Journal: Neural Regeneration Research

Article Title: Gut microbiota dysbiosis contributes to α-synuclein-related pathology associated with C/EBPβ/AEP signaling activation in a mouse model of Parkinson's disease

doi: 10.4103/1673-5374.391191

Figure Lengend Snippet: Rotenone administration induces intestinal barrier disruption in a gut microbiota-dependent manner. (A) Colonic mucus layer thickness (per section/two sections per animal, n = 3). (B) Alcian blue-stained colonic sections showing the mucus layer (arrows). The paired black arrows indicate where the mucus layer was measured. Scale bars: 50 μm ( n = 3). (C) Immunofluorescence images of colonic sections stained with anti-MUC2 antibody and 4′,6-diamidino-2-phenylindole (DAPI). Paired white arrows indicate the mucus layer. Scale bars: 50 μm. (D) Protein levels of occludin and zonula occludens-1 (ZO-1) in the colon ( n = 5). (E) Serum lipopolysaccharide (LPS) endotoxin levels ( n = 10). (F–H) mRNA expression levels of interleukin (IL-6; F), tumor necrosis factor-α (TNF-α; G), and inducible nitric oxide synthase (iNOS; H) in the colon ( n = 5). (I) Colon length ( n = 9), with representative images shown. (J) Colonic mucus layer thickness (per section/two sections per animal, n = 3). (K) Alcian blue-stained colonic sections showing the mucus layer (arrows). Paired black arrows indicate where the mucus layer was measured (scale bars: 50 μm). (L) Immunofluorescence images of colonic sections stained with anti-MUC2 antibody and DAPI. Paired white arrows indicate the mucus layer. Scale bars: 50 μm. (M) Protein levels of occludin and ZO-1 in the colon ( n = 5). (N) Serum LPS endotoxin levels ( n = 10). (O–Q) mRNA expression levels of IL-6, TNF-α, and iNOS in the colon ( n = 5). (R) Colon length ( n = 9), with representative images shown. Values are presented as mean ± standard error of the mean. * P < 0.05, ** P < 0.01, *** P < 0.001, vs . Con. Con: control; Rot: rotenone; Con + Ab: Con mice treated with antibiotics; Rot + Ab: Rot mice treated with antibiotics.

Article Snippet: The sections were cross-validated with primary antibody anti-mucin-2 (MUC2) (rabbit, 1:500, Abclonal, Cat# A14659) staining at 4°C overnight, followed by incubation with goat-anti-rabbit IgG H&L/AF594 antibody (goat, 1:1000, Bioss, Beijng, China, Cat# bs-0295G-AF594) for 1 hour at room temperature.

Techniques: Disruption, Staining, Immunofluorescence, Expressing

Journal: Neural Regeneration Research

Article Title: Gut microbiota dysbiosis contributes to α-synuclein-related pathology associated with C/EBPβ/AEP signaling activation in a mouse model of Parkinson's disease

doi: 10.4103/1673-5374.391191

Figure Lengend Snippet: Transplantation of healthy gut microbiota alleviates rotenone-induced motor deficits, intestinal inflammation, endotoxemia, and intestinal barrier impairment. (A) Rotarod test. The Y-axis shows the time spent on the rotarod. (B) Pole test. The Y-axis shows the time of descent from the top of the pole to the ground. (C) Colon length ( n = 9), with representative images shown. (D–F) mRNA expression levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and inducible nitric oxide synthase (iNOS) in the colon ( n = 5). (G) Serum lipopolysaccharide (LPS) endotoxin levels ( n = 10). (H) Colonic mucus layer thickness (per section/two sections per animal, n = 3). (I) Alcian blue-stained colonic sections showing the mucus layer (arrows). Paired black arrows show where the mucus layer was measured (scale bars: 50 μm). (J) Immunofluorescence images of colonic sections stained with anti-mucin-2 (MUC2) antibody and 4′,6-diamidino-2-phenylindole (DAPI). Paired white arrows indicate the mucus layer. Scale bars: 50 μm. Values are mean ± standard error of the mean. * P < 0.05, ** P < 0.01, *** P < 0.001, vs. Rot + PBS. Rot: Rotenone; Rot + FMT Con : Rot mice transplanted with gut microbiota from donor control mice; Rot + PBS: Rot mice treated with PBS. PBS: Phosphate-buffered saline.

Article Snippet: The sections were cross-validated with primary antibody anti-mucin-2 (MUC2) (rabbit, 1:500, Abclonal, Cat# A14659) staining at 4°C overnight, followed by incubation with goat-anti-rabbit IgG H&L/AF594 antibody (goat, 1:1000, Bioss, Beijng, China, Cat# bs-0295G-AF594) for 1 hour at room temperature.

Techniques: Transplantation Assay, Expressing, Staining, Immunofluorescence, Saline

Journal: bioRxiv

Article Title: Retention of DLK1 in the endoplasmic reticulum identifies roles for EGF domain-specific O-glycans in the secretory pathway

doi: 10.1101/2024.08.31.610613

Figure Lengend Snippet: (A) The cell surface transport assay for DLK1 was developed based on the RUSH method. Reporter DLK1 is composed of DLK1 ( black ), GFP ( green ), and streptavidin-binding peptide (SBP, red ), while Hook protein is composed of Ii ( black ) and streptavidin ( grey ). Reporter DLK1 and Hook protein were designed so that both proteins bind via SBP and streptavidin fused to the C-terminus of Reporter DLK1 and Hook protein, respectively. Upon induction with doxycycline (DOX), the Ii domain directs Hook protein to the ER where Reporter DLK1 is accumulated. Biotin competes for Hook protein, thus allowing Reporter DLK1 to be transported to the cell surface via the Golgi. (B) Expression of Reporter DLK1 in the transport assay. After DOX induction, transfected HEK293 cells were treated with biotin for 60 min and analyzed by flow cytometer. Anti-DLK1 antibody detects cell surface expression of Reporter DLK1. GFP expression reflects the total expression level of Reporter DLK1. (C) Time course for the surface expression of Reporter DLK1 after the addition of biotin. Transfected HEK293 cells were gated at a constant GFP intensity in all samples. (D) Detection of Reporter DLK1 del and Hook protein expressed in HEK293 cells. (E) Comparison of mucin type O-glycan modifications on DLK1 [249-257] and DLK1 [268-283] peptides derived from the DLK1-ECD or Reporter DLK1. The mean ± standard deviation (n=3) is presented. A list of glycopeptides detected by mass spectrometry is summarized in Table S1 (DLK1-ECD) and Table S2 (Reporter DLK1). The MS/MS spectra are presented in Fig.S7 and S8 (DLK1-ECD) and Fig.S9 and S10 (Reporter DLK1).

Article Snippet: To generate a vector expressing the human DLK1 ectodomain (ECD), the entire EGF domain and the flanking region modified by mucin-type O-glycans (amino acid position 18-302) were inserted into the pSeqTag2C-MycHis6 vector (Invitrogen) at the SfiI/NotI sites.

Techniques: Transport Assay, Binding Assay, Expressing, Transfection, Flow Cytometry, Comparison, Derivative Assay, Standard Deviation, Mass Spectrometry, Tandem Mass Spectroscopy