Figure 5B shows that these cell http://www.selleckchem.com/products/SB-203580.html lines express the long 50 UTR MDR1 mRNA and, furthermore, that TSA treatment decreased the levels of expression of the long 50 UTR MDR1 mRNA in all of them. Even more, we used K 562 cell sublines resistant to different con centrations of daunomycin that have been generated in our laboratory by selective pressure with increasing con centrations of the drug. We have previously shown that resistance to daunomycin in this sublines was related to the active expression of P glycoprotein, and that this ex pression correlates with the expression of the long 50 UTR MDR1 mRNA. As shown in Figure 5C, TSA was able to increase daunomycin accumulation in the K 562 d20, a subline that expresses P glycoprotein and the long 50 UTR MDR1 mRNA associated with the USP promoter.

This result suggests that TSA decreases P glycoprotein ex pression leading to an increased accumulation of dauno mycin in these cells. Futhermore, as shown in Figure 5C, TSA downregulated MDR1 mRNA, as determined by real time PCR in K 562 d450, a cell subline which expresses high levels of P glycoprotein and also the long 50 UTR MDR1 mRNA. Meanwhile, TSA increased the expression of the MDR1 mRNA in the parental K562 that does not express P glycoprotein and expresses the short 50 UTR MDR1 mRNA. TSA effect on RUNDC3B mRNA levels The ABCB1 gene is located in a genetic locus with the nested gene RUNDC3B in the complementary DNA strand . Based on this special localization, we investigated whether the expression of RUNDC3B mRNA was regulated by TSA and whether the expression of this mRNA was related to the expres sion of the short or the long 50UTR of the MDR1 mRNA.

As shown in Figure 6B, TSA upregulates RUNDC3B mRNA expression levels independently of the MDR1 mRNA isoform expressed in the different cell lines. Discussion In the present study, we demonstrate that the increase in MDR 1 mRNA levels induced by iHDACs inhibitors in pancreatic adenocarcinoma cell lines does not parallel an increase in Pgp protein or in Pgp activity. Anacetrapib This obser vation is important since we and others have reported that the histone deacetylase inhibitors TSA and SAHA induce two major effects in several drug resistant cell lines down regulation of Pgp and induction of apop tosis. In that sense, we have demonstrated that TSA markedly reduced Pgp expression in the L1210R drug resistant murine cell line and sensitized these cells to daunomycin, as shown in Figure 1C, where TSA treatment increases DNM accumulation in L1210R cells, suggesting that iHDACs might have a therapeutic potential against chemoresistant tumours.

This is attrib uted to decreased cytoadherence at lower body tempera ture, which assists anti malarial drugs in clearing ring stage parasites www.selleckchem.com/products/GDC-0449.html before they mature and cytoadhere. Fur ther experimental work is necessary to study if this is due to the effect of antipyretics on the trafficking of ETRAMP and KAHRP resulting in decreased PfEMP1 presentation. Activated platelets act as bridges between pRBCs and endothelial cells, allowing the binding of pRBC to the endothelium devoid of cytoadherence receptors. The MSP 1 gpIIIa interaction might play a crucial role in platelet activation, either via complex formation with MSP 6 MSP 7 or through contact with pRBCs, thus influencing systemic inflammation in CM. Antibody mediated blocking of the activity of gpIIb IIIa in vitro has revealed decreased platelet activation.

Other studies also show lowered antibody response to MSP 1 MSP 6 MSP 7 in CM patients when compared with non affected patients. Lowered antibody response to GPI anchor has also been reported in CM non survivors when com pared to the CM survivors. Hence, the MSP 1 gpIIIa interaction could indicate a novel mechanism of platelet activation by MSP 1. Platelet activation is also associated with tissue factor expression on the platelet surface. TF expression leads to amplification of the coagula tion cascade resulting in the consumption of coagulation factors. Platelet activation via MSP 1 and gpIIIa might thus also play a role in TF expression on the platelet sur face causing amplification of the coagulation cascade, leading to haemostasis dysfunction.

Haemostasis dysfunction during CM culminates in increased endothelial hemorrhage resulting in leakage of plasma proteins, proinflammatory cytokines and parasite factors across the BBB. This influx of foreign substances activates the microglial cells, resident macrophages of the brain and spinal cord. Upon activation, they release proinflammatory cytokines which damage astrocytes and glial cells that are crucial for BBB maintenance. In addition to the damage caused by this cytotoxic environ ment, it was hypothesized that the interaction between HSA and the TGF B receptors TGFBR1 and TGFBR2 could result in astrocyte dysfunction, followed by sei zures and neuronal death. When mapping of the interactome, an interesting observation was the dual behaviour of TGF B.

