Project description:Chem-seq analysis was conducted to reveal genome-wide localization of a cell penetrating peptide, SVS-1. A549 cells treated with 15 mM of biotin-PEG-GG-SVS-1 for 1 hour were passaged once to remove dead cell debris. After 24 hours incubation from the passage, the treated cells were fixed with formaldehyde to crosslink SVS-1 and chromatin, and total chromatin was subjected to fragmentation by sonication. Chromatin bound by the biotin-PEG-GG-SVS-1 was isolated with avidin-conjugated resin, followed by DNA purification. The input DNAs were also purified and employed as controls for the peak callings. The purified DNAs were subjected to library preparation for deep sequencing by the illumina platform. Sequencing was run by the NextSeq with paired end sequencing (2x76), 100 million clusters /sample.
Project description:To investigate the effects of the cell-penetrating TLR decoy peptide, M-CSF generated bone marrow derived macrophages obtained from C57BL6/J mice were pretreated with two different concentrations of the peptide followed by stimulation with LPS for 2 hours. The cells were lyzed and analyzed by RNA Sequencing.
Project description:Simple and efficient delivery of CRISPR genome editing systems in primary cells remains a major challenge. Here, we describe an engineered Peptide-Assisted Genome Editing (PAGE) CRISPR-Cas system for rapid and robust editing of primary cells. PAGE couples a cell-penetrating Cas protein with a cell-penetrating endosomal escape peptide in a 30-minute incubation that yields up to ~98% editing efficiency in primary human and mouse T cells. PAGE provides a broadly generalizable platform for next generation genome engineering in primary cells. CITATION INFORMATION: Zhang Zhen, Baxter Amy E, Ren Diqiu, Qin Kunhua, Chen Zeyu, Collins Sierra M., Huang Hua, Komar Chad A., Bailer Peter F., Parker Jared B., Blobel Gerd A., Kohli Rahul M., Wherry E. John*, Berger Shelley,*, and Shi Junwei*. Peptide-assisted genome editing permits efficient CRISPR engineering of primary T cells.
Project description:We performed transcriptional profiling of cells treated with different cell penetrating peptides for different timepoints to see the effect of each peptide on cell biology and pathways which are affected by it. In conclusion, CPPs evoke similar response, regardless of the CPP structure or chemistry, mostly afecting genes related to ribosome biogenesis, microtubule dynamics and expression of long non-coding RNAs
Project description:Here, we determined the ability of peptide nucleic acid (PNA) oligomers, coupled to different cell-penetrating peptides (CPPs), to interfere in regulatory RNA circuits of human blood-derived leukocytes. Using RNA-seq, FACS and confocal microscopy we identified octaarginin as a CPP enabling PNA delivery and sequence-dependent RNA inhibition in blood-derived myeloid cells at nanomolar concentration. At 200 nM, an R8-PNA targeting immune-regulatory microRNA-155 was delivered into nearly 100 % of human macrophages within 24 hours without apparent cytotoxicity, and globally de-repressed microRNA-155 target-mRNAs. This was not observed when coupling the PNA inhibitor to a K3 instead of the R8 peptide. We suggest that CPP choice is a fundamental success-determining factor for therapeutic RNA-inhibition in human myeloid leukocytes.
Project description:We analyzed the genome wide localization of H3K4me3, H3K27me3 and the NUP98-PHF23 (with V5 tag) fusion protein which binds H3K4me3 via its PHD finger, using ChIP-seq. Results correlated with gene expression profiles. NUP98-PHF23 bound only 1.6% of H3K4me3 marks including Hoxa/b + Meis1.
Project description:Screening for potential target proteases of vaspin revealed rapid and specific cleavage within the vaspin N-terminus, releasing short peptides with cell penetrating activity. For microarray analysis to determine the effect of a vaspin-derived peptide on gene expression in adipocytes, fully differentiated primary mouse adipocytes from subcutaneous AT were treated with 100 nM of peptide for 24 h before harvesting, RNA isolation and microarray analysis. Solvent (water) treated cells served as controls.
Project description:We and others have proposed that coactivator binding inhibitors, which block the interaction of estrogen receptor and steroid receptor coactivators, may represent a potential class of new breast cancer therapeutics. The development of coactivator binding inhibitors has been limited, however, because many of the current molecules which are active in in vitro and biochemical assays are not active in cell-based assays. Our goal in this work was to prepare a coactivator binding inhibitor active in cellular models of breast cancer. To accomplish this, we used molecular dynamics simulations to convert a high-affinity stapled peptide with poor cell permeability into R4K1, a cell-penetrating stapled peptide. R4K1 displays high binding affinity for estrogen receptor ɑ, inhibits the formation of estrogen receptor/coactivator complexes, and distributes throughout the cell with a high percentage of nuclear localization. R4K1 represses native gene transcription mediated by estrogen receptor ɑ and inhibits proliferation of estradiol-stimulated MCF-7 cells. Using RNA-Seq, we demonstrate that almost all of the effects of R4K1 on global gene transcription are estrogen receptor-associated. This chemical probe provides a significant proof-of-concept for preparing cell-permeable stapled peptide inhibitors of the estrogen receptor/coactivator interaction.
Project description:We analyzed the genome wide localization of H3K4me3, H3K27me3 and the NUP98-PHF23 (with V5 tag) fusion protein which binds H3K4me3 via its PHD finger, using ChIP-seq. Results correlated with gene expression profiles. NUP98-PHF23 bound only 1.6% of H3K4me3 marks including Hoxa/b + Meis1. Assess H3K4me3 and H3K27me3 histone marks, and correlate these marks with chromatin binding of the NP23 fusion protein using lymphoblast and myeloblast cell lines derived from NP23 leukemias.