2 Endogenous levels of total p53 protein in mature MO3

2 Endogenous levels of total p53 protein in mature MO3.13 cells 48 h after infection with (Bb, MOI-10:1) with or without the MEK1/2 inhibitor U0126 (10 M). suppression of ERK or p53 reduced apoptosis. It is possible that inflammation and apoptosis in oligodendrocytes are divergent arms of MAPK pathways, particularly the MEK/ERK pathway. contamination and to showed an upregulation of IL-6, CXCL8, IL-1 and CXCL13 as well as apoptosis of neurons and oligodendrocytes [24]. Similarly, intrathecal inoculation of into the cisterna magna of rhesus macaques resulted in elevated levels of IL-6, CXCL8, CCL2 and CXCL13 in the CSF, multifocal leptomeningitis, radiculitis and inflammatory lesions in the dorsal root ganglia (DRG) with concomitant neuronal and satellite cell death through apoptosis [25]. Subsequent studies have indicated that apoptosis of CNS neurons S63845 occurs only in the presence of microglia and [26] while oligodendrocyte apoptosis can occur in the presence alone with no other S63845 cell involvement [27]. S63845 In both these studies, an intense inflammatory environment was present, again supporting the hypothesis that neuronal or glial loss occurs in the context of an inflammatory milieu. Moreover, both inflammation and apoptosis of oligodendrocytes was mitigated strain B31 (clone 5A19) was used for all contamination assays. was routinely cultured under microaerophilic conditions in Barbour-Stoenner-Kelly (BSK-H) medium (Sigma Aldrich, St. Louis-MO) supplemented with Amphotericin (0.25 mg/mL), Phosphomycin (193 mg/mL) and Rifampicin (45.4 mg/mL) for about 5-7 days. At the end of the time period and on the day of contamination, bacterial concentration was determined using a dark-field microscope. Required numbers of bacteria were harvested at 2095 g for 30 minutes at room heat (RT) without brakes and resuspended in experimental medium containing DMEM-high glucose (Invitrogen/Life Technologies, Inc., Grand Island-NY) and 100 nM phorbol myristate acetate (PMA) (Sigma Aldrich, St. Louis-MO) and diluted further to the required multiplicity of contamination (MOI). Cell culture Cells from the human oligodendrocyte cell line MO3.13 (CELLutions Biosystems Inc., Ontario, Canada) were cultured according to the manufacturer’s protocol. Briefly, cells were produced in complete medium made up of DMEM (high glucose) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) at 37 C, 5% CO2. After confluency, cells were trypsinized, collected, and seeded at the required density (0.8 104/ well for 6-well plates, 1 105/T-75 flask and 0.5 104/well for 2-well chamber slides). After day 3, cells were allowed to differentiate for 3 days further by replacing the complete medium with medium CD247 devoid of serum and supplemented with 100 nM PMA and 1% P/S (differentiation medium). Cells produced accordingly, as per the manufacturer, stain positive for markers such as myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG), which are phenotypic markers of mature, myelinating oligodendrocytes [28]. Such differentiated and mature oligodendrocytes were used for all of the experiments described. For all of the experiments with were carried out in OPCDM without P/S and processed for ELISA and TUNEL as described in the following paragraphs. Contamination assays and pathway inhibitors For the infection assays, diluted to the appropriate MOI in either experimental medium or in OPCDM without P/S was added to the mature, differentiated MO3.13 cells or to the primary oligodendrocytes respectively, for various S63845 time intervals. Viability of after 48 h in experimental medium was confirmed by re-culturing in BSK-H medium as well as by live/lifeless bacterial viability assay kit (Invitrogen/Life Technologies, Inc., Grand Island-NY). To determine the role of various pathways, several pathway inhibitors were added 2 h prior to bacterial addition and co-incubated for the duration of contamination. The following inhibitors (EMD Millipore, Billerica, MA) were used: p38 (SB203580), MEK1/2 (U0126), JNK (SP600125), NFkB p65 (JSH 23), IKK-1/2 (inhibitor XII), p53 (pifithrins and ). Supernatants were collected at specified intervals, centrifuged at 2095 g, 10 minutes at 4 C to remove bacteria and cellular debris, aliquoted and stored at ?20 C until analysis by Multiplex Enzyme-Linked Immuno Sorbent Assay (ELISA) for chemokines and cytokines. S63845 Preparation of cell lysates Cell lysates were extracted according to the manufacturer’s protocols (Cell Signaling Technology, Boston, MA). Briefly, medium was removed, oligodendrocytes were washed once with ice-cold PBS and incubated on ice for 5 minutes in lysis buffer made up of 1mM phenyl methyl sulfonyl fluoride, a serine protease.