Journal: Science (New York, N.Y.)

Article Title: Rules of engagement for condensins and cohesins guide mitotic chromosome formation

doi: 10.1126/science.adq1709

Figure Lengend Snippet: ( A ) Representative live cell imaging of a DT40 CDK1 as _Halo-lamin B1_3xGFP-NES cell released from G 2 block with 1NM-PP1. DNA:grey; Halo-lamin B1: magenta; 3xGFP-NES: green. Seven z-sections (1 μm interval) were taken every 1.5 min. A single section is shown for each timepoint. Scale bar = 5 μm. 3XGFP enters nuclei a few minutes prior to visible nuclear lamina disruption (NEB, t= 10–11 min). Intensity of lines under the images illustrates the relative amount of the indicated complexes on chromatin at each time point. Green arrow indicates cytoplasmic GFP entering the nucleus before nuclear envelope breakdown. ( B ) Relative nuclear GFP fluorescence intensity (cytosolic GFP intensity = 1) from experiment of . ( C ) Chromatin enrichment for proteomics (ChEP) analysis of WT CDK1 as cells (SILAC analysis). Log 2 SILAC ratio normalized against G 2 is shown for cohesin (average of SMC1, SMC3, and RAD21), condensin I (CAP-H, CAP-G, and CAP-D2) and condensin II complexes (CAP-H2, CAP-G2, and CAP-D3). t= 0 is after completion of 1NM-PP1 washout, n= 6. ( D ) Estimated number of chromatin-associated cohesin, condensin I and condensin II complexes (per Mb DNA) during mitotic entry in wild type CDK1 as cells. Average iBAQ number from ChEP analysis for subunits as listed in C was normalized relative to values for Histone H4, n= 6. ( E ) Absolute quantification of SMC3 (cohesin subunit), CAP-H (condensin I subunit) and CAP-H2 (condensin II subunit) on chromatin (per Mb DNA). Protein numbers are calculated following ChEP analysis of corresponding Halo-tagged cell lines normalized using purified spike-in Halo-Histone H4 protein (n= 4).

Article Snippet: DNA fragments encoding 3x superfolder GFP or mCherry (Addgene plasmids 75385 and 75387 digested with BamHI/XhoI) and double-stranded oligos encoding BP-NLS or NES from Gg cAMP-dependent kinase inhibitor alpha were ligated into pcDNA3 (digested with BamHI/ApaI).

Techniques: Live Cell Imaging, Blocking Assay, Disruption, Fluorescence, Multiplex sample analysis, Quantitative Proteomics, Purification

Journal: Burns & Trauma

Article Title: p53 promotes the expansion of regulatory T cells via DNMT3a- and TET2- mediated Foxp3 expression in sepsis

doi: 10.1093/burnst/tkad021

Figure Lengend Snippet: Foxp3 expression is regulated by DNMT3a and TET2. ( a , b ) C57BL/6 mice were subjected to sham (n = 5) or CLP (n = 10) procedure and sacrificed after 24 h. Total mRNAs were extracted from splenetic CD4 + T cells and subjected to qPCR analysis of DNMT1, DNMT3a, DNMT3b, TET1, TET2 and TET. β-Actin was used as the loading control. The mRNA levels were normalized to the sham group ( * p < 0.01). ( c ) Total proteins were extracted from cells and used for western blotting of DNMT3a, TET2 and β-actin. The relative expressions were quantified using ImageJ and normalized to that of the loading control ( * p < 0.01, # p < 0.01). ( d ) Jurkat T cells were transfected with lentivirus containing DNMT3a mRNA or siRNA expressing plasmids or blank vector. ( e ) Jurkat T cells were transfected with lentivirus containing TET2 mRNA or siRNA expressing plasmids or blank vector. After stable expression, cells were stimulated with PBS (control) or LPS (1 μg/ml) for 24 h. Then total proteins were extracted from cells and used for western blotting analysis of Foxp3 and β-actin. The relative expression of Foxp3 was quantified using ImageJ and normalized to that of β-actin ( * p < 0.05, * * p < 0.01, # p < 0.01 vs control group in siRNA vector, ## p < 0.01 vs LPS group in siRNA vector). CLP cecal ligation and puncture, m RNA messenger ribonucleic acid, siRNA small interfering ribonucleic acid, qPCR quantitative real-time polymerase chain reaction, DNMT DNA-buffered saline, LPS lipopolysaccharide, Foxp3 forkhead/winged helix transcription factor p3

Article Snippet: Lentivirus vectors expressing the DNA fragments encoding GFP-tagged full-length DNA methyltransferase enzyme (DNMT) 3a, DNMT3a mRNA-targeted small interfering ribonucleic acid (siRNA), ten–eleven translocation 2 (TET2) and TET2 mRNA-targeted siRNA were designed, constructed, packed and purified by Obio Technology Co. Ltd (Shanghai, China).

Techniques: Expressing, Control, Western Blot, Transfection, Plasmid Preparation, Ligation, Real-time Polymerase Chain Reaction, Saline