Journal: Journal of Extracellular Vesicles

Article Title: Characterization of physical properties of tissue factor–containing microvesicles and a comparison of ultracentrifuge-based recovery procedures

doi: 10.3402/jev.v3.23592

Figure Lengend Snippet: Nanoparticle tracking analysis (NTA) of TF-containing microvesicles (1.03–1.08 g/ml) separated by density gradient centrifugation. Microvesicles were prepared from normal human plasma (A), conditioned media from MDA-MB-231 cells (B), and conditioned media from MDA-MB-231 cells expressing TF-tGFP protein (C). The plasma and conditioned media were collected and cleared of any cell debris by centrifuging at 5,400 g on a microcentrifuge, and microvesicles sedimented at 100,000 g . The microvesicles were resuspended in PBS and were fractionated by density gradient ultracentrifugation using a sucrose–OptiPrep gradient covering an approximate density range of 1.02–1.22 g/ml alongside 2 sets of DensityMarkerBeads. The samples were centrifuged at 52,000 g for 90 min at 20°C in a SW41Ti rotor on a Beckman L8-M ultracentrifuge. Following centrifugation, aliquots (0.5 ml) were sequentially removed and assessed for TF antigen. Samples containing TF antigen were then pooled (1.03–1.08 g/ml) and diluted 1:10 in PBS, and the size of the microvesicle population was analysed by NTA using a NanoSight LM10 instrument. A control sample was prepared by adding MDA-MB-231-derived microvesicles to the same pooled fractions from a blank density gradient centrifugation (D). A negative control made of the pooled fractions from a blank density gradient centrifugation showed no detectable trace (E). The illustrations are typical (n=3) of the size distributions which were determined using NTA software. The total amounts of microvesicles in the samples are not comparable. TF-containing microvesicles were immuno-purified from conditioned media of MDA-MB-231 (F) and A375 cell lines (G). The samples were incubated with a monoclonal antibody against TF (10H10; 4 µg/ml) followed by protein A-magnetic beads. The samples were washed with PBS and eluted in phosphate buffer containing NaCl (500 mM). The samples were analysed by NTA against a sample treated similarly but without the antibody.

Article Snippet: The samples and markers were placed in a SW41Ti swing-out rotor and centrifuged at 52,000 g for 90 min at 20°C on a Beckman L8-M ultracentrifuge (Beckman Coulter).

Techniques: Gradient Centrifugation, Expressing, Centrifugation, Derivative Assay, Negative Control, Software, Purification, Incubation, Magnetic Beads

Journal: Molecular metabolism

Article Title: The CARM1 transcriptome and arginine methylproteome mediate skeletal muscle integrative biology.

doi: 10.1016/j.molmet.2022.101555

Figure Lengend Snippet: Figure 1: RNA-seq analysis of skeletal muscle from CARM1 mKO mice. (A) Representative Western blots for CARM1 (short and long exposure) in the tibialis anterior (TA) muscle from male wildtype (WT) animals and age-matched, skeletal muscle-specific CARM1 knockout (mKO) littermates. Approximate molecular weights (MWs) in kilodaltons (kda) to the right. (B) Graphical summary of CARM1, PRMT1, PRMT5, and PRMT7 transcript levels in extensor digitorum longus (EDL) and soleus (SOL) muscles. Bars indicate group means, whiskers represent SEMs, points illustrate individual results, and the dotted lines denote 1. Data are displayed relative to the WT EDL. Statistical analysis was completed using a 2- way ANOVA and Tukey’s post hoc test. *, p < 0.05 vs. WT; $, p < 0.05 vs EDL (main effect of muscle type). n ¼ 29e37. (C) WT vs mKO gene fragments per kilobase of transcript per million mapped reads (FPKM) scatter plot (FDR <5%). Highlighted in red are differentially expressed genes. (D) Heat map showing differentially expressed genes with a minimum log fold change (FC) of 2.0 and < 1% false discovery rate (FDR) in TA muscle from WT and mKO littermates. n ¼ 4. Bubble plots showing the distribution and size of over-represented gene ontology (GO) terms in the upregulated (E) and downregulated gene sets (F). (G) Pie charts representing commonality in upregulated and downregulated genes among the CARM1 mKO, muscle atrophy and muscular dystrophy biosets following a BaseSpace Correlation Engine meta-analysis. White portion ¼ only altered in CARM1 mKO dataset; grey area ¼ modified in CARM1 mKO and muscle atrophy data; striped region ¼ changed in CARM1 mKO and muscular dystrophy data; grey striped area ¼ commonly modified genes in all conditions. (H) Graphical summary of cholinergic receptor nicotinic alpha 1 subunit (CHRNA1), forkhead box 01 (FOXO1), cholinergic receptor nicotinic gamma subunit (CHRNG), CARM1, and fibroblast growth factor binding protein 1 (FGFBP1) mRNA levels in mKO TA muscles displayed relative to WT mice. Bars indicate group means, whiskers represent SEMs, points illustrate individual results, and the dotted lines denote 1. Statistical analysis was completed using a student’s t-test. *, p < 0.05 vs. WT. n ¼ 7e8. (For interpretation of the references to color/colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Previous work has implicated CARM1 in muscle atrophy [13,15,19,24e26], thus we more closely examined our results by comparing the data to publicly available biosets relating to muscle atrophy and muscular dystrophy using the Illumina BaseSpace Correlation Engine [49].

Techniques: RNA Sequencing, Western Blot, Knock-Out, Muscles, Binding Assay