 h. g Viral titers of pCMV-CREB5-Flag-transfected or pCMV-8-Flag control and EV-A71 infected cells. All experiments were repeated three times. * P < .05; ** P < .01