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

Development of a Method and Validation for the Quantitation of FruArg in Mice Plasma and Brain Tissue Using UPLC–MS/MS

Aged garlic extract (AGE) is a popular nutritional supplement and is believed to promote health benefits by exhibiting antioxidant and anti-inflammatory activities and hypolipidemic and antiplatelet effects. We have previously identified <i>N</i>-α-(1-deoxy-d-fructos-1-yl)-l-arginine (FruArg) as a major contributor to the bioactivity of AGE in BV-2 microglial cells whereby it exerted a significant ability to attenuate lipopolysaccharide-induced neuroinflammatory responses and to regulate the Nrf2-mediated antioxidant response. Here, we report on a sensitive ultraperformance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) protocol that was validated for the quantitation of FruArg in mouse plasma and brain tissue samples. Solid-phase extraction was used to separate FruArg from proteins and phospholipids present in the biological fluids. Results indicated that FruArg was readily absorbed into the blood circulation of mice after intraperitoneal injections. FruArg was reliably detected in the subregions of the brain tissue postinjection, indicating that it penetrates the blood–brain barrier in subnanomolar concentrations that are sufficient for its biological activity

The S-Nitrosylation Status of PCNA Localized in Cytosol Impacts the Apoptotic Pathway in a Parkinson’s Disease Paradigm

<div><p>It is generally accepted that nitric oxide (NO) or its derivatives, reactive nitrogen species (RNS), are involved in the development of Parkinson’s disease (PD). Recently, emerging evidence in the study of PD has indicated that protein S-nitrosylation triggers the signaling changes in neurons. In this study, SH-SY5Y cells treated with rotenone were used as a model of neuronal death in PD. The treated cells underwent significant apoptosis, which was accompanied by an increase in intracellular NO in a rotenone dose-dependent manner. The CyDye switch approach was employed to screen for changes in S-nitrosylated (SNO) proteins in response to the rotenone treatment. Seven proteins with increased S-nitrosylation were identified in the treated SH-SY5Y cells, which included proliferating cell nuclear antigen (PCNA). Although PCNA is generally located in the nucleus and participates in DNA replication and repair, significant PCNA was identified in the SH-SY5Y cytosol. Using immunoprecipitation and pull-down approaches, PCNA was found to interact with caspase-9; using mass spectrometry, the two cysteine residues PCNA-Cys81 and -Cys162 were identified as candidate S-nitrosylated residues. In addition, the evidence obtained from in vitro and the cell model studies indicated that the S-nitrosylation of PCNA-Cys81 affected the interaction between PCNA and caspase-9. Furthermore, the interaction of PCNA and caspase-9 partially blocked caspase-9 activation, indicating that the S-nitrosylation of cytosolic PCNA may be a mediator of the apoptotic pathway.</p></div

LPS induces Nrf2 and HO-1 protein expression in BV-2 microglial cells.

<p>Confluent cells were serum starved for 4 h prior to stimulation with LPS (100 ng/ml) and assay for Nrf2 and HO-1 expression at different times. (A, B, C). Time- dependent increase in Nrf2 and HO-1 protein expression after stimulation with LPS (100 ng/ml). Results are means ± SEM of three independent experiments. (D, E, F) Dose-dependent increase of Nrf2 and HO-1 protein expression by LPS after a 6 h incubation time. Results are means ± SEM of three independent experiments and were analyzed by one-way ANOVA followed by Bonferroni post-tests. ***p<0.001 vs. no LPS control.</p

Localization of PCNA in SH-SY5Y cells.

<p>(A) Localization of PCNA in the cytosolic and nuclear fractions of SH-SY5Y cells was examined by Western blot with antibodies against PCNA, Lamin A (nuclei marker), and aldose reductase (AR, cytosolic marker). (B) Localization of PCNA in SH-SY5Y cells was observed by confocal microscopy. Signal for aldose reductase served as cytosolic marker. (C) Effect to the localization of PCNA in SH-SY5Y cells by rotenone treatment was monitored by confocal microscopy with immunofluorescence using the PCNA antibody. DAPI was used as a nuclei stain.</p

Phenotype changes in SH-SY5Y cells in response to rotenone treatment.

