Journal: Cell reports
Article Title: Dissecting gene activation and chromatin remodeling dynamics in single human cells undergoing reprogramming.
doi: 10.1016/j.celrep.2024.114170
Figure Lengend Snippet: Figure 3. Global chromatin de-compaction during heterokaryon reprogramming (A) Representative Voronoi density rendering visual- izations of H2B STORM images of the somatic nu- cleus of control and heterokaryons at different time points after fusion. (B) Quantification of chromatin compaction changes during early heterokaryon reprogramming. Voronoi density values at the 95th percentile corresponding to H2B localizations from somatic nuclei of controls and heterokaryons at different time points after fusion. n = two–four biological replicates. Smaller dots represent individual values, and colored dots are the mean of biological replicates. Two-tailed t test was used to calculate p values and statistical signif- icance using the mean values of biological replicates. (C) Fold change Voronoi density relative to control. The percentage of decrease compared with control is shown for 48 h. Data correspond to the mean and SEM of two–four biological replicates. (D) Homokaryon generation strategy. hFBs were stained against CD90 using antibodies labeled with different fluorophores and fused with PEG. Fused cells containing both fluorescent markers were sor- ted as homokaryons. (E) Representative Voronoi density rendering visual- izations of H2B STORM images of the somatic nu- cleus of unfused fibroblast control and fibroblast homokaryon 48 h after fusion. (F) Voronoi density values at the 95th percentile cor- responding to H2B localizations of fibroblast nuclei at control and homokaryons at 48 h after fusion. n = one biological replicate. Smaller dots are individual values; black dots are the mean values of biological replicates. Error bars are the standard deviations. Two-tailed t test was used to calculate p values and statistical significance. Images in (A) and (E) are color-coded based on Vor- onoi density values from low (blue) to high (red).
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-H2B Proteintech Cat# 15857-1-AP, RRID:AB_10664929 Rabbit polyclonal anti-H3K9me3 Thermo Fisher Scientific Cat# 720093, RRID:AB_2532803 Rabbit monoclonal anti-H3K27me3 Thermo Fisher Scientific Cat# MA5-11198, RRID:AB_11000749 Rabbit monoclonal anti-H3K4me3 Thermo Fisher Scientific Cat# MA5-11199, RRID:AB_10977872 Rabbit monoclonal anti-H3K9ac Thermo Fisher Scientific Cat# MA5-11195, RRID:AB_10986969 Mouse monoclonal anti-Lamin A/C, unconjugated, Clone JOL2 Abcam Cat# ab40567, RRID:AB_775967 Mouse monoclonal anti-TRA-1-60 (Podocalyxin), Biotin Thermo Fisher Scientific Cat# 13-8863-82, RRID:AB_891594 R-phycoerythrin (PE) conjugated 5E10 mouse monoclonal anti-CD90 (THY1) BD Biosciences Cat# 561970, RRID:AB_10894194 APC CD90 (Thy-1) Monoclonal Antibody (eBio5E10 (5E10)) Thermo Fisher Scientific Cat# 17-0909-42, RRID:AB_11042579 Alexa Flour 647 Rat monoclonal anti-CD324 (E-Cadherin) BioLegend Cat# 147307, RRID:AB_2563954 Chemicals, peptides, and recombinant proteins ESGRO Recombinant Mouse LIF Protein EMD Millipore ESG1107 Polyethylene Glycol 1500 (PEG 1500) Roche 10783641001 Dimethyl sulfoxide (DMSO) Millipore Sigma D2650 SAv-HRP (Streptavidin Horseradish Peroxidase) Biolegend 05210 Deionized formamide Ambion AM9342 Dextran sulfate sodium salt Sigma-Aldrich D8905-5G 2-Mercaptoethanol 14.3 M Sigma-Aldrich 516732 Tween 20 Sigma-Aldrich P1379-100ML Glucose Oxidase from Aspergillus niger Sigma-Aldrich G2133-50KU Catalase from bovine liver Sigma-Aldrich C100-50MG D-(+)-Glucose, anhydrous,99%, 500g Alfa Aesar A16828 Cysteamine Sigma-Aldrich 30070-10G Paraformaldehyde aqueous 16% solution (w/v), EM grade Electron Microscopy Sciences 1570 Bovine Serum Albumin (BSA) Fisher BP1600-100 Triton X-100 Fisher BioReagentsTM BP151-100 DAPI Solution (1 mg/mL) Thermo ScientificTM 62248 SSC (20X), RNase-free Ambion AM9763 Sodium Chloride Fisher Chemical S271-500 RNaseOUTTM Recombinant Ribonuclease Inhibitor Invitrogen 10777019 RNase A, DNase and protease-free (10 mg/mL) Thermo ScientificTM EN0531 RNaseZAPTM Sigma R2020-250ML Nuclease-free water Ambion AM9932 0.05% Trypsin-EDTA (1X) Gibco 25300–054 Accutase Innovative Cell Technologies, Inc. AT104 Anti-Anti (100X) Antibiotic-Antimycotic Gibco 15240–062 GlutaMAXTM-l (100X) Gibco 35050–061 Sodium Pyruvate (100mM) Gibco 11360–070 MEM Amino Acids (100X) Gibco 11140–050 DPBS (1X) Gibco 14190–136 DPBS (10X) Gibco 14200–075 DMEM (1X) Gibco 11965–084 (Continued on next page) Cell Reports 43, 114170, May 28, 2024 15
Techniques: Control, Two Tailed Test, Staining, Labeling
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