Journal: Journal of Clinical Pathology

Article Title: Unusual fusion gene rearrangements in patients with nodular fasciitis: a study of rare and novel USP6 fusion partners with a review of the literature

doi: 10.1136/jcp-2023-208768

Figure Lengend Snippet: Patient characteristics and clinical findings in USP6::MYH9 fusion cases

Article Snippet: Thin histological sections (3 μm thick) were used, and each sample was stained using the following antibodies and protocols: anti-smooth muscle actin (SMA) mouse monoclonal antibodies (clone 1A4, BioSB— Bioscience for the World, dilution 1:75; pretreatment: heating up to 99°C in a pH 6 buffer in a water bath); anti-H-caldesmon mouse monoclonal antibodies (clone BSB-19|, BioSB, dilution 1:100; pretreatment: heating up to 99°C in a pH 9 buffer in a water bath); anti-desmin mouse monoclonal antibodies (clone D33, BioSB, dilution 1:100; pretreatment: heating up to 99°C in a pH 9 buffer in a water bath); anti-S100-β rabbit monoclonal antibodies (clone EP32, BioSB, dilution 1:300; pre-treatment: heating up to 99 ◦C in a pH 9 buffer in a water bath); anti-Ki-67 mouse monoclonal antibodies (clone MIB-1, BioSB, dilution 1:150; pretreatment: heating up to 99°C in a pH 6 buffer in a water bath).

Techniques:

Journal: PLoS ONE

Article Title: Expression of MYCN in Multipotent Sympathoadrenal Progenitors Induces Proliferation and Neural Differentiation, but Is Not Sufficient for Tumorigenesis

doi: 10.1371/journal.pone.0133897

Figure Lengend Snippet: (A) Spheres grown from postnatal mouse adrenal glands cells express markers of neural crest stem cells (NCSCs), sympathoadrenal progenitors (SAPs), and sympathetic neurons (SNs). mRNA levels were analyzed by qRT-PCR and are shown in relation to TATA-Box binding protein-1 (TBP1). Expression data from postnatal mouse superior cervical ganglia (SCG) are included for comparison. Data are the mean values from four experiments run in triplicate. *p<0.05,**p<0.01,***p<0.001,****p < .0001, unpaired Student’s t-test. (B) The majority of sphere cells show nuclear SOX10 reactivity by immunoperoxidase labeling. Cytoplasmic nestin expression with linear morphology was observed by immunofluorescence microscopy in spheres. TH expression was prominent at the periphery of spheres. Representative images from 3 independent experiments. Scale bars equal 20 microns. (C) Sphere differentiation was induced by transfer to poly-d-lysine and fibronectin-coated plates in medium with low CEE content. Immunofluorescent images show the three cell types observed in differentiated spheres: TuJ1/TH-reactive neurons, SMA-reactive myofibroblasts, and S100-reactive Schwann cells. Scale bars 50 μM upper left panel and 25 μM all other panels. (D) A shift in differentiation potential was observed from primary spheres, grown directly from adrenal gland cells by differential plating, to secondary spheres, the products of dissociated primary spheres. Differentiated secondary spheres showed a reduced frequency of bipotent and neuron-containing colonies, and were more likely to show myofibroblast-only colonies (primary spheres n = 3, secondary spheres n = 4). Primary and secondary spheres were grown at clonal density.

Article Snippet: Primary antibodies used to assess differentiation by immunocytochemistry included those against neuronal class III β-tubulin (TUJ1)(Covance, MMS-435P, 1:400), S100 (Immunostar, 22520, 1:1), smooth muscle actin (Sigma, A5228, 1:200), and tyrosine hydroxylase (Millipore, AB152, 1:400) following block/permeabilization with 10% goat serum and 0.1% Triton X-100.

Techniques: Quantitative RT-PCR, Binding Assay, Expressing, Comparison, Labeling, Immunofluorescence, Microscopy

Journal: Cells

Article Title: Micro-RNAs Shuttled by Extracellular Vesicles Secreted from Mesenchymal Stem Cells Dampen Astrocyte Pathological Activation and Support Neuroprotection in In-Vitro Models of ALS

doi: 10.3390/cells11233923

Figure Lengend Snippet: MSC-derived EVs reduce astrogliosis in astrocytes from the spinal cords of adult SOD1 G93A mice. ( a ) Representative Western blots (WB) of immunoreactive bands for vimentin, GFAP, and S100β; ( b – d ) quantitative representation of WB densitometric expression signals for ( b ) vimentin, ( c ) GFAP, and ( d ) S100β normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as housekeeping protein, in WT astrocytes, untreated SOD1 G93A astrocytes, and SOD1 G93A astrocytes treated for 24 h with MSC-derived EVs. Vimentin, GFAP, and S100β expression are significantly increased in untreated SOD1 G93A astrocytes vs. WT astrocytes (F (2,12) = 10.998, p = 0.004; F (2,8) = 17.903, p = 0.001; F (2,13) = 9.653, p = 0.005, respectively), while the exposure to EVs significantly reduces their expression (F (2,12) = 10.998, p = 0.01; F (2,8) = 17,903, p = 0.02; F (2,13) = 9.653, p = 0.01, respectively). Data are presented as means ± SEM of n = 3–6 independent experiments; statistical significance (* vs. WT and # vs. SOD1 G93A astrocytes) for p < 0.05 at least (one-way ANOVA, followed by Bonferroni post hoc test). ( e ) Representative confocal microscopy immunocytochemical images for GAPDH (red fluorescence), vimentin (green fluorescence), an’ 4′,6-diamidin-2-fenilindolo (DAPI, blue fluorescence) in WT astrocytes, SOD1 G93A astrocytes and EVs-treated SOD1 G93A astrocytes. Scale bar: 100 µm. ( f – h ) Quantitative representation of protein expression was calculated as the relative fluorescence intensity of the protein of interest co-localized with the stable reference protein GAPDH. The quantitative analyses of the relative co-localization intensity of fluorescence correlated with the protein expression level were performed by calculating the Manders and Costes co-localization coefficients [ , ], and normalizing the results with respect to the fluorescence intensity of the stable housekeeping protein GAPDH. ( f ) Vimentin, ( g ) GFAP and ( h ) S100β expression are significantly increased in untreated SOD1 G93A astrocytes vs. WT astrocytes [F (2,6) = 98.542, p < 0.001; F (2,6) = 47.483, p < 0.001; F (2,6) = 61.283, p < 0.001, for vimentin, GFAP, and S100β, respectively], while the exposure to MSC-derived EVs significantly reduces their expression [F (2,6) = 98.542, p < 0.001; F (2,6) = 47.483, p < 0.001; F (2,6) = 61.283, p < 0.02, for vimentin, GFAP and S100β, respectively]. Data are presented as means ± SEM of n = 3 independent experiments, run in triplicate; statistical significance for p < 0.05 at least (* vs. WT and # vs. SOD1 G93A astrocytes; one-way ANOVA, followed by Bonferroni post hoc test).

Article Snippet: Membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal anti-GFAP (1:1000, Merck, Milan, Italy, Cat# G4546), mouse monoclonal anti-Vimentin antibody (1:1000; Merck, Milan, Italy, Cat# V2258), mouse monoclonal anti-S100β antibody (1:100; Merck, Milan, Italy, Cat# MAB079-1), rabbit recombinant monoclonal anti-NLRP3 antibody (1:100, Abcam, Cambridge, UK, Cat# ab210491), rabbit polyclonal anti-Nrf2 antibody (1:500, Abcam, Cambridge, UK, Cat# ab31163), rabbit polyclonal anti-NQO1 antibody (1:1000, Abcam, Cambridge, UK, Cat# ab34173), and mouse monoclonal anti-GAPDH antibody (1:1000, Merck, Milan, Italy, Cat# G8795).

Techniques: Derivative Assay, Western Blot, Expressing, Confocal Microscopy, Fluorescence