Journal: Frontiers in Immunology

Article Title: Bifunctional Small Molecules Enhance Neutrophil Activities Against Aspergillus fumigatus in vivo and in vitro

doi: 10.3389/fimmu.2019.00644

Figure Lengend Snippet: Bifunctional compounds enhance phagocytosis of A. fumigatus conidia by zebrafish neutrophils expressing human FPR1. (A) (i) Diagram of experimental approach: Calcofluor-stained A. fumigatus conidia (blue) are co-delivered with treatments into the circulation via the Duct of Cuvier at 3 days post-fertilization. Imaging is performed at 2 h post-infection at a distal site, the caudal venous plexus, which is rich in leukocytes. (ii) Example image of 3 dpf irf8 -MO treated Tg( mpx :EGFP) larva (GFP-labeled neutrophils) with mosaic expression of human FPR1 (traced by mCherry, red fluorescence), 2 h following delivery of calcofluor-labeled (blue fluorescent) A. fumigatus conidia. A GFP/mCherry co-labeled leukocyte containing phagocytosed conidia is indicated in magnified panel (open white arrowhead). Off-target expression of the transgene was also observed in tissues including the somites (full white arrowhead). (B) Treatment with C-001 and C-016 resulted in significantly increased phagocytosis of conidia by GFP/mCherry+ (human FPR1-expressing) neutrophils compared to GFP(only) wild-type cells. Each point represents an infected larva. N ≥ 40 larva scored per condition. Data collated from multiple experiments. Statistics: two-tailed T -test. * p ≤ 0.05, *** p ≤ 0.001.

Article Snippet: Human formyl-peptide receptor (FPR1) was sub-cloned from pBGSA FPR1-EGFP (Addgene ID:62604) into a middle entry vector and combined with existing 5′ (lyzC promoter) and 3′ (T2A-mCherry) vectors using standard Gateway approaches.

Techniques: Expressing, Staining, Imaging, Infection, Labeling, Fluorescence, Two Tailed Test

Journal: Scientific Reports

Article Title: Matrin-3 is essential for fibroblast growth factor 2-dependent maintenance of neural stem cells

doi: 10.1038/s41598-018-31597-x

Figure Lengend Snippet: Identification of NSC maintaining phosphorylated proteins by 2D-DIGE and mass spectrometry analysis. ( A ) The experimental strategy for 2D-DIGE. ( B ) The 2D-DIGE map and 3D-images of up- or downregulated proteins. The asterisks indicate the up- or downregulated phosphoproteins. The red frame indicates the Matrin-3 protein group. Bottom panels (spots 871 and 889): 3D-images of Matrin-3 protein expression at 0 and 60 min after FGF2 stimulation following 6-h FGF2 deprivation. ( C ) The identified major proteins are listed. The spot numbers correspond to the annotation shown in ( B ). Detailed results of the protein identification are shown in Table .

Article Snippet: A total of 2 μg of the plasmid encoding EGFP and mouse Matrin-3-siRNA (Santa Cruz Biotechnology, CA, USA) was cotransfected.

Techniques: Mass Spectrometry, Expressing

Journal: Scientific Reports

Article Title: Matrin-3 is essential for fibroblast growth factor 2-dependent maintenance of neural stem cells

doi: 10.1038/s41598-018-31597-x

Figure Lengend Snippet: In vitro and in vivo expression of Matrin-3 and phospho-Matrin-3 (Ser208). ( A ) The expression of Matrin-3 and phospho-Matrin-3 (Ser208) in NSCs after FGF2 deprivation (-FGF2) and restimulation (+FGF2) on 1D-WB. ( B , C ) Mobility changes in phospho-Matrin-3 (P-Ser208-Matrin-3) and Matrin-3 after FGF2 deprivation and restimulation on 2D-WB. The lanes indicate the FGF2 stimulation times. ( B ) The blue arrowhead indicates the alkaline shift by dephosphorylation. The yellow arrowhead indicates the acidic shift by phosphorylation. ( C ) Red arrowheads indicate phospho-Matrin-3 (P-Ser208-Matrin-3) appearance. ( D ), (a) Expression of Matrin-3, (b) phospho-Matrin-3 (P-Ser208-Matrin-3), and (c) ATM from embrynic and adult mouse cerebra on 1D-WB. ( E ) Immunostaining of phospho-Matrin-3 (P-Ser208-Matrin-3), Ki67, and Tuj1 in the E14 mouse cortex (serial sections). CP; cortical plate, IZ; intermediate zone, SVZ; subventricular zone, VZ; ventricular zone. Actin is used as a control. Bar, 100 μm. ( F ) HE, DAPI staining and immunostaining of phospho-Matrin-3 (P-Ser208-Matrin-3) of the murine adult hippocampal dentate gyrus (same sections). DG; dentate gyrus, CA; cornet d’Ammon. Bars: 500 μm (upper panel), 200 μm (bottom panel); n = 5. Dotted line, subgranular zone (SGZ).

