Statistical analyses were performed using one-way ANOVA and Dunnett’s test (Graphpad software) against scrambled control (Scr). Error bars represent standard deviation (SD) of triplicate data. (H) Virus titres of other human enteroviruses (Echovirus 7, Coxsackievirus A6 and EV71 strain 41) in the supernatant of siRNA-treated cells were analysed via viral plaque assay. Statistical analyses were performed using one-way ANOVA and Dunnett’s test (Graphpad software) against untreated control. (G) Virus titres in the supernatant of cells treated with individual siRNAs within siRNA SMARTpool were analysed via viral plaque assay. The band intensities representing MINK protein expression level were quantitated with reference to actin control bands (for each individual siRNA) and PTC. (F) Band intensity of MINK gene knockdown verification in deconvolution assay. Western blot analysis was performed to detect protein expression levels of MINK, with β-actin as the loading control. (E) Verification of gene knockdown efficiency of individual siRNA within the siRNA SMARTpool directed against MINK at 45nM. The intensities of protein bands were quantitated using ImageJ Gel Analysis program. The band intensities representing MINK protein expression level were quantitated with reference to actin control bands (for each individual concentration) and PTC. (D) Band intensity of MINK gene knockdown verification. MINK protein expression was observed to decrease in a dose-dependent manner across siRNA concentration. Parallel transfection of scrambled siRNA served as a knockdown control. (C) Verification of gene knockdown efficiency of MINK siRNA SMARTpool at concentrations ranging from 0nM to 45nM. Error bars represent standard deviation (SD) of triplicate data and values obtained were normalised against the transfection control. Virus titres in the supernatant of siRNA-treated cells were analysed via viral plaque assay. (B) Cell viability of siRNA-treated cells was measured in relation to untreated cells using alamarBlue assay after 72h incubation. Cells in both the negative and transfection controls were not infected with EV71 while cells in the positive control were infected with EV71 in the absence of siRNA. The images were taken at 10X magnification. Immunofluorescence detection of EV71 VP2 proteins (green) with the nuclei stained with DAPI (blue) is shown. (A) siRNA-treated EV71 infected cells were fixed at the same time-points and intracellular viruses were detected by immunofluorescence assay. Silencing of MINK significantly reduced EV71 replication in a siRNA concentration-dependent manner. Values obtained in the graph were normalised against the mean of the transfection control (EV71-infected cells treated with only the transfection reagent). siRNA controls utilised included non-targeting siRNAs as well as siRNAs targeting several housekeeping genes. First 6 bars represent siRNA controls used while the other bars represent the host serine/threonine kinases targeted in the screening. As such, 6 genes have been identified as positive hits from the primary screen. 40% reduction in viral antigen positive cells was considered as the acceptable level of virus inhibition and positive hits are genes which resulted in a percentage of viral antigen positive cells of less than 60% upon the knockdown of these genes. Human serine/threonine kinase siRNA library screen.Įffect of gene knockdown by siRNA on EV71 replication analysed from primary screen.
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