by Agol, V. first infects the oropharynx and the digestive tract and then spreads to the central nervous system (CNS), in which it targets mostly motor neurons. Studies with mouse models have shown that PV-infected motor neurons in the spinal cord die by apoptosis (10, 19). PV-induced apoptosis therefore seems to play a major role in the tissue injury occurring in the CNS. PV triggers apoptosis in vitro in tissue cultures of human colon carcinoma (Caco-2) cells (4), promonocytic cells (U937) (29), dendritic cells (41), murine Resminostat hydrochloride L cells expressing CD155 (21, 36), HeLa cells (8, 39), and cultures of mixed mouse primary nerve cells (12) from the cerebral cortexes of mice transgenic for CD155. Analyses of the apoptotic pathways induced following PV infection in several cell lines have demonstrated that mitochondria are key actors of PV-induced apoptosis. In particular, mitochondrial outer membrane permeabilization (MOMP) following PV infection leads to a loss of mitochondrial transmembrane potential and the release of proapoptotic molecules, including cytochrome family, has recently been investigated. PV activates the PI3K/Akt survival signaling Mouse monoclonal to ERBB3 pathway in IMR5 cells. We began by determining whether PV infection of IMR5 neuroblastoma cells resulted in Akt activation. IMR5 cells were infected with PV as previously described (6). Briefly, the growth medium (Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum) was discarded. The virus was then added to monolayers at a multiplicity of infection (MOI) of ten 50% tissue culture infective dose units Resminostat hydrochloride (TCID50) per cell (this MOI was used for all assays performed in this study). Adsorption was allowed to proceed for 30 min at 37C in humidified air containing 5% CO2. The cells were then washed twice with serum-free medium to remove unbound particles and incubated with fresh Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37C. The virus was allowed to grow for the indicated times. Time zero postinfection (p.i.) corresponds to the inoculation time point. Mock-infected cells were used as negative controls. As previously described (6), both adherent and detached cells were taken into account in all experiments. Kinetics of Akt phosphorylation at Ser473, which is required for full Akt activation (3), was investigated in mock- and PV-infected cells. Whole-cell lysates were analyzed at the indicated times p.i. by Western blotting with a specific anti-phospho (Ser473)-Akt antibody (Fig. ?(Fig.1A).1A). We checked for equal protein loading on the total Akt Western blot. The amount of phosphorylated Akt increased until 30 min p.i. and then decreased; at 4 h p.i., the amount of phosphorylated Akt present was similar to that in mock-infected cells analyzed at the same time point. To check that the virus stock used in this study did not contain host-derived components that may activate the Akt signaling pathway, we depleted the virus suspension of PV using an anti-PV antibody and infected cells with either the depleted or nondepleted suspension system. As opposed to cells contaminated using the nondepleted share, no Akt activation (30 min p.we.) was discovered in cells Resminostat hydrochloride treated using the depleted suspension system (Fig. ?(Fig.1A,1A, bottom level still left). We also examined that poliovirus purified by isopycnic CsCl gradient centrifugation (9) could promote Akt activation (30 min p.we.) at an performance very similar to that attained with the Resminostat hydrochloride trojan preparations found in this research (Fig. ?(Fig.1A,1A, more affordable right -panel). We after that looked into whether Akt activation in response to PV an infection happened through the PI3K pathway by dealing with IMR5 cells with a particular PI3K inhibitor, wortmannin (5), at concentrations of 100 nM and 500 nM 2 h before these were mock or trojan contaminated. The concentrations from the inhibitor were preserved through the adsorption PV and period infection. Cell lysates had been gathered 30 min after an infection and put through Traditional western blot evaluation for the recognition of Akt phosphorylation (Fig. ?(Fig.1B,1B, best -panel). Wortmannin inhibited Akt phosphorylation at both concentrations without changing total Akt amounts. The activation of Akt in response to PV an infection was illustrated by immunofluorescence.
