ABSTRACT: Naïve T cells (CD4+CD62LhiCD44loCD25-) were purified from spleens of young (2 months; n=3) and aged (22 months; n=3 pools of five mice) mice and were either frozen or activated for 24 hrs.
Project description:Differential gene expression profile of CD4+ T cells from 10 months old Wt, miR-155-/-, miR-146a-/- and DKO mice spleens. Wt, miR-155-/-, miR-146a-/- and DKO mice were aged 10 months, CD4+ T cells were sorted from mice spleens for analyses.
Project description:CD4+YFP+ Treg cells were sorted from the peripheral lymph nodes and spleens of Foxp3-Cre mice (8-10-weeks-old), Foxp3-Cre;Bim-fl/fl mice (8-10-weeks-old), or Foxp3-Cre mice (older than 18-months-old), respectively. Detailly, peripheral lymph nodes and spleens were harvested from mice, and grinded into single cells, then the CD4+-T cells were isolated by Mouse CD4 T Lymphocyte Enrichment Set-DM (BD Biosciences, 558131), followed with FACS sorting (SONY Cell Sorter, SH800S) to purify CD4+YFP+ Treg cells with purities >99%. RNA samples were prepared with the miRNeasy Mini Kit (Qiagen, 21704), then reverse transcribed, amplified, labeled (Affymetrix GeneChip pico kit, 703308), and hybridized to Clariom D Arrays, mouse (Affymetrix, 902931).
Project description:Kaposi’s Sarcoma Herpesvirus (KSHV) is the causative agent of Kaposi’s Sarcoma (KS) and isassociated with primary effusion lymphoma (PEL), multicentric Castleman’s disease (MCD) and two inflammatory diseases. KSHV-associated cancers are primarily associated with genes expressed during latency, while other pathologies are associated with lytic gene expression. The major lytic switch of the virus, RTA, interacts with cellular machinery to co-opt the host ubiquitin proteasome system to evade the immune response as well as activate the program of lytic replication. Through SILAC labeling, ubiquitin remnant enrichment and mass spectrometry, we have analyzed the RTA dependent ubiquitin-modified proteome. We identified RTA dependent changes in the populations of polyubiquitin chains, as well as changes in ubiquitinated proteins in both cells expressing RTA and naturally infected cells following lytic reactivation. We observed an enrichment of proteins that are also reported to be SUMOylated, suggesting that RTA, a SUMO targeting ubiquitin ligase, may function to alleviate a SUMO dependent block to lytic reactivation. RTA targeted substrates directly through a ubiquitin ligase domain dependent mechanism as well as indirectly through cellular ubiquitin ligases, including RAUL. Our ubiquitome analysis revealed an RTA dependent mechanism of immune evasion. We provide evidence of inhibition of TAP dependent peptide transport, resulting in decreased HLA complex stability. The results of this analysis increase our understanding of mechanisms governing the latent to lytic transition in addition to the identification of a novel RTA dependent mechanism of immune evasion.Kaposi’s Sarcoma Herpesvirus (KSHV) is the causative agent of Kaposi’s Sarcoma (KS) and is associated with primary effusion lymphoma (PEL), multicentric Castleman’s disease (MCD) and two inflammatory diseases. KSHV-associated cancers are primarily associated with genes expressed during latency, while other pathologies are associated with lytic gene expression. The major lytic switch of the virus, RTA, interacts with cellular machinery to co-opt the host ubiquitin proteasome system to evade the immune response as well as activate the program of lytic replication. Through SILAC labeling, ubiquitin remnant enrichment and mass spectrometry, we have analyzed the RTA dependent ubiquitin-modified proteome. We identified RTA dependent changes in the populations of polyubiquitin chains, as well as changes in ubiquitinated proteins in both cells expressing RTA and naturally infected cells following lytic reactivation. We observed an enrichment of proteins that are also reported to be SUMOylated, suggesting that RTA, a SUMO targeting ubiquitin ligase, may function to alleviate a SUMO dependent block to lytic reactivation. RTA targeted substrates directly through a ubiquitin ligase domain dependent mechanism as well as indirectly through cellular ubiquitin ligases, including RAUL. Our ubiquitome analysis revealed an RTA dependent mechanism of immune evasion. We provide evidence of inhibition of TAP dependent peptide transport, resulting in decreased HLA complex stability. The results of this analysis increase our understanding of mechanisms governing the latent to lytic transition in addition to the identification of a novel RTA dependent mechanism of immune evasion.