On the one hand, it has a protective effect on the host during the pathogenesis of CM due to its anti inflammatory prop erty. During the release of TGF B mediated by para site derived products such as PfTRAP, TGF B down regulates the proinflammatory cytokine TNF and up reg ulates the anti inflammatory GSK-3 cytokine IL 10. On the other hand, activated platelets locally release TGF B that synergizes with TNF in creating a proinflammatory con dition leading to BBB disruption. Increased activa tion of TGF B during early stages of the disease leads to reduced parasite clearance time.

It free overnight delivery is estimated, after 60 years of age, more than 50% people suffers from colon polyps. The phenotypes of colon polyps include hyperplastic polyps, inflammatory polyps and adenomas polyps. Certain types of colon polyps grow large and fast and become cancerous. Aden omas polyps account about 50% colon polyps. How the polyp epithelium differentiate into cancer tissue is still unclear. P53 protein is a cancer suppressor protein, it is encoded by the TP53 gene in human. P53 protein is a crucial regu lator of cell cycle and apoptotic process in the cell, it func tions in the cancer prevention. The gene expression disorders of p53, including mutations in exon 7, codon 245, conserved areas, and the L3 struc tural domain, are associated with the pathogenesis of colon cancer.

To date, the factors causing p53 suppression are still to be investigated. Recent studies indicate the ubiquitin E3 ligase A20 plays a critical role in the immune regu lation as well as in associating with the pathogenesis of cancer. By promoting the tolerogenicity in dendritic cells, A20 plays a role in the induction of immune toler ance, which is a crucial drawback in cancer prevention in the body. A20 and other ubiquitin E3 ligases may be involved in the suppression of p53 function. In this study, we found that the adenomas and hyperplastic colon polyps had high levels of A20, which was signifi cantly correlated with the tumorigenesis of colon polyps. Methods Reagents The antibodies of A20, p53 were purchased from Santa Cruz. The reagents for real time RT PCR, Western blotting, A20 over expression and immune precipi tation were purchased from Invitrogen.

The HEK293 cells were purchased from China Cell Line. MG132 was purchased from Sigma Al drich. Recombinant A20 and p53 proteins were purchased from R D Systems. Patients Patients with colon cancer, non cancer colon polyp and IBS were recruited into this study from 2005 to 2012 at our department. The diagno sis was carried out by their physicians and pathologists. After diagnosis, the colon polyps were removed by their surgeons under colonoscopy. The colon cancer tissue and polyp epithelium were collected in the operation room. Biopsies from IBS patients were obtained under colonoscopy. The tissue was processed for the RNA and protein extraction immediately after collection, the extracts were stored at 80 C until use.

The using human tissue in this study was approved by the Human Research Ethic Committee of the China PLA General Hospital. The written, informed con sents were obtained from each patient. Follow up All the patients with colon polyps were required to do follow up visits every three months after the colonos copy surgery. Quantitative real time RT PCR Total RNA was extracted from the collected cancer tis sue and polyp epithelium using Trizol reagent according to AV-951 the manufacturers instructions.

However, there are no VIT or vWA domains found in these proteins. www.selleckchem.com/products/INCB18424.html vPARP is associated with vaults, very large cytoplasmic ribonucleoprotein particles first described in the 1980s whose function is unclear. Vaults have a patchy taxonomic distribution within eukaryotes. Our analysis suggests that the phylogenetic distribution of vPARP is also limited, members of Clade 5A with the vPARP domain structure are found only in ani mals that have been shown to contain vaults, while Clade 5B proteins are found in Dictyostelium, which also contains vaults. However, although vaults have been identified in trypanosomes, no evidence of proteins sharing the domain structure of vPARP can be found in this group of organisms, although such pro teins may be present in species with currently unse quenced genomes.

mART activity may be ancient Clade 6 proteins are found in Opisthokonts, Excavates, and Plantae. Based on its position as sister group to all other clades of PARPs and the distribution of species containing Clade 6 PARPs within the eukaryotes, it is likely that the last common eukaryotic ancestor had at least one Clade 6 like protein encoded in its genome. This clade is charac terized by N termini with no known functional domains and C terminal extensions beyond the PARP catalytic domain of varying lengths. Almost all of these proteins contain a PfamB 2311 domain immediately before their PARP catalytic domain, although the function or significance of this domain is unknown, supporting the placement of these proteins in a single clade. Another characteristic of Clade 6 members is changes within the PARP catalytic domain.

