Journal: JCI insight

Article Title: Gut mucosal cells transfer α-synuclein to the vagus nerve.

doi: 10.1172/jci.insight.172192

Figure Lengend Snippet: Figure 2. Human A53T α-synuclein protein transfers from gut cells to adjoining vagal neurons. (A) Intestinal organoids were prepared from an SNCAA53T mouse in which CCK-containing cells express enhanced green fluorescent protein (eGFP), and vagal nodose ganglia neurons were isolated from an Snca–/– mouse lacking endogenous α-synuclein. (B) Representative images of organoids and neurons grown in coculture for 5 days, with eGFP-positive cells (green) in the organoid and β-tubulin III (Tuj1, cyan) highlighting neuronal processes. (C) Representative high-magnification α-synuclein (red) staining of an eGFP-positive EEC. Red arrow indicates localization to a PGP9.5-positive (cyan) process in an Snca–/– mouse neuron. (D and E) Representative images with neuron-specific β-tubulin III (cyan). Surface and (adjacent) intracellular confocal slices are shown. Scale bars are 30 μm for B, 3 μm for C, and 5 μm for D and E.

Article Snippet: Primary antibodies used for immunostaining included rabbit CCK (73), rabbit α-synuclein (Abcam catalog ab138501, RRID:AB_2537217, at 1:1,000), guinea pig PGP9.5 (Abcam catalog ab10410, RRID:AB_297150, at 1:100), chick β-tubulin III (Tuj1; Neuromics catalog CH23005, RRID:AB_2210684, at 1:100), and chick GFP (Abcam catalog ab13970, RRID:AB_300798, at 1:1,000).

Techniques: Isolation, Staining

Journal: Cells

Article Title: Neuronal Differentiation of GBM-Initiating Cells Combined with Elimination of Undifferentiated Cells Preserves Motor Function

doi: 10.3390/cells15060539

Figure Lengend Snippet: ISX9 induced GICs to differentiate into neurons. ( A ) Proliferation of GICs, E6 (closed circle) and E16 (open circle), cultured in the presence of various concentrations of ISX9, was determined using the MTT assay. ( B , C ) Dose-dependent ISX9 effects on βIII tubulin ( B ) and Ki67 ( C ) in GICs, E6 (closed circle), and E16 (open circle). ( D ) Heat map with hierarchical clustering of the top 1000 genes in GICs, E6, and E16, cultured with DMSO or ISX9 (30 μM) for 3 days. ( E ) Enriched pathways for clusters A and D. ( F ) Immunoreactivity of hGICs cultured in the presence of ISX9 (30 μM) for 3 days, for hSynapsin (green) and MAP2 (red). The higher magnification images are shown on the bottom right of each figure surrounded by white dotted lines. All nuclei are labeled with Hoechst33342 (Hoechst, blue). Scale bar: 20 μm. ( G ) Quantitative data of ( F ). ** p < 0.01, *** p < 0.001. Statistical significance was determined using the t -test. Error bars indicate ±SD.

Article Snippet: The following Abs were used to detect antigens: mouse monoclonal anti-human mitochondria (1:200, hMito, Novus Biologicals, NBP2-32980-0.1 mg), mouse monoclonal anti-hSynapsin I/II/III as a pre-synapse marker (1:1000, Biolegend), mouse monoclonal anti-βIII tubulin (1:200, R&D Systems, MAB1195), chicken monoclonal anti-MAP2 (1:5000, Novus Bio), chick monoclonal anti-βIII tubulin (1:1000, Novus Bio, NB100-1612), rabbit polyclonal anti-cleaved Caspase 3 (1:1000, Casp3, Cell Signaling Technology, Danvers, MA, USA, 9661), rabbit polyclonal anti-MPZL2 (1:100, EVA1, Proteintech, Rosemont, IL, USA, 11787-1-AP), rabbit polyclonal anti-neurofilament M (1:100, NFM, Sigma-Aldrich, AB1987), and rabbit polyclonal anti-postsynaptic density protein 95 (1:500, PSD95, Proteintech) as a post-synapse marker.

Techniques: Cell Culture, MTT Assay, Labeling

Journal: Cells

Article Title: Neuronal Differentiation of GBM-Initiating Cells Combined with Elimination of Undifferentiated Cells Preserves Motor Function

doi: 10.3390/cells15060539

Figure Lengend Snippet: Examination of optimal method that induces neurons from as many GICs as possible and eliminates undifferentiated GICs. GICs were cultured under three culture conditions, both ISX9 and BRQ simultaneously, ISX9 followed by BRQ, and BRQ followed by ISX9, with either the maximum concentration that induced neuronal differentiation or half the concentration, as shown in and previously . The surviving cells were counted ( A ) and immunolabeled for the GIC marker EVA1 ( B ), two neuronal markers, βIII tubulin ( C ) and MAP2 ( D ), and the proliferation marker Ki67 ( E ). The table shows the concentrations of the chemicals used in each experiment. The arrows indicate the sequential chemical treatments. Error bars indicate ±SD. Statistical significance was determined using one-way ANOVA. Error bars indicate ±SD.

Article Snippet: The following Abs were used to detect antigens: mouse monoclonal anti-human mitochondria (1:200, hMito, Novus Biologicals, NBP2-32980-0.1 mg), mouse monoclonal anti-hSynapsin I/II/III as a pre-synapse marker (1:1000, Biolegend), mouse monoclonal anti-βIII tubulin (1:200, R&D Systems, MAB1195), chicken monoclonal anti-MAP2 (1:5000, Novus Bio), chick monoclonal anti-βIII tubulin (1:1000, Novus Bio, NB100-1612), rabbit polyclonal anti-cleaved Caspase 3 (1:1000, Casp3, Cell Signaling Technology, Danvers, MA, USA, 9661), rabbit polyclonal anti-MPZL2 (1:100, EVA1, Proteintech, Rosemont, IL, USA, 11787-1-AP), rabbit polyclonal anti-neurofilament M (1:100, NFM, Sigma-Aldrich, AB1987), and rabbit polyclonal anti-postsynaptic density protein 95 (1:500, PSD95, Proteintech) as a post-synapse marker.

Techniques: Cell Culture, Concentration Assay, Immunolabeling, Marker