Phosphorylation of ERK-Dependent NF-κB Triggers NLRP3 Inflammasome Mediated by Vimentin in EV71-Infected Glioblastoma Cells

Figures

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  Figure 1. EV71 infection induced activation of NF-κB signaling. U251 cell was infected with EV71 for two and twenty-four hours, (a) Western immunoblotting analysis of ERK phosphorylation expression level at different times after infection. (b) p-ERK and GAPDH ratio by gray analysis of Western immunoblotting. (c,d) Inhibition of phosphorylation of ERK by knockdown of vimentin. (a,c) Vimentin knockdown U251 cells (KD-VIM) and wild type U251 cells (U251) were infected with EV71 for two and twenty-four hours. Western immunoblotting analysis ERK phosphorylation expression level. (d) p-ERK and β-actin ratio by gray analysis of Western immunoblotting. *** p < 0.0001.

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  Figure 2. EV71 induced the activation of NF-κB in U251 cell. (a,c) NF-κB (p65) and IKBα were examined after EV71 infection, comparing with P65 and IKBα expressions at zero hours, two hours and twenty-four hours, respectively. IKBα and NF-κB (P65) expression level detected by Western immunoblotting analysis. After infection, P65 was up-regulated dramatically in the nucleus, while the level of IKBα decreased within twenty-four hours (p < 0.01). (b,d) IKBα/NF-κB (p65) and β-actin ratio analysed by gray analysis of Western immunoblotting. *** p < 0.0001.

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  Figure 3. Phosphorylation of ERK mediates the activation of NF-κB signaling pathway in enterovirus 71 infection. (a) Inhibition of phosphorylation of ERK declined the level of NF-κB (p65) in the nucleus after EV71 infection. NF-κB (P65) expression level both in Control and ERK inhibitor (PD098059) groups by using Western immunoblotting analysis. (b) NF-κB (P65) and GAPDH ratio analyzed between the ERK inhibitor (PD098059) and control groups by gray analysis of Western immunoblotting. * p < 0.05, ** p < 0.01 and *** p < 0.0001.

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  Figure 4. Role of vimentin in EV71 (VP1)-induced NF-κB activation. (a,b) Role of vimentin in EV71-induced NF-κB activation in nucleus. The levels of NF-κB (p65) translocation to the nucleus were examined in nuclear fractions at zero hours, two hours and twenty-four hours. (a) The levels of NF-κB (P65) were detected by Western immunoblotting. (b) NF-κB (P65) and β-actin ratio analyzed by gray analysis of Western immunoblotting. (c–e) Role of vimentin in VP1-induced NF-κB activation. (c) The levels of IKBα and NF-κB (P65) detected by Western immunoblotting analysis, respectively. (d,e) IKBα or NF-κB (P65) and GAPDH ratio analyzed by gray analysis of Western immunoblotting, respectively. ** p < 0.01, *** p < 0.0001.

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  Figure 5. Vimentin and NF-κB signaling pathway are essential for the EV-71-induced inflammasome activation. (a) The expressions of NLRP3 and caspase-1 protein detected by Western blotting between VIM+/+ cells and VIM−/− cells. (b,c) Optical density analysis of NLRP3 and Caspase-1 by Western blotting. (d) Western blot detected the NLRP3 and caspase-1 protein expression among VIM+/+ cells, VIM−/− cells and CAPE cells. (e,f) NLRP3 or Caspase-1 and β-actin ratio analyzed by gray analysis of Western immunoblotting. * p < 0.05.

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  Figure 6. Vimentin mediates the CPE of EV-71 infection. (a) Quantitative real-time PCR assay of EV71 replication in VIM-WT and VIM-KD U251 cells. (b) The expression of vimentin in VIM-WT U251cells is much higher than that in VIM-KD U251 cells, analyzed by gray analysis of Western immunoblotting. (c) Morphological profiles of U251 cells (left: VIM+/+ cells infected with EV-71; middle: VIM−/− cells infected with EV-71; right: uninfected U251 cells). ** p < 0.01, *** p <0.0001.

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  Figure 7. A proposed schematic of EV71-mediated NLRP3 inflammasome activation via VIM-ERK-NF-κB pathway in U251 cells.