None of the Clade 6 proteins we identified contain the final glutamic acid of the HYE catalytic triad, although they mostly retain the histidine and tyrosine. This might lead to an inability to catalyze poly ation. In fact, the human proteins in this clade have been predicted to have mono ation activity based on structural models, although this awaits experimental confirmation. None of the Clade 6 PARPs have been functionally characterized. Clade 6 can be subdivided into five groups. Clade 6A contains fungal proteins exclusively. These proteins consist Dacomitinib of a long N terminal region containing no known functional domains, a PfamB 2311 domain, the PARP catalytic domain, and a C terminal extension containing an UBCc. The UBCc domain is the catalytic domain contained in E2 Ub conjugating enzymes. These enzymes carry Ub and transfer it either directly to a substrate in cooperation with an E3 enzyme or to the E3 Ub ligase. An active cysteine residue characterizes the UBCc domain and is found in Clade 6A proteins. In addition, these proteins also share a number of residues conserved across a range of UBCc and UBCc like domains.

The mixture was then transferred to a streptavi din different coated plate. The bound NF B transcription factor subunits p50 and p65 were detected with specific primary antibodies. A horseradish peroxidase conjugated second ary antibody was then used for chemiluminescent detec tion. The relative light unit values were measured using a LUMIstar Omega microplate reader. Statistical Analysis Differences between groups were assessed by using one way ANOVA with the SPSS 13. 0 program, with a probalility value of P 0. 05 considered indic ative of statistically significance. Results MTT Cell Viability Studies To exclude the possibility that changes in the barrier func tion resulted from cell death and the subsequent forma tion of holes in the monolayer, we tested the cytotoxic effects of 100 nM Tat on D407 cells.

As shown in Figure 1, the average absorbance at 490 nm did not differ signifi cantly between the control and treatment groups, indicat ing that the exposing cells to 100 nM Tat for 24 72 hours did not decrease cell viability relative to controls. HIV 1 Tat Induces destruction of barrier function in RPE The TER appeared to be somewhat affected by the serum, so we reduce the serum concentration of the medium to 1% from day 3 when cells reached confluence, and meas ured the TER every other day. The TER of D407 cells grad ually increased on the subsequent days, peaking at day 8 and then remaining stable for 1 week. Mennel suggested that obtaining stable values on 2 subse quent days indicated the formation of a tightly coupled cell monolayer, and hence we decided to begin treating the cells with 100 nM Tat from day 10.

The TER of D407 cells was measured at 1, 2, 3, 12, 24, 48, and 72 hours after treatment with 100 nM Tat. A reduc tion in the TER was first evident after 3 hours of treatment. Continuous culturing of cells for longer peri ods further reduced the TER, with a maximum effect after 24 hours of treatment that was maintained to 72 hours. The TER of control groups remained unchanged throughout the experiment. The permeability to sodium fluorescein, which has a low molecular weight, is regarded as a reliable marker of para cellular permeation. The permeability values of cells as measured at 20, 40, and 60 min after treatment with 100 nM Tat for 24 hours were all significantly higher than those of cells in the standard medium and the Hi Tat con tained medium, indicating that treating D407 cells with 100 nM Tat for 24 hours induced a loss of junctional integrity.

HIV Dacomitinib 1 Tat Induces Genes and Proteins Expression of TJs in RPE The real time quantitative reverse transcriptase polymer ase chain reaction demonstrated that occludin and clau din 1 to 4 were expressed in D407 cells, whereas there was no expression of claudin 5, similar to those from studies on claudins in another RPE cell line ARPE19.

Whole genome expression studies have shown that some of these programs are needed for basal homeo static cellular functions, while others are specific for cog nitive functions. The composition and regulation of most transcriptional programs however may depend on the strength and duration of training. Its well known, for ex ample, that practice or repeated training of a skill or con cept can improve memory for the subject. Multiple training sessions required to form strong memory traces may, therefore, be associated with increased gene expres sion or the reinforcement of existing transcriptional pro grams, such as those necessary for structural changes to strengthen synaptic circuits. How this is induced at the level of chromatin and which genes are targeted by epigenetic processes remains poorly understood.

