Journal: bioRxiv

Article Title: Vaccinia virus induces EMT-like transformation and RhoA-mediated mesenchymal migration

doi: 10.1101/2023.02.04.527154

Figure Lengend Snippet: a , Representative super-resolution images of a control Vero cell (left) and a VACV-infected Vero cell (right) expressing LifeAct-mRuby. Scale bars represent 10 μm. b , Representative actin stress fiber images (top) and fluorescent quantitative (bottom left) and width (bottom right) of analysis stress fibers in control and VACV-infected cells by phalloidin staining. Scale bar represents 5 μm. c , Representative immunofluorescence images of F-actin (phalloidin, green) and p-MLC (red) in the control and VACV-infected cell. Scale bar represents 20 μm. d , e , Immunoblotting (d) and quantitative (e) analysis of MLC and p-MLC in Vero cells during VACV infection (n=3). f , g , Montage of frames from live-cell imaging (f) and relative fluorescent intensity analysis (g) of control and VACV-infected Vero cells expressing LifeAct-mRuby. The dotted lines represent the cell outlines (blue: control cells; red: VACV-infected cells). Scale bar represents 40 μm. h , Schematic diagram of the process of VICM accompanied by stress fibers depolymerization, leading protrusion formation, and rear contraction. i , j , Time-lapse actin heatmap (i) and plot profile of gray value (j) along white dotted line in a control cell in two hours. Scale bar represents 20 μm. k, l , Time-lapse actin heatmap (k) and plot profile of gray value (l) along white dotted line in a VACV-infected cell in two hours. Scale bar represents 20 μm. m , n , MSD (m) and representative images of actin cytoskeleton distribution (n) of Vero-LifeAct-mRuby cells during VACV infection after 250 nM jasplakinolide, 1 μM cytochalasin D, 10 μM CK666, and 1 μM latrunculin A treatment. Scale bar represents 20 μm. Data are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (unpaired t-test).

Article Snippet: Jasplakinolide, cytochalasin D, latrunculin A, Y15, tetracycline, ML-7 hydrochloride, and ML-9 were purchased from MedChemExpress.

Techniques: Control, Infection, Expressing, Staining, Immunofluorescence, Western Blot, Live Cell Imaging

Journal: Advanced Science

Article Title: Adhesion‐Related Macrophages Regulate Metabolic Homeostasis Through CAV‐1 Dependency

doi: 10.1002/advs.202520936

Figure Lengend Snippet: ARMs acquire adipocyte RNA. (A) Venn diagram illustrating the overlap of genes that are upregulated in RNA‐Seq but either not upregulated or not detected in ATAC‐Seq. (B) KEGG pathway analysis of exogenous genes in ARMs, depicting the six most significant pathways. (C) Violin plots of foreign ARM gene expression in different cell types from single nuclear sequencing data of eWAT. (D) Representative electron micrographs of the cell–cell contact between bead‐purified ARMs and their adhered adipocytes isolated from 8‐week HFD‐fed mice. Scale bars: 5 and 1 µm. (E) RNA‐seq gene expression levels of Cytoskeletal remodeling. (F) Representative RNAscope images showing uptake of adipocyte‐derived Adipoq mRNA by CFSE‐labeled ARMs after 30 min of coculture. Scale bars: 10 and 2 µm. (G) Schematic of the ex vivo assay used to quantify adipocyte‐derived RNA acquired by ARMs or SMs in (H). Mouse eWAT was digested to obtain the stromal vascular fraction (SVF), from which ADSCs were purified and differentiated into mature adipocytes. Adipocyte RNA was labeled with RNASelect and cocultured with ARMs or SMs from eWAT of HFD‐fed mice for 1 h. After trypsin digestion into a single‐cell suspension, adipocyte‐derived RNA signals in ARMs or SMs were analyzed by flow cytometry. (H) Percentage of RNASelect + macrophages after coculture of RNASelect + adipocytes with ARMs or SMs in the presence of the actin‐depolymerizing agent cytochalasin D ( n = 3 per group). (I,J) Percentage of RNASelect + ARMs after coculture with RNASelect + adipocytes: (I) ARMs isolated from HFD WT and Cav‐1 −/− mice; (J) ARMs after coculture with TNF‐α‐pretreated adipocytes ( n = 3 per group). Data are expressed as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, NS = not significant.

Article Snippet: Macrophages and adipocytes were pretreated with DMSO or 50 μ m Cytochalasin D (Topscience, T3229) for 1 h. For TNF‐α stimulation, adipocytes were treated with TNF‐α (20 ng/mL) for 16 h prior to coculture.

Techniques: RNA Sequencing, Gene Expression, Sequencing, Purification, Isolation, RNAscope, Derivative Assay, Labeling, Ex Vivo, Single Cell, Suspension, Flow Cytometry