Additional file 2: Figure S2. of Cytosolic phospholipase A2 plays a crucial role in ROS/NO signaling during microglial activation through the lipoxygenase pathway

cPLA2 protein expression level decreased significantly after siRNA knockdown. Representative blot demonstrating protein levels of cPLA2 and β-actin in BV-2 cells between groups: (1) control, (2) BV-2 cells were transfected with negative control siRNA for 24 h, and (3) BV-2 cells were transfected with siRNA against cPLA2 for 24 h

Additional file 1: Figure S1. of Cytosolic phospholipase A2 plays a crucial role in ROS/NO signaling during microglial activation through the lipoxygenase pathway

High concentrations of LPS and IFNγ were toxic to primary microglia at 24 h post-stimulation. Primary microglial cells were treated with various concentrations of either (A) LPS or (B) IFNγ. Twenty-four hours later, cell viability was measured with the WST-1 protocol as described in the text. Results were expressed as the mean ± SEM (n = 3) and significant difference compared with the control group was determined by one-way ANOVA followed by Dunnett’s post-tests, **P < 0.01, ***P < 0.001

TNFα alters occludin and cerebral endothelial permeability: Role of p38MAPK

<div><p>Occludin is a key tight junction (TJ) protein in cerebral endothelial cells (CECs) playing an important role in modulating blood-brain barrier (BBB) functions. This protein (65kDa) has been shown to engage in many signaling pathways and phosphorylation by both tyrosine and threonine kinases. Despite yet unknown mechanisms, pro-inflammatory cytokines and endotoxin (lipopolysaccharides, LPS) may alter TJ proteins in CECs and BBB functions. Here we demonstrate the responses of occludin in an immortalized human cerebral endothelial cell line (hCMEC/D3) to stimulation by TNFα (10 ng/mL), IL-1β (10 ng/mL) and LPS (100 ng/mL). Exposing cells to TNFα resulted in a rapid and transient upward band-shift of occludin, suggesting of an increase in phosphorylation. Exposure to IL-1β produced significantly smaller effects and LPS produced almost no effects on occludin band-shift. TNFα also caused transient stimulation of p38MAPK and ERK1/2 in hCMEC/D3 cells, and the occludin band-shift induced by TNFα was suppressed by SB202190, an inhibitor for p38MAPK, and partly by U0126, the MEK1/2-ERK1/2 inhibitor. Cells treated with TNFα and IL-1β but not LPS for 24 h resulted in a significant (p < 0.001) decrease in the expression of occludin, and the decrease could be partially blocked by SB202190, the inhibitor for p38MAPK. Treatment with TNFα also altered cell morphology and enhanced permeability of the CEC layer as measured by the FITC-dextran assay and the trans-endothelial electrical resistances (TEER). However, treatment with SB202190 alone could not effectively reverse the TNFα -induced morphology changes or the enhanced permeability changes. These results suggest that despite effects of TNFα on p38MAPK-mediated occludin phosphorylation and expression, these changes are not sufficient to avert the TNFα-induced alterations on cell morphology and permeability.</p></div

Schematic description of transendothelial permeability assays.

<p>(A) Diagram depicting method for Dextran assay protocol. (B) Diagram depicting the measurement using the TEER protocol.</p

TNFα-mediated occludin band-shift and phosphorylation of ERK1/2 and p38MAPK in hCMEC/D3 cells.

<p>Cells were treated with or without TNFα (10 ng/mL) at 5, 10, 15, 30, 60 min (A). Cell lysates were collected and occludin, P- ERK, T-ERK, P-P38, T-P38 and β-actin expression pattern was analyzed by immunoblotting assay. Quantification of the proportion of occludin upper band were determined through PI_upper/PI_total/PI_β-actin and then normalized to control (B). phospho-ERK1/2 (C) and phospho-p38MAPK (D) were similarly quantified and plotted. Results are mean ± SD from 3 or more experiments and data are analyzed by one-way ANOVA followed by Bonferroni post-tests (**P<0.01, ***P<0.001 compared with no treatment control).</p

Effects of TNFα, IL-1β and LPS on morphology of hCMEC/D3 cells.

