Prohibitin plays a critical role in Enterovirus 71 neuropathogenesis
(a) SK-N-SH cells were transfected with PHB siRNA at various concentrations for 48 hours. The efficiency of siRNA knockdown was verified by Western blot. PHB-knockdown SK-N-SH cells were infected with EV71 at M.O.I. 1. Viral titers in the culture supernatant were determined by plaque assay at 48 h.p.i. Statistical analysis was performed using two-tailed student’s t-test (** p<0.005, *** p<0.005). Non-targeting siRNA (NTC) served as control. (b) SK-N-SH cells were pre-treated with anti-PHB antibodies followed by EV71 infection at M.O.I. 1. Culture supernatant was harvested at 48 h.p.i. for viral titer determination by plaque assay. IgG isotype antibodies served as control. Statistical analysis was performed using one-way ANOVA with Dunnett’s post-test (***, p<0.0005; ****, p<0.0001). (c) SK-N-SH cells were reverse-transfected with 50 nM PHB siRNA, siNTC or left untreated prior to transfection with 0.25 μg of lucEV71 RNA. Luminescence signal was read at 48 h.p.t. Statistical analysis was performed using one-way ANOVA with Dunnet’s post test (**, p<0.005). (d) SK-N-SH cells were first infected with EV71 at M.O.I. 1 followed by Roc-A treatment for 48 hours. At 48 h.p.i. the culture supernatant was collected for viral titer determination by plaque assay, and the cell lysate was harvested for Western blot analysis. Statistical analysis was performed using one-way ANOVA with Dunnett’s post-test (**, p<0.005). Relative band quantification (below Western blot) was determined by ImageJ, by normalizing to loading control, β-actin. Error bars represent mean ± standard deviation. Cellular cytotoxicity was assessed using alamarBlue assay. One representative from two independent experiments is shown.