Journal: PLoS ONE

Article Title: Syntaxin Binding Protein 1 Is Not Required for Allergic Inflammation via IgE-Mediated Mast Cell Activation

doi: 10.1371/journal.pone.0058560

Figure Lengend Snippet: A, Wild type, STXBP1 mutant, or heterozygous mice were identified by PCR using DNA from liver cells. B , After 4–5 weeks of culture, liver-derived mast cells (LMC) were examined by toluidine blue staining to determine mast cell purity. A representative photomicrograph is shown (original magnification = ×100). C , LMCs were sensitized with anti-TNP IgE, stained with FITC-conjugated anti-IgE or anti-c-kit antibodies, and examined by flow cytometry. D, STXBP1 +/+ and STXBP1 −/− LMCs were sensitized with anti-TNP IgE and stimulated with TNP-BSA (10 ng/ml) for various times. Whole cell lysates were analyzed by Western blotting for STXBP1 and Actin. E, Sec1/Munc18 (SM) family gene expression profiles were identified by RT-PCR using mRNA from wild type mouse bone marrow-derived mast cells (BMMC), and wild type (STXBP1 +/+ ) or STXBP1 −/− LMCs, with or without TNP-BSA stimulation. All members of SM family are expressed in wild type BMMCs and LMCs. Similar results were observed in 2 independent experiments.

Article Snippet: FITC-conjugated rat anti-mouse IgE (IgG1), FITC-rat IgG1 and FITC-conjugated rat anti-mouse CD117 (c-kit) were purchased from Cedarlane Laboratories (Burlington, ON, Canada).

Techniques: Mutagenesis, Derivative Assay, Staining, Flow Cytometry, Western Blot, Gene Expression, Reverse Transcription Polymerase Chain Reaction

Journal: Genes & Diseases

Article Title: The bidirectional effects of APPswe on the osteogenic differentiation of MSCs in bone homeostasis by regulating Notch signaling

doi: 10.1016/j.gendis.2024.101317

Figure Lengend Snippet: APPswe inhibits the self-renewal, proliferation, and migration of MSCs. (A, B) The effect of APPswe on the self-renewal ability of MSCs was detected by colony-forming unit-fibroblast assays. (C, D) Cell cycle changes in MSCs treated with APPswe were detected by flow cytometry. (E) The expression of the proliferation-related gene Ki67 in different groups was detected by cell immunofluorescence. (F) The expression levels of proliferation-related proteins in MSCs after treatment with APPswe were detected by western blotting. (G) Transwell assays were used to detect the effect of APPswe treatment on the cell migration ability of MSCs. (H) The effect of APPswe on MSC migration was detected by a wound healing test. (I) The expression levels of F-actin in the different treatment groups were detected by FITC-phalloidin staining. (J) Western blot analysis was performed to detect the expression levels of migration-related proteins in the different groups. n = 3; ∗ p < 0.05; ∗∗ p < 0.01, ∗∗∗ p < 0.001. APPswe, Swedish mutant amyloid precursor protein; MSC, mesenchymal stem cell.

Article Snippet: FITC anti-rat CD45, PE anti-rat CD90, and PE anti-rat CD29 antibodies were obtained from Elabscience Biotechnology (Wuhan, China).

Techniques: Migration, Flow Cytometry, Expressing, Immunofluorescence, Western Blot, Staining, Mutagenesis

Journal: Genes & Diseases

Article Title: The bidirectional effects of APPswe on the osteogenic differentiation of MSCs in bone homeostasis by regulating Notch signaling