<p>The dose (A) and time (B) responses of NO generated in SH-SY5Y cells that were treated with rotenone were analyzed. 18h treatment (A) and 500nM rotenone (B) were used for the gradient experiments. The NO contents are represented as the ratio of the intensity of DAF-FM fluorescence in the rotenone-treated group compared with the vehicle-treated group (average ratio ± SEM, n = 3, *P<0.05). Apoptosis assessment by flow cytometry for SH-SY5Y cells treated with or without 500nM rotenone for 16h (C). Dot plot showed annexin V-FITC in x-axis and PI in y-axis. Cells in the fourth quadrant undergoing early stage apoptosis are annexin V-positive/PI negative. And cells at late stage apoptosis or necrotic cells are both annexin V-FITC and PI positive. The left represents the untreated cells as the control. The apoptotic rates are shown as the average ratio ± SEM (n = 3).</p

Effects of quercetin on induction of Nrf2 and HO-1 proteins in the presence and absence of LPS.

<p>BV-2 microglial cells were cultured in 12-well plate. After confluent, cells were serum starved for 3 h followed by adding quercetin at different concentrations for 1 h and followed by stimulation with LPS (100 ng/ml) for 6 h. (A) A representative blot from 3–5 experiments. (B, C) Bar graphs represent Nrf2/actin and HO-1/actin ratios. Results are expressed as the mean ± SEM (n = 3–5) and analyzed by two-way ANOVA with Bonferroni post-tests (see text for details). “a” denotes significant differences between LPS+quercetin vs. quercetin alone; “b” denotes significant differences between LPS+quercetin vs. LPS alone; “c” denotes significant differences as compared to 0 μM quercetin.</p

Effects of quercetin and cyanidin on cell viability and NO production induced by LPS in BV-2 microglial cells.

<p>(A, D) <i>Chemical structures of quercetin and cyanidin</i>. (B, E) <i>NO production</i>. Cells cultured in 96 well-plate were serum starved for 3 h followed by treating with quercetin or cyanidin for 1 h and followed by stimulation with LPS (100 ng/ml) for 16 h. Aliquots of the culture medium was removed for measurement of NO by the Griess protocol. Results are expressed as the mean ± SEM (n = 3–5) and significant difference from the LPS-stimulated group was determined by one-way ANOVA followed by Bonferroni post-tests. *p<0.05, **p<0.01, ***p < 0.001. (C, F) <i>Cell viability</i>. For assay of cell viability, after aliquots of the culture medium were taken for assay of NO, 10 μl of WST-1 reagent was added and incubated for 2 h. Absorbance was read at 420–480 nm. Results are expressed as the mean ± SEM from three independent experiments. Data were analyzed by two-way ANOVA and no significant effects were found.</p

Effects of the S-nitrosylation status of PCNA on the interactions of PCNA and caspase-9.

<p>(A) Diagonal CoIP using two antibodies against PCNA and caspase-9 in SH-SY5Y cells. The interactions of PCNA and caspase-9 were negatively correlated with the NO contents in SH-SY5Y cells. L-NMMA was used for the inhibition of nNOS, and rabbit IgG was used as a negative control for immunoprecipitation. (B) The S-nitrosylation of recombinant PCNA, identified by BST/Western blot, using SNOC as a NO donor. (C) Comparison of the sensitivity of the potential cysteine residues of recombinant PCNA to S-nitrosylation under different NO stress levels. The sensitivity of cysteine residues to NO modification is represented as the ratios of the S-nitrosylated peptides identified by LC-MS/MS to the sum of the corresponding peptides, which include all S-nitrosylated and non-S-nitrosylated peptides at certain sites (n = 3, *P<0.05). (D) Effects of the PCNA mutants under NO stress on the interactions of PCNA and caspase-9. In the pull-down experiment, the wild-type PCNA and three PCNA mutants, PCNA-C81A,-C162A and-C81A/C162A, were treated with SNOC and incubated with the HeLa cytosol, followed by enrichment with nickel-agarose beads and detection with Western blot using an antibody against caspase-9. The left panel shows the Western blot image, and the right panel presents the interaction of caspase-9 with different SNOC-modified recombinant PCNAs. The relative immune-recognition intensities were estimated based on the ratios of the specific band volume against the total band volumes for caspase-9 in the upper panel (n = 3, *P<0.05 versus WT PCNA). (E) Comparison of the S-nitrosylated status of PCNA at Cys81 in SH-SY5Y cells with and without rotenone treatment. The S-nitrosylation status of PCNA at Cys81 is represented as the ratios of the S-nitrosylated Cys81 peptide to the sum of the peptides that contained Cys81, which were identified by LC-MS/MS (n = 3, *P<0.05).</p