Journal: Cell Death & Disease

Article Title: Reg4 deficiency aggravates pancreatitis by increasing mitochondrial cell death and fibrosis

doi: 10.1038/s41419-024-06738-y

Figure Lengend Snippet: A Co-localization of REG4 (green) with EXTL3 (red) in mice pancreas sections using immunofluorescence microscopy ( n = 3). B Co-immunoprecipitation of REG4 and EXTL3 in mice pancreas ( n = 2). C Knockdown of Extl3 by siRNA (20 nM) in the context of the MIN6 cell line and treated with cerulean (200 μg/ml) for 6 h. Western-blot analysis for PUMA, Cleaved-Caspase 3, Caspase 3, LC3, and β-actin ( n ≥ 3 for each treatment). D Quantification of ( C ). β-actin was used as the internal reference. Randomized one-way ANOVA for ( C ) (ns not significant; ** P < 0.01).

Article Snippet: The mouse pancreatic islet cells MIN6 were obtained from the Wuhan Servicebio Technology Co., Ltd.

Techniques: Immunofluorescence, Microscopy, Immunoprecipitation, Knockdown, Western Blot

Journal: PLoS ONE

Article Title: Modified human glucagon-like peptide-1 (GLP-1) produced in E . coli has a long-acting therapeutic effect in type 2 diabetic mice

doi: 10.1371/journal.pone.0181939

Figure Lengend Snippet: MIN6 cells were cultured to a density of 6×10 4 cells/mL, then seeded into a 96-well flat plate (200 μL per well) and re-cultured for 24 hours. Control: cells treated with Dulbecco's phosphate buffered saline solution; Trypsin: cells treated with 0.25% trypsin; Trypsin + 6×mGLP-1: 10 μg/mL 6×mGLP-1 digested by 0.25% trypsin; GLP-1 standard: chemically synthesized native human GLP-1; Trypsin + GLP-1: 10 μg/mL GLP-1 digested by 0.25% trypsin. Cell density, measured as optical density at A490, was determined 48 h after the addition of the test compounds. The data represent the mean ± standard deviation (n = 6). The experiment was repeated three times and the results were statistically analyzed using one-way ANOVA method following Tukey post-hoc hypothesis test. The symbols a, b, and c refer to the significance level, and different symbol means statistical significance (at 5%, 1%, and 0.1% level) between treatments, or else, no significance.

Article Snippet: Based on the method described by Brandsma et al . [ ], with minor modifications, a mouse pancreatic tumor β cell line (MIN6) (iCell Bioscience Inc, Shanghai, China) was cultured and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% (v/v) fetal bovine serum, 100 U/mL penicillin, 0.1 mg/mL streptomycin and 50 μmol/L 2-mercaptoethanol at 37°C in an incubator with 5% CO 2 and 95% humidified air.

Techniques: Cell Culture, Control, Saline, Synthesized, Standard Deviation

Journal: International Journal of Nanomedicine

Article Title: Small Intestinal Endocrine Cell Derived Exosomal ACE2 Protects Islet β-Cell Function by Inhibiting the Activation of NLRP3 Inflammasome and Reducing β-Cell Pyroptosis

doi: 10.2147/IJN.S450337

Figure Lengend Snippet: Exosome isolation, identification and tracing. ( A ) Representative images of the isolated exosomes observed by TEM. Bar = 100μm. ( B ) WB showed the biomarkers level of CD 9, ALIX and TSG 101 in the isolated exosomes. ( C ) The size and amount of extracellular particles measured by NTA. ( D ) PKH67-labeled exosomes were absorbed by Min6 observed by a confocal microscope (Nikon, Tokyo, Japan). Bar = 50μm. The NCEXO ( E ) and PAEXO ( F ) tracing in vivo was evaluated by the in vivo imaging system (Bruker MI SE 7.2 software, Bruker, German). The fluorescence images were taken at day 1, 2, 3, 5.