<p>(A) Cells were treated with or without TNFα (10 ng/mL), IL-1β (10 ng/mL) and LPS (100 ng/mL) for 24 hours and observed under bright field microscope as described in text. Representative pictures were taken from different areas in the field. (B) Ten cells from each picture were randomly selected for measurement using the Image J protocol. Results are mean ± SD from 3 experiments. One-way ANOVA with Bonferroni post-test showed a significant difference (p<0.01) between control and TNFα. (C) Representative bright field photomicroscope pictures to assess effects of MEK1/2 and p-p38MAPK inhibitors on TNFα-induced morphological changes. Cells were pretreated with U0126 (2 μM) and SB202190 (2 μM) for 15 min prior to treatment with TNFα (10 ng/mL) for 24 h. (D) Results are analyzed as in (B). Data are expressed as the mean ± SD of three experiments. Two-way ANOVA showed a significant main effect of TNFα (p = 0.0013). The effects of the inhibitors were not significant.</p

Effects of TNFα, IL-1β and LPS on occludin band-shift in hCMEC/D3 cells.

<p>Cells were treated with or without TNFα (10 ng/mL) (A), IL-1β (10 ng/mL)(B) and LPS (100 ng/mL) (C) for 15, 30 min and 1, 2, 4, 6 hours. Cell lysates were collected and occludin and β-actin expression patterns were analyzed by immunoblotting assay as described in text. The density of upper band versus total upper and lower and normalized with β-actin was determined by measuring protein intensity as PIupper/PItotal/PIβ-actin. Results are mean ± SD from 4 or more experiments and data are analyzed by one-way ANOVA followed by Bonferroni post-tests (***P<0.001 compared with no treatment control).</p

Effects of TNFα on tyrosine and threonine phosphorylation of occludin.

<p>hCMEC/D3 cells were treated with or without 10 ng/mL TNFα for 15 min. Cell lysates were mixed with anti-tyrosine or anti-threonine antibody conjugated with protein A beads and then phosphorylated proteins in cell lysates were pulled down. Occludin and β-actin expression pattern was analyzed by immunoblotting assay.</p

Effects of TNFα, IL-1β and LPS on p-ERK1/2 and p-p38MAPK expression in hCMEC/D3 cells.

<p>Cells were treated with or without TNFα (A, B, C), IL-1β (D) and LPS (E) for 15, 30 min and 1, 2, 4, 6 hours. Cell lysates were collected and phospho-ERK1/2 (P- ERK), total ERK1/2 (T-ERK), phospho-p38MAPK (P-P38), total p38MAPK (T-P38) and β-actin expression pattern were analyzed by immunoblotting assay. Quantification of phospho-proteins was determined through assay of PIphospho/PItotal/PIβ-actin and then normalized to control. Results of (B) and (C) are mean ± SD from 4 or more experiments and data are analyzed by one-way ANOVA followed by Bonferroni post-tests **P<0.01, ***P<0.001 compared with no treatment control). Results of (D) and (E) are representation of two repeated experiments.</p

Effects of TNFα, IL-1β and LPS on paracellular permeability as measured by the Dextran and TEER assays.

<p>(A) For the Dextran assay, cells were cultured in inserts for 24 h followed by applying fluorescent FITC-Dextran beads as described in Methods. (B) TEER was determined using an endothelial volt/ohm meter for TEER-EVOM2 as described in Methods. Permeability were determined through FI24h/FI0min (Fluorescence Intensity) and then normalized to control. Results are mean ± SD from 4 or more experiments and data are analyzed by one-way ANOVA followed by Bonferroni post-tests (*P<0.05, ***P<0.001 compared with no treatment control). (C and D) Assessing effects of p-ERK1/2 and p-p38MAPK inhibitors on TNFα-induced changes on paracellular permeability as measured by the Dextran (C) and TEER assays (D). Data are expressed as the mean ± SD of four or more experiments. The results were analyzed by two-way ANOVA, and a significant main effect of TNFα was revealed (p<0.0001 for each). The effects of the inhibitors were not significant.</p