Echo30 infection induced TRIO-RhoA signaling activation in neuronal cell.

<p>Human neuroblastoma SK-N-SH cells at 80% confluence were infected with Echo30 with 2% FBS contained MEM at 48 hrs. (A) Confirmation results of 2-DE using immunoblot blot analysis. TRIO protein expression was significantly increased in Echo30-infected SK-N-SH cells. (B) Immnoblot analysis for RhoA signaling. Echo30 infection induced activation of RhoA and RhoA targeted signaling in SK-N-SH cell. All data are representative of three independent experiments.</p

Echo30 infection induced neuronal cell death.

<p>Human neuroblastoma SK-N-SH cells at 80% confluence were infected with Echo30 with 2% FBS contained MEM at 48 hrs. (A) The effect of Echo30 infection on SK-N-SH cells. Echo30 infected SK-N-SH cells significantly increase cytopathic effect. (B) Measurement of cell viability by WST-1 assay. The viability of SK-N-SH cells was decreased by Echo30 infection (mean±S.E.M, *<i>p</i><0.05). (C) Measurement of sub-G1 phase using flow cytometry analysis. Echo30 infection increase sub-G1 phase at cell cycle distribution. (D) Expression of apoptotic proteins using immunoblot analysis. The expression level of pro-apoptotic protein was increased by Echo30 infection. All data are representative of three independent experiments.</p

Protein identification of differentially expressed cellular proteins in Echo30-infected SK-N-SH cells at 48 hrs by two-dimensional gel electrophoresis (2-DE).

<p>(A) The arrows indicated the protein spots identified as differentially regulated at greater than four-fold changes (<i>p</i><0.05). (B) The spot density was represented relative to the control (non-infected SK-N-SH cells).</p

Identification of differentially expressed proteins in Echo30-infected SK-N-SH cells at 48 hrs by MALDI-TOF.

*<p>n/a⁺: Detected only in SK-N-SH cells infected with Echo30.</p

Echo30 infection enhanced NO production through TRIO-RhoA signaling activation in neuronal cells.

<p>(A) Echo30-infected SK-N-SH cells increase NOS family (eNOS, iNOS, nNOS) protein levels using immunoblot analysis. Echo30 infection enhanced NOS family protein levels. (B) Measurement of cellular NO production by griess reagent system. Echo30-infected SK-N-SH cells significantly increase cellular NO level (mean±S.E.M, *<i>p</i><0.05). (C) The effect of Rho kinase inhibitor Y27632 on NOS family induction by Echo30 infection. Pre-treatment of cells with the Y27632 prevent NOS family induction by Echo30 infection. (D) The effect of Y27632 on NO production by Echo30 infection. Pre-treatment of cells with the Y27632 blocked NO production by Echo30 infection (mean±S.E.M, *<i>p</i><0.05). All data are representative of three independent experiments.</p

Rho kinase inhibitor prevented neuronal cell death by Echo30 infection.

<p>(A) The effect of Y27632 on the cytopathic effect by Echo30 infection. The pretreatment of Y27632 inhibited Echo30 infection-inducing cytopathic effect in SK-N-SH cells. (B) The effect of Y27632 on cell viability by Echo30 infection determined by WST-1 assay. Y27632 inhibited Echo30 infection-inducing SK-N-SH cell death. (mean±S.E.M, *<i>p</i><0.05). (C) The effect of Y27632 on cell cycle analysis by Echo30 infection using FACS analysis. Y27632 inhibited increase of sub-G1 phase by Echo30 infection in SK-N-SH cells. (D) The effect of Y27632 on RhoA signaling by Echo30 infection using immunoblot analysis. Y27632 prevented Echo30 infection induced activation of RhoA signaling. (E) Graphical representation of densitometric analysis of the immnoblot bands for TRIO-RhoA signaling (mean±S.E.M, **<i>p</i><0.05, *<i>p</i><0.1). All data are representative of three independent experiments.</p