RNA-seq of DARPin F10 expressing cells as compared to Wt A549
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ABSTRACT: De novo synthesized protein DARPin F10 can target HDAC6 ZnF domain, and inhibits Influenza A virus and ZIKA virus infection. Our data shows it interferes with the virus uncoating step. To exclude the possibility that F10 upregulates interferon pathways, we checked the transcription of the cell
Project description:GFP-tagged DARPin F10 and CTR are expressed in A549 cells, then GFP positive cells are sorted by GFP positive with FACS. Cells are pelleted and total RNA is extracted. The aim is to check DARPin F10 effects on gene expression.
Project description:THP-1 cells were infected by IAV virus strain X31 with a MOI=10, and cells were collected at 3 timepoints:0 hr, 24 hr and 48 hr post-infection. Each timepoint has 3 biological replicates.
Project description:The mouse melanoma cell line B16-F10 provided by American Type Culture Collection (ATCC® CRL-6475™) were treated with DMSO, G007-LK, WNT or G007-LK+WNT, done in triplicates for a total of 12 samples.
Project description:Identifying the HDAC6 targeting DARPin F10 interacting protein in human cells. Proteins are expressed as GFP fused protein and captured by GFP-trap beads. DARPin CTR is a control DARPin that binds to nothing.
Project description:To elucidate the mechanisms by which the four carboxylic acids—3-phenyllactic acid, lactic acid, L-pyroglutamic acid, and malic acid—inhibit melanin production in B16-F10 cells, a comparative proteomic analysis was conducted to assess changes in protein expression.
Project description:Analysis of gene expression profile of B16-F10 murine melanoma cells exposed to hypoxic conditions (1% oxygen) or hypoxia mimicry (cobalt chloride) for 24 hours. Gene expression profiles were analyzed using MG-U74Av2 oligonucleotide microarrays. Data analysis revealed 2541 probesets (FDR<5%) for 1% oxygen experiment and 364 probesets (FDR<5%) for cobalt chloride, that showed differences in expression levels. Analysis of hypoxia-regulated genes (1% O2) by stringent Family-Wise Error Rate estimation indicated 454 significantly changed transcripts (p<0.05). The most upregulated genes were Lgals3, Selenbp1, Nppb (more than ten-fold increase). Both hypoxia and hypoxia-mimicry induced HIF-1 regulated genes. However, unsupervised analysis (Singular Value Decomposition) revealed distinct differences between gene expression induced by these two experimental conditions. We investigated transcriptional activity of B16-F10 murine melanoma cells cultured for 24h under hypoxic (nominal 1% oxygen; 9 experimental samples and 6 controls) and hypoxia-mimicking conditions (cobalt chloride, 100 M-NM-<M or 200 M-NM-<M, 2 samples each and 2 controls).
Project description:The objective of this experiment was to identify cellular targets of F10-mediated phosphorylation through Stable Isotope Labeling of Amino acids in Cell culture (SILAC). 293-F10 cells were propagated in heavy (H) or light (L) media for 10 cellular doublings. Mass spectrometry was used to confirm that the majority of tryptic peptides showed >90% incorporation of the heavy isotope. H-labeled cells were treated with doxycycline to induce expression of F10 for 10 hours, and then these cells were mixed with uninduced, L-labeled cells in a 1:1 ratio. Three biological replicates were obtained from separately passaged cultures and doxycycline inductions. Cells were lysed and post-nuclear supernatant proteins were precipitated and subjected to tryptic digestion. Tryptic peptides were desalted, enriched for phosphopeptides, and analyzed in triplicate by LC-MS/MS. The resulting 9 data files were pooled and phosphoprotein identification and quantification was completed using MaxQuant software (version 1.2.2.5). Differential phosphorylation in response to F10 induction was determined using an outlier score for log protein ratios. The Benjamini-Hochberg test was then used to correct for the biased statistical spread of highly abundant proteins.
Project description:Three types of stimuli -- heat shock, Lipofectamine 2000 and benzyl alcohol -- induce activity of some stress genes (hsp) in mouse B16-F10 cells. Besides hsp genes induction, each stimulus causes gene expression changes of different sets of genes. We used microarrays to analyze global gene expression changes in mouse B16-F10 cells treated with elevated temperature (heat shock, HS), with Lipofectamine 2000 (LA) or with 40mM benzyl alcohol (BA). In order to study how lipofection may affect cellular homeostasis, we used Affymetrix microarrays to analyze the whole transcriptome of mouse B16-F10 cells treated with Lipofectamine 2000. To find out which genes are affected, we compared the cells treated with Lipofectamine (LA1, LA2, LA3), the cells treated with 40mM benzyl alcohol (BA1, BA2, BA3), heat-shocked cells (HS1, HS2, HS3) and control, untreated cells (C1-5). RNA was extracted from cells 30 min after treatment.