doi: 10.1016/j.gendis.2024.101317

Figure Lengend Snippet: Effects of APPswe-C on the proliferation and migration of MSCs. (A, B) The effect of APPswe-C on the self-renewal of MSCs was detected by a colony-forming unit-fibroblast assay. (C, D) The effect of APPswe-C on the cell cycle was detected by flow cytometry. (E) The expression of the proliferation gene Ki67 was detected by cellular immunofluorescence. (F) The expression levels of proliferation-related proteins after APPswe-C treatment were detected by western blotting. (G, H) The cell migration of each group after APPswe-C treatment was detected by Transwell assay. (I, J) The effect of APPswe-C treatment on cell migration was detected by a wound healing test. (K) The expression of F-actin in the cells of each group after APPswe-C treatment was detected by FITC-phalloidin staining. (L) The expression levels of cell migration-related proteins were detected by western blot. MSCs were treated with APPswe-C for 24 h and then with BMP2 for 48 h to detect the expression levels of related proteins. n = 3; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. APPswe-C, Swedish mutant amyloid precursor protein without an intracellular segment; MSC, mesenchymal stem cell; BMP2, bone morphogenetic protein 2.

Article Snippet: FITC anti-rat CD45, PE anti-rat CD90, and PE anti-rat CD29 antibodies were obtained from Elabscience Biotechnology (Wuhan, China).

Techniques: Migration, Flow Cytometry, Expressing, Immunofluorescence, Western Blot, Transwell Assay, Staining, Mutagenesis

Journal: The Journal of Experimental Medicine

Article Title: Rcan1 negatively regulates FcεRI-mediated signaling and mast cell function

doi: 10.1084/jem.20081140

Figure Lengend Snippet: Egr1 binds to and transactivates the Rcan1 promoter. (A) RNA from TNP-BSA–treated BMMCs was analyzed by real-time quantitative PCR for Egr1 and Rcan1. Egr1 and Rcan1 expression was normalized to endogenous control GAPDH. The data are expressed as relative mRNA levels compared with the mean expression level in BMMCs treated with TNP-BSA for 15 min (=1; Egr1) or for 60 min (=1; Rcan1), because at this time point Egr1 or Rcan1 showed the highest expression level, respectively ( n = 3 experiments). (B) The location and sequence of the Egr1 binding site on the Rcan1 promoter. (C) BMMCs were transfected with various plasmids generated from pGL4 containing different lengths of the Egr1 binding sequence and the control reporter plasmid pRL-TK. All constructs start at −67 bp relative to the ATG codon. The 5′ end of each construct is shown at the left. Firefly and Renilla activities were sequentially quantified using a dual-luciferase reporter assay system. Results are means ± SEM ( n = 3). (D) Nuclear proteins from untreated (NT) or TNP-BSA–treated BMMCs (TNP-BSA 1 h) were subjected to EMSA. An Egr1 DNA probe was synthesized based on the Egr1 binding sequences on the Rcan1 promoter (E). A mutant Egr1 DNA probe with two nucleotide mutations was used for a competition assay (Em). Strong TNP-BSA–induced Egr1 binding was seen and was competed by the specific cold probe (E) but not by the mutant probe (Em). Results are representative of three independent experiments. (E) ChIP assay for the association of Egr1 with Rcan1 promoter in vivo. BMMCs were stimulated with TNP-BSA for 60 min or left unstimulated. Protein–DNA complexes were extracted and precipitated with anti-Egr1 antibody (Egr1) or control IgG (IgG). DNA from samples before immunoprecipitation (IP) was used as an input control (Input). PCR was performed using primers based on the Rcan1 promoter sequence. Amplified DNA was resolved in agarose gel. Specific Egr1 binding to the Rcan1 promoter was seen in TNP-stimulated cells (lane 8) but not in unstimulated cells (lane 7). Results are representative of three independent experiments. M, molecular marker.

Article Snippet: Antibodies to p38 MAPK and actin were purchased from Santa Cruz Biotechnology, Inc. FITC-conjugated rat anti–mouse CD117 (c-kit) mAb and FITC–rat IgG2a were purchased from Cedarlane Laboratories Ltd. FITC-conjugated rat anti–mouse IgE (IgG1) and FITC–rat IgG1 were purchased from BD.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Sequencing, Binding Assay, Transfection, Generated, Plasmid Preparation, Construct, Luciferase, Reporter Assay, Synthesized, Mutagenesis, Competitive Binding Assay, In Vivo, Immunoprecipitation, Amplification, Agarose Gel Electrophoresis, Marker