With the emergence of the post genomic era, recent studies in the field of learning and memory have investi gated the implication of chromatin remodeling in cognitive processes. Several studies have revealed that chromatin re modeling plays a critical role in memory formation. Chromatin remodeling is a complex molecular and structural process that involves the dynamic regulation of nucleosomes through different epigenetic mechanisms including histone posttranslational modifications, DNA methylation and RNA interference. In the ro dent brain, several histone PTMs are rapidly induced and are associated with altered gene transcription following training.

Acetylation of lysine 9 and 14 on H3, of lysine 5, 8 and 12 on H4, and of lysine 5, 12, 15, and 20 on H2B, in creases in the hippocampus following contextual fear con ditioning, a well established behavioral paradigm for the establishment of contextual fear memory. Moreover, inhibition of histone deacetylases by HDAC inhibitors such as suberoylanilide hydroxamic acid, sodium butyrate, valproic acid or trichostatin A can enhance memory and rescue deficits in contextual memory in rodents. Although these studies provide strong evidence that his tone acetylation is modulated by memory formation, a global assessment of histone acetylation at the level of the genome and the mechanism with which it regulates gene expression in memory processes is lacking.

Using a genome wide approach, we examined the distribution of H4K5ac, a mark of active chromatin implicated in tran scriptional re activation of post mitotic cells through gene bookmarking, and its role in regulating Cilengitide transcrip tional activity following the establishment of contextual fear memory in the adult mouse. We propose that gene bookmarking may also be relevant in the hippocam pus following learning, whereby genes may be primed for rapid induction through activity induced histone acetyl ation. Using chromatin immunoprecipitation followed by deep sequencing and bioinformatics analysis, we show that H4K5ac in the hippocampus is prevalent throughout the genome and is a mark characteristic of ac tively transcribed genes.

Other viral infections including the 1918 influenza virus, hepatitis C virus and Ebola virus suppress type I IFN gene expression, leading to exten sive viral Pacritinib order replication and increased pathogenesis. IRF3 plays an important role in typeI IFN gene expression and the present study demonstrated that IRF3 gene expression was suppressed during H PRRSV infection. This result is in agreement with a previous study reporting that PRRSV NSP1b inhibited IRF3 and NF B transactivation, and down regulated IFN b gene expression. This suggested that NSP1b mediates subversion of the host innate immune response and plays an important role in PRRSV pathogenesis. Further more, influenza A NSP1 can suppress innate immunity by inhibiting activation of IRF3, and subsequently dis rupting the induction of a b interferon.

Many viruses induce apoptosis in infected cells but some can block the apoptosis pathway, leading to pro longed life of the cell and an increase in the yield of progeny virions. H PRRSV up regulated expression of anti apoptotic genes and down regulated expression of some pro apoptotic genes in H PRRSV infected lungs. MCL1, BFL 1, putative inhibitor of apopto sis, ADM and IL10 were up regulated. MCL1 and BFL 1 belong to the BCL 2 subfamily, which negatively regu lates apoptosis and blocks the apoptosis pathway, ADM is an anti apoptotic peptide, and IL10 protects cells against apoptosis. The pro apoptotic genes APR 1, p53 protein, SARP 3, and NDK H 5 were down regulated to prevent the occurrence of apoptosis.

Batimastat These findings indicate that H PRRSV could induce an anti apoptotic state to prolong the life span of infected cells and increase the yield of progeny virions. IL10 could have an important role in the regulation of the immune response to PRRSV. Up regulation of IL10 gene expression has been demonstrated in PRRSV infected porcine leukocytes, alveolar macrophages, den dritic cells, and in vivo in PRRSV infected pigs. Incubation of freshly isolated CD14 positive cells with IL10 during differentiation increased susceptibility to PRRSV infection and was correlated with up regulation of CD163 on the cell surface. This suggests that IL10 plays an important role in CD163 up regulation and susceptibility to PRRSV during differentiation of macrophages in vivo. CD163 alone can confer PRRSV replication on a non permissive pig cell line and its expression on macrophages in vivo could determine the efficiency of replication and subsequent pathogenicity of PRRSV. It is possible that internalization of H PRRSV via CD163 on the target cells could induce expression of IL10 and subsequently induce the expres sion of CD163 on neighboring undifferentiated mono cytes, increasing overall susceptibility to PRRSV.

