Fig 2: ARFGAP1 is required for EV-A71 replication. (A) RD cells were treated with siRNA targeting ARFGAP1 or control siRNA for 72 h and infected with EV-A71 (MOI = 1) for another 8 h. The cell lysates were analyzed by immunoblotting with the indicated antibodies. (B) RD cells were treated with siRNAs targeting ARFGAP1 or control siRNA for 72 h and then infected with EV-A71 (MOI = 1) for 6 h. Relative levels of EV-A71 RNA and ARFGAP1 mRNA were quantified by qPCR and normalized to GAPDH mRNA. Data represent the averages from at least three independent experiments ± SD. ∗∗, P < 0.001. (C) RD cells were treated with siRNA targeting ARFGAP1 or control siRNA for 72 h. Cells were then incubated with viruses for 30 min on ice. The cells were washed with cold PBS and then incubated at 37°C for 1 h to allow virus entry before treated with trypsin to remove any viruses that bound to cell surface. ns, no significance; ∗∗∗, P < 0.0001. (D) RD cells were treated with siRNA targeting ARFGAP1 or control siRNA for 72 h and infected with EV-A71 (MOI = 1) for another 20 h. Virus from the supernatant was tittered. ∗, P < 0.05. (E) RD cells were infected with EV-A71 (MOI = 1) for 1 h and then treated with QS11 or DMSO for 5 h. Total RNA was isolated, and the levels of viral RNA were determined by qPCR. The cell viability was measured by cellular ATP level. ns, no significance; ∗, P < 0.01; ∗∗∗, P < 0.0001. (F) RD cells were infected with EV-A71 (MOI = 1) for 1 h and then treated with QS11 or DMSO for 7 h. The cell lysates were analyzed by immunoblotting with the indicated antibodies. (G) RD cells were infected with EV-A71 (MOI = 1) or mock infected for 8 h and then stained with mouse dsRNA antibody and rabbit ARFGAP1 antibody. DAPI staining indicates the nucleus (blue).