Project description:In order to identify genes expressed by cells that leave the spleen, the spleens were harvested from untreated reconstituted humanized mice (N=4) and a single cell suspension was prepared . The cultures were treated with either teplizumab (anti human CD3 hOKT3g1(Ala-Ala)) or hIg for 18 hrs in vitro. Splenocytes were also harvested 18 hrs after reconstituted mice (N=4) were treated with teplizumab in vivo. The humanized mice used where NOD/SCID IL2gc-/- (NSG) reconstituted with human CD34+ at birth.
Project description:In natural habitats, plants are often exposed to multiple stresses. Most studies, however, for plant abiotic stress responses analyzed those to individual stress but not combined stresses. In this report, we performed comparison analyses of gene expression to individual stresses, salt, osmotic and heat, and to a combination of these three stresses, which mimics arid conditions. We show here that the combined stress treatment induces unique gene expression pattern but not a simple reflection of the additive effects of individual stresses. First, the number of genes induced by combined stresses (150 mM NaCl, 200 mM mannitol and 35°C heat) was much smaller when compared to the sum of those induced by individual stress treatments, while the number of genes downregulated by multiple stresses was larger. A large number of genes induced by mannitol were not induced by multiple stresses, while those induced by salts were less affected in combined stress treatments. In addition, 125 genes, including13 for transcription factors, were found to be induced specifically by combined stress treatments. We report here that the plant response to a multi-stress environment represents an output of complex interactions between different stress aspects and signaling events of the various components of this environmental situation, and the determinative factor in this response is to avoid/minimize the antagonistic effects and to magnify the synergistic features of the imposed environmental challenges. Based on our results, we propose that genes that are highly induced by multiple treatments may be candidate for engineering stress tolerant crop plants. Wild-type plants of Arabidopsis thaliana were treated with three abiotic stresses, high salinity (150mM and 300mM), high osmotic pressure (200mM and 400mM mannitol) and heat (35°C), individually or simultaneously. Plants were treated with salt or mannitol for 16 hrs (first 1 hr and last 7 hrs are in light and other time in dark) or with heat for 4 hrs (in light) before sampling. There are three biologcal replicates.
Project description:Oxaliplatin resistance was induced in 2 colorectal cancer cell lines (LoVo-92, wt-p53 and LoVo-Li, functionally inactive p53) and one ovarian cancer cell line (A2780, wt-p53). Resistance was induced by weekly exposure to oxaliplatin for 4 hrs or 72 hrs with increasing concentrations for a period of 7 months
Project description:Oxaliplatin resistance was induced in 2 colorectal cancer cell lines (LoVo-92, wt-p53 and LoVo-Li, functionally inactive p53) and one ovarian cancer cell line (A2780, wt-p53). Resistance was induced by weekly exposure to oxaliplatin for 4 hrs or 72 hrs with increasing concentrations for a period of 7 months.
Project description:Spleens were harvested from B6.Sle1.Yaa at 2, 4, and 7 months of age or from LCMV infected mice 8 days post infection. RNA-seq was employed to assess transcriptional changes in these cells throughout disease progression.
Project description:The cells harvested for microarray analysis were high-dose rate (HDR) irradiated, low-dose rate (LDR) irradiated or unirradiated T-47D cells. The HDR-irradiated cells were harvested 24h after a dose of 0.3 Gy at 35 Gy/h, a time where hyper-radiosensitivity (HRS) had returned. The HRS-deficient LDR-irradiated cells were harvested 2 months after a dose of 0.3 Gy at 0.3 Gy/h.