microsomes from mouse liver and small intestine (cd1 strain; male; aged ~11 weeks)  (Sekisui XenoTech)

Journal: Frontiers in Neuroscience

Article Title: Sulfate reducing bacteria induce α-synuclein in intestinal and neuronal cells and tissues and inhibit tyrosine hydroxylase in neuronal cells

doi: 10.3389/fnins.2025.1672793

Figure Lengend Snippet: DSV induces increase in number of cells with α-synuclein aggregates and synuclein expression in intestinal enteroendocrine STC-1 cells (A) . Cells were infected for 24 h. with various DSV MOIs. Cells were then fixed and processed for immunofluorescence using an antibody that specifically recognizes aggregated α-synuclein. (B) Percentage of cells positive for α-syn aggregates were counted and values were compared to control (uninfected) cells. Data represent mean ± SEM. (C) Cells were infected with DSV and harvested for western blotting to probe for α-syn expression. Actin was used as loading control. (D) Western blot images were quantified using Fiji Image J. Data represent mean ± SEM as a fold change difference compared to control. Control values were set as 1. The blot shown is a representative image of multiple independent experiments that were conducted atleast three times. The blot was cut to allow probing for two proteins on the same gel, actin for the upper bands while the lower bands were used for probing synuclein.** p < 0.01, * p < 0.05.

Article Snippet: Mouse small intestinal enteroendocrine cells (STC-1) and Human neuronal (SH-Sy5y) cells were purchased from ATCC (Manassas, VA).

Techniques: Expressing, Infection, Immunofluorescence, Control, Western Blot

Journal: Frontiers in Neuroscience

Article Title: Sulfate reducing bacteria induce α-synuclein in intestinal and neuronal cells and tissues and inhibit tyrosine hydroxylase in neuronal cells

doi: 10.3389/fnins.2025.1672793

Figure Lengend Snippet: Schematic representation of how DSV may be contributing to the pathogenesis of PD. In the setting of dysbiosis, DSV overgrowth may occur in the intestine. Higher number of DSV could then induce increase in α-synuclein aggregates in the intestinal cells such as enteroendocrine cells (EEC) that are known to produce α-syn. In addition, DSV causes secretion of α-syn aggregates from EEC. DSV is also known to disrupt intestinal tight junction barrier leading to leaky gut which, in turn, leads to the translocation of bacteria in the bloodstream. Extracellular α-synuclein aggregates along with DSV could be carried by vagus nerve and/or blood circulation to the brain. In the brain, the transported α-synuclein may further spread to neurons and seed endogenous α-syn aggregation in these cells. Gut-derived α-syn could also activate microglia to produce and secrete TNF-a which in turn could induce α-syn aggregation in the neurons. Furthermore, DSV translocated through leaky gut could directly access neurons to cause α-syn aggregation in these cells. DSV may also activate microglia to secrete TNF-a which in turn could induce α-syn aggregation in neuronal cells. In the neurons, DSV further directly inhibits tyrosine hydroxylase (TH) either by an unknown mechanism and/or mediates its inhibitory effects on TH via its induction of α-syn. Figure was generated using Biorender.com .

Article Snippet: Mouse small intestinal enteroendocrine cells (STC-1) and Human neuronal (SH-Sy5y) cells were purchased from ATCC (Manassas, VA).

Techniques: Translocation Assay, Bacteria, Derivative Assay, Generated

Journal: Heliyon

Article Title: Calycosin prevents NLRP3-induced gut fibrosis by regulating IL-33/ST2 axis

doi: 10.1016/j.heliyon.2024.e30240

Figure Lengend Snippet: NLRP3-IL-33/ST2 signaling in intestinal epithelial cells promotes activation and fibrosis-related factors of intestinal stromal cells . (A–B) The relative protein levels of Collagen-1, TIMP and α-SMA in Cajal cells cultured with the media from MODE-K cells with IL-33 knockdown (Control: MODE-K cells; MOCK: MODE-K cells transfected with negative control shRNA; shIL33: MODE-K cells transfected with specific shRNA against IL33) were detected by carrying out a Western Blotting assay (n = 3). (C–D) The no. of Cajal cells across the membrane was counted by performing a Transwell assay (n = 3). (E–F) The relative levels of α-SMA and collagen-I expression in CA-treated Cajal cells were detected by carrying out a Western Blotting assay (n = 3). (G–H) The relative expressions of Collagen-1, TIMP and α-SMA in Cajal cells with shIL33, ovNLRP3 or combination were detected by carrying out a Western Blotting assay (n = 3). Error bars indicate SEM. * indicates P < 0.05 vs. controls.

Article Snippet: To construct the co-culture system in vitro , immortalized mouse intestinal epithelial cells MODE-K and mouse small intestine mesenchymal cells Cajal were purchased from Procell Life Science&Technology Co.,Ltd. (Wuhan, Hubei, China) and were cultured in DMEM (Hyclone, Hangzhou, China) supplemented with 10 % FBS, 2 mM l -glutamine, 1 % sodium pyruvate and 100 units/mL penicillin G, 100 μg/mL streptomycin sulfate (Beyotime, Hangzhou, China).

Techniques: Activation Assay, Cell Culture, Knockdown, Control, Transfection, Negative Control, shRNA, Western Blot, Membrane, Transwell Assay, Expressing

Journal: Antibody Therapeutics

Article Title: Effects of Fc glycosylation on the activity of WNT mimetic agonistic antibodies

doi: 10.1093/abt/tbae002

Figure Lengend Snippet: N297 mutations disrupt WNT mimetic activity. (A) Diagram of the WNT mimetic molecule FA-L6. (B) Mutation of N297 to G significantly disrupts the activity of FA-L6 WNT mimetic. (C) Mutations of N297 significantly disrupt the activity of FA-L6 WNT mimetic, whereas the LALAPG mutation does not. (D) Activity of FA-L6 Fc mutations tested in a mouse small intestinal organoid growth assay. Representative images of mouse small intestinal organoids treated with Basal media, Basal media +1 μM IWP2 or with Basal media +1 μM IWP2 plus 1 nM FA-L6 WT, 10 nM FA-L6 WT, 1 nM FA-L6 LALAPG, 10 nM FA-L6 LALAPG, 1 nM FA-L6 N297A, 10 nM FA-L6 N297A, 1 nM FA-L6 N297G and 10 nM FA-L6 N297G. Bars: 100 μm. (E–G) The removal of the N297G Fc domain by IdeS (E) rescues the molecule’s STF activity (F). (G) SDS-PAGE of IdeS digested FA-L6 N297G. (H and I) Removal of the Fc domain from FA-L6 WT does not affect the molecule’s activity. (H) STF activity of untreated and IdeS treated FA-L6 WT. (I) SDS-PAGE of IdeS digested FA-L6 WT.

Article Snippet: Mouse small intestinal organoids (STEMCELL technologies Cat# 70931) were maintained in mouse IntestiCult Organoid Growth Medium (STEMCELL technologies Cat# 06005) and split at a 1 to 8 ratio after fully grown for 7 days to set up the growth assay in 48-well tissue culture plates.

Techniques: Activity Assay, Mutagenesis, Growth Assay, SDS Page