Project description:Transcription profiles of BV2 microglial cell lines: unstimulated, stimulated with LPS or transfected with constitutively active Stat1 and Stat3.
Project description:To comprehensively study the intracellular signaling changes and explore the underlying mechanisms of baicalein-mediated microglial responses, we employed systemic proteomic approach, using target-free SWATH mass spectra (SWATH-MS), focusing on intracellular proteins. BV2 cells were treated with LPS followed by the addition of vehicle or baicalein and they were cultured for a total of 48 hours before being collected for proteomic analysis.
Project description:Murine BV2 microglia cells were transfected either with siRNA negative control or siRNA against caspase-3 for 48h. Later on some the of the BV2 transfected cells were co-cultured with C6 glioma cells during 6h. We used the SA Biosciences Mouse Wound Healing PCR Array (PAMM-121Z) to quantitate gene expression of relevant genes related to the wound healing process Glioma cells recruit and exploit microglia, resident immune cells of the brain, for their proliferation and invasion capability. The underlying molecular mechanism used by glioma cells to transform microglia into a tumor-supporting phenotype remains elusive. Here, we report that glioma-induced microglia conversion is coupled to a reduction of basal microglial caspase-3 activity, increased S-nitrosylation of mitochondria-associated caspase-3 through inhibition of thioredoxin-2 activity, and demonstrate that caspase-3 inhibition regulates microglial tumor-supporting function. Further, we identified nitric oxide synthase-2 (NOS2) activity originating from the glioma cells as a driving stimulus in the control of microglial caspase-3 activity. Repression of glioma NOS2 expression in vivo led to reduction in both microglia recruitment and tumor expansion, whereas depletion of the microglial caspase-3 gene promoted tumor growth. This study provides evidence that the inhibition of Trx2-mediated denitrosylation of SNO-procaspase-3 is part of the microglial pro-tumoral activation pathway initiated by glioma cancer cells. qPCR gene expression profiling. Three independent experiments of siControl BV2 monoculture, siCaspase3 BV2 monoculture, siControl BV2 cocultured 6h with C6 glioma cells and siCaspase3 BV2 cocultured 6h with C6 glioma cells. Equal amount total RNA from each culture was used for the gene expression analysis. Please note that the raw data for three independent experiments (prior to averaging the data) is provided in the 'Raw_Data_File_with_the_Ct_values_for_3_indep_experiments.txt'.
Project description:Mouse microglia (BV2 cells) were stimulated with a TLR4 ligand (E. coli LPS, 1 µg/ml) for 4h. The Agilent SurePrint G3 Mouse Gene Expression Microarray (G4852A) was used for the analysis, which provides full coverage of genes and transcripts with the most up-to-date content, including mRNAs and lincRNAs (http://www.chem.agilent.com/store/en_US/Prod-G4852A/G4852A). BV2 cells were grown to 80% confluence for four groups: the siRNA control (Group A, cells treated with a non-specific scrambied siRNA control), the LPS-stimulated (Group B, cells treated with the siRNA control plus LPS stimulation), lincRNA-Cox2 siRNA (Group C, cells treated with an siRNA to lincRNA-Cox2), and lincRNA-Cox2 siRNA/LPS stimulated (Group D, cells treated with the lincNRA-Cox2 siRNA plus LPS stimulation). Cells were treated with the siRNAs for 24h, followed by additional culture for 4h in the presence or absence of LPS (E. coli LPS, 1 µg/ml). Total RNAs were prepared with the RNeasy Mini kit (Qiagen) according to the manufacturer’s instruction (Ambion).
Project description:We conducted small RNA sequencing and bioinformatics analysis of GCH1-KD BV2 microglial cells treated with adenovirus. Their RNA was extracted and analyzed, and the results were verified by quantitative real-time polymerase chain reaction (qRT-PCR). This study explored the miRNAs and mRNAs regulated by GCH1 and revealed a possible mechanism of GCH1 in microglial activation.