The third component of the yeast adaptation response to HMF involves selleck chem Vorinostat degradation of selleck compound damaged proteins and protein modifications mainly regulated by transcription factor genes RPN4 and HSF1. Chemical stress causes damage to protein conformation leading to protein unfolding and aggregation. Small heat shock pro teins, acting as chaperones, assist in folding or refolding nascent or denatured proteins and enzymes to maintain a functional conformation. In this study, we found HSP26 and SSA4 encoding chaperones were significantly induced to counteract HMF stress damage to proteins. The deletion mutation of SSA4 displayed a significant longer lag phase under the HMF challenge, indicating its important role in adaptation and tolerance to HMF.

While the presence of chaperones provides positive con tribution to protein protection, severe or prolonged stress condition can result in irreversible protein damage. Misfolded or damaged proteins, especially aggregated proteins are highly toxic to cells. Degra dation of misfolded and damaged proteins by the ubi quitin mediated proteasome pathway plays an important genes of multiple functional categories are associated with the yeast adaptation to the inhibitor HMF during the lag phase. Transcription factor genes YAP1, PDR1, PDR3, RPN4, and HSF1 were identified as key regulatory genes for yeast global adaptation. Functional enzyme coding genes, for example ARI1, ADH6, ADH7, and OYE3, as well as gene interactions involved in the bio transformation and regulated by YAP1, are directly involved in the conversion of HMF into the less toxic compound FDM.

PDR genes encode plasma Anacetrapib membrane proteins and function as transporter of ATP binding cassette proteins. The large number of induced PDR genes observed by our study suggests a hypothesis of the important PDR function of pumping HMF and endogenous toxic metabolites to maintain cell viability. Important PDR gene functions include specific transpor ter ATPase gene RSB1, toxin transporter genes TPO1 and TPO4, and multiple cellular transport facilitator role in maintaining normal cell function and viability. Denatured proteins are targeted via the cova lent attachment of ubiquitin to a lysine side chain, and polyubiquitinated proteins are finally delivered to protea some to be degraded.

selleck inhibitor We observed that at least 14 ubi quitin related and proteasome genes were induced by HMF, indicating their important functions in adaptation to the HMF stress.

Strains with deletion Entinostat mutations in these genes were sensitive to HMF with an extended lag phase, for example, genes OTU1 and SHP1. It was suggested that the degradation of pro teins by the ubiquitin mediated proteasome pathway has regulatory roles on cell cycle, metabolic adaptations, selleck bio gene regulation, development, and differentiation.

The purity after sorting was greater than 95%. Tumor and MDSC conditioned medium preparation EMT6 cells and 4T1 cells were incu bated for 72 hours on a 24 well plate and the culture supernatants were collected. To obtain MDSC CM, FACS sorted splenic MDSCs were cultured for 24 hours. 4T1 MDSC CM and EMT6 MDSC CM were prepared by cultivating MDSCs in 50% further info 4T1 CM or EMT6 CM for 24 hours. A volume of 4T1 MDSC CM containing 1 ng of IL 6, or the same volume of EMT6 MDSC CM or MDSC CM, was added to the 4T1 and EMT6 cell cultures. To some cultures, the following signaling inhibitors were added. Stat3 inhibitor peptide, PI3K inhibitor, NF B inhibitor, JNK inhibitor, p38 MAPK inhibitor, and ERK inhibitor. Immunofluorescence microscopy Tissues were fi ed in 4% PFA and embedded in paraffin.

Sections were stained with H E for histopathological analysis. To investigate IL 6, IL 6Ra, and Adam17 e pression levels in MDSCs, sections were stained with anti Gr 1 mAb and other appropriate antibodies. The following primary antibodies were used anti mouse IL 6, anti mouse IL 6Ra, anti mouse Adam17 and anti mouse Gr 1. The following secondary antibodies were used Ale a 488 conjugated anti rabbit IgG and Ale a 594 conjugated anti rat IgG. Image acquisition and processing was performing using a confocal fluorescence microscope and an FV10 ASW 2. 0 Viewer. ELISA EMT6 and 4T1 cells were plated on a 24 well plate. The cells were permitted to grow for 24 or 48 hours. Supernatants were collected and assayed for IL 6 and soluble IL 6Ra levels by ELISA.

For IL 6 detection, anti mouse IL 6 was used as the capture antibody, biotinylated anti mouse IL 6 in 0. 1% BSA in PBS T as the detection antibody and recombi nant IL 6 as the standard. To detect soluble IL 6Ra, we used anti mouse IL 6Ra as the capture antibody, biotinylated anti mouse IL 6Ra as the detection Batimastat antibody and recombinant IL 6Ra as the standard. RNA analysis RNA was isolated from sorted splenic MDSC using the RNeasy kit. cDNA was generated from 1 ug of total RNA by reverse tran scriptase from Moloney Murine Leukemia Virus, and subjected to PCR. PCR products were analyzed by 1. 5% agarose gel electrophoresis. Western blot analysis Cells were harvested in lysis solution containing 50 mM Tris HCl, 1% NP40, 150 mM NaCl, 2 mM EDTA, 100 uM PMSF, a protease inhibitor cocktail, and a phos phatase inhibitor.

After incubation on ice for 30 minutes, cellular debris was removed by centrifuga chronic myelocytic leukemia tion. Proteins were separated by SDS PAGE and then transferred to a polyvinylidene difluoride membrane. After blocking with 5% skim milk, the membranes were probed with an appropriate antibody. Blots were developed with an enhanced chemilumines cence Western blotting detection system. The following antibodies were used anti b actin, anti phospho Stat3, anti Stat3, anti I B, anti phospho JNK, anti phospho ERK, and anti phospho p38. Invasion assay Matrigel matri solution was applied to each Transwell.

5 M, 2 10?2 M and 10?2 M, respectively, and were diluted selleck chemicals llc appropriately with cell culture medium. For in vivo studies, DON and SCM 198 were dissolved in 0. 9% sodium chloride so lution containing 1% sodium carbo ymethylcellulose. Lyophilized AB1 40 was first dissolved in sterilized distilled water followed by dilution with calcium free PBS to a final concentration of 1 mg ml. This solution was aggre gated at 37 C for 7 days before its application in in vitro e periment or in the surgery. Cell culture Cerebral corte of newborn SD rats was separated and cut into small pieces after removing meninges and blood vessels to prepare mi ed glial cells. Trypsinization with 0. 125% trypsin was stopped with DMEM F12 medium containing 10% FBS, 100 units ml penicillin, 100 ug ml streptomycin and 5 ug ml plasmo cin.

The tissue was gently pipetted to obtain a single cell suspension, which was then transferred to a new centri fuge tube after standing at room temperature for one to two minutes. This procedure was repeated three or four times. Then cells were centrifuged at 200 g for 5 minutes, resuspended in fresh DMEM F12 medium and plated according to different protocols. Twenty one days later, microglial cells were purified by mild trypsiniza tion method. For primary astrocyte culture, cortical mi ed glial cells from SD rats were cultured for two weeks. When cells became confluent, astrocytes were purified by shaking at 350 rpm at 37 C for 12 hours. The purity of primary microglia and astrocytes were confirmed with a mouse monoclonal CD11b antibody and a mouse mono clonal glial fibrillary acidic protein antibody, respectively.

Cerebral corte from fetuses of 17 to 18 days of gesta tion was used to prepare neurons, as described previ ously with minor modifications. Preparation of single cell suspension of neurons was the same with that of mi ed glial cells. Cells were maintained in neurobasal medium supplemented with 2% B27, 0. 5 mM L glutam ine, 100 units ml penicillin and 100 Anacetrapib ug ml streptomycin. Medium was changed 24 hours after plating and every three days thereafter. Neurons cultured for 10 to 14 days were used in the e periments. The purity of neurons was confirmed using a rabbit polyclonal MAP2 antibody. Immortalized murine BV 2 microglial cell line was first generated by Blasiet al. and retains many morpho logical and functional properties of primary microglia.

Cells were maintained in DMEM supplemented with 10% FBS, 100 units ml selleck compound penicillin and 100 ug ml streptomycin, and were passed twice a week. Microglia neuron co culture Microglia neuron co culture assay was performed as ac cording to Yuekui Li et al. with minor modifica tions. Neurons and BV 2 cells were separately seeded into 24 well or 6 well format transwell plates. BV 2 cells were pretreated with or without 0. 1 to 10 uM SCM 198 or 100 uM IBU for 2 hours and were stimulated with 1 ug ml LPS for another 2 hours.