Article Snippet: A total of 2 μg of the plasmid encoding EGFP and mouse Matrin-3-siRNA (Santa Cruz Biotechnology, CA, USA) was cotransfected.

Techniques: In Vitro, In Vivo, Expressing, De-Phosphorylation Assay, Immunostaining, Staining

Journal: Scientific Reports

Article Title: Matrin-3 is essential for fibroblast growth factor 2-dependent maintenance of neural stem cells

doi: 10.1038/s41598-018-31597-x

Figure Lengend Snippet: Significance of Matrin-3 for maintaining NSCs in vitro and in vivo . ( A ), (a) In vitro Matrin-3-siRNA induces extension of cellular processes of GFP + NSCs. Red arrowheads indicate extension of cellular processes. (b) Matrin-3-siRNA reduced neurosphere-forming stem cells. Bar, 100 μm. (c) The number of GFP + neurospheres per dish was counted. ** P < 0.01, * P < 0.05 (one-way ANOVA plus Bonferroni/Dunn post-hoc test). Error bars, SE (5 separate experiments). ( B ), (a) Co-immunostaining for GFP and nestin, Ki67, or Tuj1 of NSCs in vitro . Cells are co-immunostained for GFP and nestin, Ki67 (blue arrowheads), or Tuj1 (white arrowheads). (b) Statistical measurements of neuronal differentiation in Matrin-3-knockdown cells. The number of nestin + , Ki67 + and Tuj1 + cells in GFP + cells in one view was counted. ** P < 0.01, * P < 0.05 (one-way ANOVA plus Bonferroni/Dunn post-hoc test). Error bars, SE (5 separate experiments). ( C ) In utero knockdown of Matrin-3 in the SVZ and VZ layer. (a) GFP + cells in SVZ and VZ areas at E17.5. The cells are counterstained with DAPI. Bar, 500 μm. (b) Immunostaining of Matrin-3 shows the reduced expression in the Matrin-3 siRNA-treated tissue compared to the control siRNA-treated tissue. The dotted line surrounds the electroporated area. (c) HE staining revealed the disordered layer structure. Blue arrowheads indicate the SVZ/VZ area. (d) Co-immunostaining for GFP and nestin, Ki67, or NeuN in the VZ. Yellow arrowheads indicate nestin + , Ki67 + and NeuN + cells in GFP + cells. Bar, 50 μm; n = 4–5.

Article Snippet: A total of 2 μg of the plasmid encoding EGFP and mouse Matrin-3-siRNA (Santa Cruz Biotechnology, CA, USA) was cotransfected.

Techniques: In Vitro, In Vivo, Immunostaining, In Utero, Expressing, Staining

Journal: Scientific Reports

Article Title: Matrin-3 is essential for fibroblast growth factor 2-dependent maintenance of neural stem cells

doi: 10.1038/s41598-018-31597-x

Figure Lengend Snippet: ATM phosphorylates Ser208 of Matrin-3 in the nucleus of NSCs. ( A ) Extension of cellular processes of NSC is induced by ATM inhibition (KU55933) in vitro . The FGF2 deprivation model (-FGF2) is used as an indicator of neuronal differentiation. Red arrowheads indicate extension of cellular processes. ( B ), (a) Phospho-mutant Matrin-3(Ser208Ala) inhibits the formation of neurospheres.P-Ser208-Matrin-3, Flag and DAPI images are merged. Bar, 100 μm. (b) Statistical measurements of Flag + neurospheres in per dish were performed. ** P < 0.01 (Student’s t test). Error bars, SE (5 separate experiments). ( C) , (a) Flag-Matrin-3 and Flag-Matrin-3-Ser208Ala plasmids were transfected into NSCs in vitro . Phospho-mutant Matrin-3 inhibits Matrin-3 nuclear localisation and induces neuronal differentiation. Flag, Tuj1, and DAPI images are merged. Tuj1 + /Flag + cells are observed to assess the nuclear translocation of Matrin-3 and neural differentiation. White arrowheads indicate incidence of Tuj1 + in Flag + cells. (b) The bar graph indicates Tuj1 + /Flag + cells. ** P < 0.01 (Student’s t test). Error bars, SE (5 separate experiments). ( D ) Phosphorylation of Matrin-3 is necessary to regulate NSCs and to maintain self-renewal ability and the undifferentiated state.

Article Snippet: A total of 2 μg of the plasmid encoding EGFP and mouse Matrin-3-siRNA (Santa Cruz Biotechnology, CA, USA) was cotransfected.

Techniques: Inhibition, In Vitro, Mutagenesis, Transfection, Translocation Assay