Article Snippet: After specific cell treatment, the Min6 cell apoptosis rate was performed using an Annexin V and PI apoptosis kit (Proteintech, PF00005) according to the manufacturer’s protocol and then measured by flow cytometry (Becton, Franklin Lake, NJ, USA).

Techniques: Isolation, Labeling, Microscopy, In Vivo, In Vivo Imaging, Software, Fluorescence

Journal: International Journal of Nanomedicine

Article Title: Small Intestinal Endocrine Cell Derived Exosomal ACE2 Protects Islet β-Cell Function by Inhibiting the Activation of NLRP3 Inflammasome and Reducing β-Cell Pyroptosis

doi: 10.2147/IJN.S450337

Figure Lengend Snippet: ACE2-riched NCEXO regulates proliferation, apoptosis and inflammation of islets β-cells in vitro. ( A and B ) WB analysis revealed that NCEXO exhibited higher levels of ACE2 compared to PAEXO. ( C ) Viability assessment of Min6 cells under varying concentrations of exosomes using CCK-8. ( D ) Flow cytometry analysis of Min6 cells apoptosis rate. Unit (%) ( E ) Min6 cell proliferation was determined with an EDU Kit. Bar = 100μm ( F and H ) Protein expression of Bax, Bcl-2, Caspase-3, Cleaved Caspase-3, NFκB and P- NFκB in MIN6 cells and densitometric quantification. ( G ) Real-time PCR analysis of Bax, Bcl-2, Caspase-3, NFκB, PDX1 of Min6 cell. ( I ) Min6 cell insulin secretion ability was measured by GSIS. *P < 0.05, **P < 0.01 and ***P < 0.001; # P < 0.05 NCEXO group vs PA group.

Article Snippet: After specific cell treatment, the Min6 cell apoptosis rate was performed using an Annexin V and PI apoptosis kit (Proteintech, PF00005) according to the manufacturer’s protocol and then measured by flow cytometry (Becton, Franklin Lake, NJ, USA).

Techniques: In Vitro, CCK-8 Assay, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction

Journal: International Journal of Nanomedicine

Article Title: Small Intestinal Endocrine Cell Derived Exosomal ACE2 Protects Islet β-Cell Function by Inhibiting the Activation of NLRP3 Inflammasome and Reducing β-Cell Pyroptosis

doi: 10.2147/IJN.S450337

Figure Lengend Snippet: The protective effect of NCEXO primarily stems from its ACE2. ( A ) Protein expression of NLRP3, ASC, Caspase-1, Cleaved Caspase-1, Pro-ILβ, IL1β, IL18, GSDMD and N- GSDMD in mouse pancreatic islets and densitometric quantification. ( B ) Quantitative Real-time PCR was used to measure the level of NLRP3, ASC, Caspase-1, IL1β, IL18, GSDMD of mouse pancreatic islets. ( C ) Quantitative Real-time PCR was used to measure the level of NLRP3, ASC, Caspase-1, IL1β, IL18, GSDMD of Min6 cell. ( D ) The densitometric quantification of Protein expression of NLRP3, ASC, Caspase-1, Cleaved Caspase-1, Pro-ILβ, IL1β, IL18, GSDMD and N- GSDMD in mouse pancreatic islets. ( E ) Quantitative Real-time PCR was used to measure the level of FOXO1, NKX6.1, PDX1, ACE2, NFκB, MAFA of Min6 cell. ( F ) Effect of A779/A1-7 on Min6’cells proliferation measured by EDU. Bar = 100μm ( G-I ) Protein expression of FOXO1, NKX6.1, PDX1, IκB, P-IκB, NFκB, P- NFκB, NLRP3, ASC, Caspase-1, Cleaved Caspase-1, Pro-ILβ, IL1β, IL18, GSDMD and N- GSDMD in Min6 cell. *P < 0.05, **P < 0.01 and ***P < 0.001.

Article Snippet: After specific cell treatment, the Min6 cell apoptosis rate was performed using an Annexin V and PI apoptosis kit (Proteintech, PF00005) according to the manufacturer’s protocol and then measured by flow cytometry (Becton, Franklin Lake, NJ, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction