Journal: BMC Cardiovascular Disorders

Article Title: β1 adrenoceptor antibodies induce myocardial apoptosis via inhibiting PGC-1α-related pathway

doi: 10.1186/s12872-020-01492-3

Figure Lengend Snippet: List of utilized primers for qRT-PCR

Article Snippet: Antibodies against β1AR (1:1000; cat. bs-0498R, Bioss), VEGF (1:800; cat. AF5131, Affinity), PGC-1α (1:1000, cat. bs-1832R, Bioss), and caspase3 (1:1000; cat. bs-0081R, Bioss) were used as primary antibodies.

Techniques:

Journal: BMC Cardiovascular Disorders

Article Title: β1 adrenoceptor antibodies induce myocardial apoptosis via inhibiting PGC-1α-related pathway

doi: 10.1186/s12872-020-01492-3

Figure Lengend Snippet: β1AA inhibited the PGC-1α related factor expression. β1AA significantly inhibited the mRNA expression of PGC-1α ( a ) and VEGF ( b ). And the effect of β1AA on mRNA expression of PGC-1α could also be mimicked by isoprenaline. However, isoprenaline promoted the mRNA expression of VEGF (n = 5). c ELISA test for secreted VEGF in the supernatant of the medium (n = 5). d Raw data of results of WB test. e Relative expression content of PGC-1α, VEGF and caspase3 (n = 5). β1AA increased the protein expression of caspase3, and inhibited the protein expression of PGC-1α and VEGF. The effects of β1AA could be inhibited by atenolol. And the effect of β1AA on protein expression of PGC-1α could also be mimicked by isoprenaline. However, isoprenaline promoted the protein expression of VEGF expression rather than inhibition as β1AA did. **, P ≤ 0.01. n.s., no statistical significance. Group data presented by mean ± SEM

Article Snippet: Antibodies against β1AR (1:1000; cat. bs-0498R, Bioss), VEGF (1:800; cat. AF5131, Affinity), PGC-1α (1:1000, cat. bs-1832R, Bioss), and caspase3 (1:1000; cat. bs-0081R, Bioss) were used as primary antibodies.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: BMC Cardiovascular Disorders

Article Title: β1 adrenoceptor antibodies induce myocardial apoptosis via inhibiting PGC-1α-related pathway

doi: 10.1186/s12872-020-01492-3

Figure Lengend Snippet: Overexpression of PGC-1α rescued the inhibitory effect of β1AA on VEGF expression. a Raw data of results of WB test. β1AA increased the protein expression of β1AR and caspase3, and inhibited the protein expression of VEGF. But the overexpression of PGC-1α inhibited the effect of β1AA. b Overexpression of PGC-1α increased the VEGF mRNA expression and rescued the inhibitory effect of β1AA. c ELISA test for secreted VEGF in the supernatant of the medium. Overexpression of PGC-1α also rescued the inhibitory effect of β1AA on the VEGF secretion. d and e overexpression of PGC-1α increased the NRF1 and ERRα mRNA expression and rescued the inhibitory effect of β1AA. **, P ≤ 0.01. Group data presented by mean ± SEM

Article Snippet: Antibodies against β1AR (1:1000; cat. bs-0498R, Bioss), VEGF (1:800; cat. AF5131, Affinity), PGC-1α (1:1000, cat. bs-1832R, Bioss), and caspase3 (1:1000; cat. bs-0081R, Bioss) were used as primary antibodies.

Techniques: Over Expression, Expressing, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: AXL-GAS6/PROS1 Interaction: A Critical Switch Between Aberrant- and Healthy Repair Following Alveolar Lung Injury

doi: 10.1101/2025.04.07.647567

Figure Lengend Snippet: (A) IPF signaling pathway was one of the increasingly upregulated pathways over time as revealed by IPA analysis. The gene set contributing to this pathway included distinct groups of early and late profibrotic associated genes (marked by black rectangles) that were serially and time dependently upregulated. Timepoint order forced, genes hierarchical clustered. (B- D) Fold change of mRNA expression levels of TAM RTK family, Axl , Mertk and Tyro3 over the course of repair. (E-F) Fold change of expression level of TAM RTK ligands, Gas6 and Pros1 over the course of repair. Data are shown as mean ± SEM of n= 3-7. P ≤ 0.05 (*); P ≤ 0.01 (**); P ≤ 0.001 (***); P ≤ 0.0001 (****) compared with stuffer control.

Article Snippet: GAS6 was measured in human BALF or SAEC supernatant using the human GAS6 DuoSet ELISA kit (R&D Systems, #DY885B) and the human PROS1 ELISA Kit (Aviva Systems Biology, #OKBB010207).

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: AXL-GAS6/PROS1 Interaction: A Critical Switch Between Aberrant- and Healthy Repair Following Alveolar Lung Injury

doi: 10.1101/2025.04.07.647567

Figure Lengend Snippet: (A) Schematic of the e xperimental setup to study AXL-GAS6/PROS1 interaction using aFRET in mouse lung tissue sections. Lungs were isolated on day 4 post DT. aFRET is a coincidence assay, labels both receptor (AXL) and ligands (GAS6 or PROS1) (see Methods for details). FRET occurs in the range of 1-10 nm. Scheme was created in BioRender. Geillinger-kästle, K. (2025) https://BioRender.com/h45x771 (B) Representative image of AXL-GAS6 interaction. (C) Representative image of AXL-PROS1 interaction. (B-C) Upper figures showed the mean of the donor lifetime (Ƭ) in the absence of acceptor. Lower figures showed the interaction state between AXL-GAS6/ PROS1 for each coincidental region (marked by yellow and black circles). Different coincidental regions possessed different interaction state. (D) Box and whiskers plots showed the heterogeneity of AXL-GAS6/PROS1 interaction states represented as FRET efficiency (Ef%). Ef% below 4% represents the Förster Radius (R 0 at 5.83nm) and not proteins, interactive state as marked by red dashed line. Data are shown as box and whiskers plot of n=5-6. Each dot represents median of Ef% for each mouse. P ≤ 0.05 (*). (E) Correlation plot between Ef% AXL-GAS6 and AXL expression. (F) Correlation plot between Ef% AXL-PROS1 and AXL expression. (G-H) Surface plasmon resonance response curve showing the binding of rhGAS6 and rhPROS1 in different concentration to rhAXL. (I-J) Concentration dependent binding of rhGAS6 and rhPROS1 to rhAXL. Different colored lines in (G-H) correspond to different colored dots in (I-J), which indicate different concentrations of rhGAS6 and rhPROS1. (K-L) GAS6 and PROS1 concentration in human BALF of non- diseased control, ILD/IPF-, and COPD patients. Data are shown as mean ± SEM of n= 4-8. P ≤ 0.01 (**); P ≤ 0.001 (***).

Article Snippet: GAS6 was measured in human BALF or SAEC supernatant using the human GAS6 DuoSet ELISA kit (R&D Systems, #DY885B) and the human PROS1 ELISA Kit (Aviva Systems Biology, #OKBB010207).

Techniques: Isolation, Expressing, SPR Assay, Binding Assay, Concentration Assay, Control

Journal: bioRxiv

Article Title: AXL-GAS6/PROS1 Interaction: A Critical Switch Between Aberrant- and Healthy Repair Following Alveolar Lung Injury

doi: 10.1101/2025.04.07.647567

Figure Lengend Snippet: (A) 7664 is a tissue section from a healthy lung whereas 1015 and 1033 are sections from IPF patients with various advanced degrees of IPF. The panels show representative images mapping the interactive states of GAS6 and PROS1 with AXL (pseudo-colour heat map of (Ef%) on the expression level of GAS6 and PROS1 (fluorescence images in grey scale). The individual violin plots quantify the heterogeneity of interactive states for both ligands and the receptor. The dotted line at 4% Ef represents the Förster Radius (R 0 ). Values below 4% are not representative of ligand-receptor interactions. (B) Global violin plots of all coincident regions (per pixel) of AXL-GAS6 and AXL-PROS1. Each global violin plot represents 2x10 6 data points. To determine the p values for AXL-GAS6 and AXL-PROS1, using non-parametric Mann- Whitney U test, we utilised 1000 randomly generated data points of the 2x10 6 . Healthy lung has similar distribution of AXL-GAS6 and AXL-PROS1 interactions, whereas the two patient samples show, in one case (1015) no interaction of AXL-GAS6 or AXL-PROS1 and another case (1033) a significantly higher distribution of AXL-PROS1 versus AXL-GAS6. The p values between AXL-GAS6 and AXL-PROS1 are 1.9x10 -3 (7664), 2.4x10 -1 (1015) and 8.2x10 - respectively. The dotted line at 4% Ef represents the Förster Radius (R 0 ).

Article Snippet: GAS6 was measured in human BALF or SAEC supernatant using the human GAS6 DuoSet ELISA kit (R&D Systems, #DY885B) and the human PROS1 ELISA Kit (Aviva Systems Biology, #OKBB010207).

Techniques: Expressing, Fluorescence, MANN-WHITNEY, Generated

Journal: bioRxiv

Article Title: AXL-GAS6/PROS1 Interaction: A Critical Switch Between Aberrant- and Healthy Repair Following Alveolar Lung Injury

doi: 10.1101/2025.04.07.647567

Figure Lengend Snippet: AXL is predominantly expressed in basal and aberrant basaloid cells, promoting proliferation via AXL-GAS6 signaling. (A) UMAP plots visualize AXL and its ligands, GAS6 and PROS1 normalized expression in the epithelial cell compartment of an integrated scRNA-seq IPF atlas. (B) Quantification of AXL and its ligands expression in epithelial cells of IPF patients and the non-diseased controls. (C) Expression of AXL and its ligands within the epithelial cells compartment in the lung of PF patients and the non-diseased controls. (D-E) AXL and its ligands expression in SAEC under submerged culture condition. (F) Representative western blot image validating knockout of AXL via CRISPR/Cas9 in 2 different donors. AXL (140 KDa, red bands), loading control ß-Actin (42 KDa, green bands), and M is protein marker. (G) Quantification of AXL expressions of each donor before and after CRISPR/Cas9 mediated knockdown. Experiments were done in duplicate. (H) Proliferation of AXL WT and AXL KD SAEC as assessed by BrdU in all donors 48 hours post seeding. (I) Correlation between AXL expression in (G) and SAEC proliferation in (H). (J) Proliferation of AXL WT and (K) AXL KD SAEC as assessed by BrdU 48 hours post treatment with rhGAS6/ rhPROS1/ combination of both proteins. WT: Wildtype; KD: Knockdown. Data are shown as mean ± SEM of n= 4 – 7. P > 0.05 (ns/ non-significant); P ≤ 0.05 (*); P ≤ 0.001 (***).

Article Snippet: GAS6 was measured in human BALF or SAEC supernatant using the human GAS6 DuoSet ELISA kit (R&D Systems, #DY885B) and the human PROS1 ELISA Kit (Aviva Systems Biology, #OKBB010207).

Techniques: Expressing, Western Blot, Knock-Out, CRISPR, Control, Marker, Knockdown

Journal: bioRxiv

Article Title: AXL-GAS6/PROS1 Interaction: A Critical Switch Between Aberrant- and Healthy Repair Following Alveolar Lung Injury

doi: 10.1101/2025.04.07.647567

Figure Lengend Snippet: (A) SAEC basal cells were allowed to differentiate for 23 days. On day 23 post ALI, 5 ng/ml rhTGF-ß was added to the culture. On day 26 post ALI, rhTGF-ß was added together with rhGAS6, rhPROS1, or a combination of both. Analysis was performed on day 29 post ALI. Scheme was created in BioRender. Geillinger-kästle, K. (2025) https://BioRender.com/k46g355 . (B) GAS6 and (C) PROS1 concentration in the SAEC cell culture supernatant post rhTGF-ß treatment compared to the BSA control. (D) Epithelial barrier integrity as measured by FITC-dextran permeability assays. (E) Fold change of AXL mRNA expression as measured by qPCR. (F) Correlation between slope measured by FITC- dextran permeability assay (D) and ΔCT of AXL (r= -0.9519 R 2 = 0.9062) measured by qPCR (E). Data are shown as mean ± SEM of n= 4 – 5. P > 0.05 (ns/ non-significant); P ≤ 0.05 (*); P ≤ 0.01 (**).

Article Snippet: GAS6 was measured in human BALF or SAEC supernatant using the human GAS6 DuoSet ELISA kit (R&D Systems, #DY885B) and the human PROS1 ELISA Kit (Aviva Systems Biology, #OKBB010207).

Techniques: Concentration Assay, Cell Culture, Control, Permeability, Expressing, FITC-Dextran Permeability Assay

Journal: bioRxiv

Article Title: AXL-GAS6/PROS1 Interaction: A Critical Switch Between Aberrant- and Healthy Repair Following Alveolar Lung Injury

doi: 10.1101/2025.04.07.647567

Figure Lengend Snippet: After injury, the normal repair process involves an initial inflammatory response, followed by AXL- GAS6 interaction, which promotes cell proliferation. Once sufficient proliferation is achieved, AXL-PROS1 binding occurs, halting proliferation and triggering cell differentiation. This balance between proliferation and differentiation ensures proper repair, leading to healthy alveoli. However, dysbalanced AXL signaling due to repeated injury and persistently high TGF- ß levels, can result in aberrant repair and irreversible fibrosis (IPF). This may occur due to high-affinity interaction between AXL and GAS6 prevents PROS1 from effectively binding to AXL. As a result, excessive proliferation occurs with insufficient differentiation, leading to aberrant repair and fibrosis. Scheme was created in BioRender. Geillinger-kästle, K. (2025) https://BioRender.com/q77c168.xss

Article Snippet: GAS6 was measured in human BALF or SAEC supernatant using the human GAS6 DuoSet ELISA kit (R&D Systems, #DY885B) and the human PROS1 ELISA Kit (Aviva Systems Biology, #OKBB010207).

Techniques: Binding Assay, Cell Differentiation

Journal: World Journal of Diabetes

Article Title: What can we learn from β-cell failure biomarker application in diabetes in childhood? A systematic review

doi: 10.4239/wjd.v12.i8.1325

Figure Lengend Snippet: Overview of the included studies with their corresponding quality assessment

Article Snippet: Xu et al [ ], 2017 , China , Antioxidant biomarkers; NF-KB, Bax, cleaved Caspase-3 and Bcl2 , Level of antioxidant SOD, MDA and GSP levels; Beta cell function was measured (HOMA). , SOD, MDA and GSP → kits Nanjing Jiancheng Biotechnology Institute (Nanjing, China). Antibodies for Bax, Bcl-2, cleaved Caspase-3 and NF-KB → Santa Cruz Biotechnology (Santa Cruz, CA, United States). Nuclear and cytoplasmic protein → ProteoJETTM Cytoplasmic and Nuclear Protein Extraction kit; (Fermentas International Inc., Burlington, ON, Canada). Protein content → BCA protein assay kit (Pierce Biotechnology, Rockford, United States) , Cells were scraped, pelleted by centrifugation at 250 g for 5 min, mixed with cell lysis buffer, homogenized, and set on ice for 10 min. Then, themixture was centrifuged at 500 g for 7 min at 4 C, and then the supernatant was further centrifuged at 20000 g for 15 min at 4 °C , Immunochemistry; Western Blot; ELISAs , Animal experiment; Male Sprague–Dawley rats; Five experimental groups: Normal control group; Model control group; Ginseng oligopeptides (GOP): Low-dose group (GOP-L); Medium-dose group (GOP-M); High-dose group (GOP-H) , Not exclusion criteria were specified , Not applied , Not applied , This study demonstrated that treatment with ginseng oligopeptides partially reversed abnormal oral glucose test tolerance in rats induced with T2DM and demonstrated an amelioration of the pancreatic damage and increased insulin content , Oral glucose tolerance, plasma glucose, serum insulin, ALT, AST, serum urea nitrogen, total cholesterol, triglycerides , T2DM prediction.

Techniques: Control, Biomarker Discovery, Permeability, High Molecular Weight, Infection, Adhesive, Expressing, Transgenic Assay, Functional Assay, In Situ, Clinical Proteomics, In Vivo

Journal: World Journal of Diabetes

Article Title: What can we learn from β-cell failure biomarker application in diabetes in childhood? A systematic review

doi: 10.4239/wjd.v12.i8.1325

Figure Lengend Snippet: Overview of the articles included with the study characteristics

Article Snippet: Xu et al [ ], 2017 , China , Antioxidant biomarkers; NF-KB, Bax, cleaved Caspase-3 and Bcl2 , Level of antioxidant SOD, MDA and GSP levels; Beta cell function was measured (HOMA). , SOD, MDA and GSP → kits Nanjing Jiancheng Biotechnology Institute (Nanjing, China). Antibodies for Bax, Bcl-2, cleaved Caspase-3 and NF-KB → Santa Cruz Biotechnology (Santa Cruz, CA, United States). Nuclear and cytoplasmic protein → ProteoJETTM Cytoplasmic and Nuclear Protein Extraction kit; (Fermentas International Inc., Burlington, ON, Canada). Protein content → BCA protein assay kit (Pierce Biotechnology, Rockford, United States) , Cells were scraped, pelleted by centrifugation at 250 g for 5 min, mixed with cell lysis buffer, homogenized, and set on ice for 10 min. Then, themixture was centrifuged at 500 g for 7 min at 4 C, and then the supernatant was further centrifuged at 20000 g for 15 min at 4 °C , Immunochemistry; Western Blot; ELISAs , Animal experiment; Male Sprague–Dawley rats; Five experimental groups: Normal control group; Model control group; Ginseng oligopeptides (GOP): Low-dose group (GOP-L); Medium-dose group (GOP-M); High-dose group (GOP-H) , Not exclusion criteria were specified , Not applied , Not applied , This study demonstrated that treatment with ginseng oligopeptides partially reversed abnormal oral glucose test tolerance in rats induced with T2DM and demonstrated an amelioration of the pancreatic damage and increased insulin content , Oral glucose tolerance, plasma glucose, serum insulin, ALT, AST, serum urea nitrogen, total cholesterol, triglycerides , T2DM prediction.

Techniques: Biomarker Discovery, Multiple Displacement Amplification, Colorimetric Assay, Control, Enzyme-linked Immunosorbent Assay, Diagnostic Assay, Ultrasound Gel, Cell Counting, Clinical Proteomics, Marker, Incubation, Lysis, Flow Cytometry, Medications, Gas Chromatography-Mass Spectrometry, Fat, Activity Assay, Centrifugation, Infection, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Derivative Assay, Immunohistochemistry, Functional Assay, Expressing, Next-Generation Sequencing, Sequencing, Software, Cytometry, Luminex, Staining, Blocking Assay, In Situ, Fluorescence, In Vivo, Immunofluorescence, Concentration Assay, Agglutination, Sandwich ELISA, Cell Function Assay, Protein Extraction, Bicinchoninic Acid Protein Assay, Western Blot

Journal: bioRxiv

Article Title: Human Platelet-derived Lysates and Extracellular Vesicles Restore Mitochondrial Function and Redox Balance in Neuronal Models

doi: 10.1101/2025.09.11.675564

Figure Lengend Snippet: PCs were centrifuged to remove residual RBC contaminants and the platelet count was measured. 2. PCs were centrifuged to pelletize the platelets and resuspend them in PBS. 3. The suspended platelet pellet underwent three freeze-thaw cycles followed by centrifugation to obtain PPL that was heat-treated, then cooled down and centrifuged to generate HPPL. 4. PEVs were isolated from the corresponding supernatant by differential centrifugation and ultracentrifugation, resuspended in PBS. The TEM image illustrates the desired spherical PEVs obtained following the described protocol. Note: HPPL and PEVs were aliquoted and stored at -80 ° C until use. Abbreviations: PC: Platelet concentrate, RBCs: Red blood cells, PBS: Phosphate buffer saline, PPL: Platelet pellet lysate, HPPL: Human platelet pellet lysate, PEVs: Platelet derived extracellular vesicles. Created in https://BioRender.com .

Article Snippet: Immunostaining was performed by incubating the cells overnight at 4 °C with primary antibodies targeting PGC-1α (1:50, sc-518038, Santa Cruz), Mitofusin-1 (1:100, ab126575, Abcam), and TOMM20 (1:100, ab186735, Abcam).

Techniques: Centrifugation, Isolation, Saline, Derivative Assay

Journal: bioRxiv

Article Title: Human Platelet-derived Lysates and Extracellular Vesicles Restore Mitochondrial Function and Redox Balance in Neuronal Models

doi: 10.1101/2025.09.11.675564

Figure Lengend Snippet: (A) mitochondrial membrane potential assessed by JC-1 fluorescent probe captured using advance fluorescence microscopy at 40× magnification; scale bar: 100 μM (B) mitochondrial permeability assessment by MitoTracker red staining and counterstained with DAPI to visualize mitochondria viewed under confocal microscope, 20 μM, 63X magnification (C) Quantification of JC-1 monomer mean fluorescence intensity (D) Mean fluorescence intensity quantification of JC-1 aggregation (E) Mean fluorescence intensity quantification of MitoTracker red measured from 6 different fields from 3 independent experiments for each group (F) Ultrastructure of SH-SY5Y cells mitochondria observed under TEM, representative images captured at 12X magnification, scale bar: 1 μm. Blue arrows represent organized dense mitochondrial cristae, red arrows show collapsed mitochondrial matrix with abnormal cristae, black arrow defines mitochondrial membrane integrity and yellow arrow shows damaged mitochondrial membranes. (G) Quantification of mitochondrial area (um 2 ) in SH-SY5Y cells (H) Measurement of the cristae quality score from 25 mitochondria from two biological replicates. Values are expressed as mean ± SD; ****P < 0.0001, ***P < 0.001 vs control; #### P< 0.0001, ### P< 0.001, ## P< 0.01, # P< 0.05 vs rotenone group.

Article Snippet: Immunostaining was performed by incubating the cells overnight at 4 °C with primary antibodies targeting PGC-1α (1:50, sc-518038, Santa Cruz), Mitofusin-1 (1:100, ab126575, Abcam), and TOMM20 (1:100, ab186735, Abcam).

Techniques: Membrane, Fluorescence, Microscopy, Permeability, Staining, Control

Journal: bioRxiv

Article Title: Human Platelet-derived Lysates and Extracellular Vesicles Restore Mitochondrial Function and Redox Balance in Neuronal Models

doi: 10.1101/2025.09.11.675564

Figure Lengend Snippet: (A) Representative immunofluorescence images showing PGC-1α, white arrows depict strong functional nuclear fluorescence, red arrows shows reduced overall fluorescence intensity, green arrows shows distinct fluorescence intensity in cytoplasm, Scale bar 10 μm, magnification 63X (B) Quantification of immunofluorescence signal intensity from 25 individual mitochondria from random fields across treatment groups (C) PGC-1α protein levels determined by ELISA in N2A cells across different treatment groups (D) Representative images showing co-localization of MFN1 and TOMM20 in N2A cells, Scale bar 10 μm, magnification 63X (E) MFN1 fluorescence intensity and its colocalization with TOMM20 across treatment groups from 30 individual mitochondria per group, selected from random fields. Colocalization between MFN1 and TOMM20 shown using Pearson’s correlation coefficient (r), calculated by Coloc-2 plugin in ImageJ (Fiji). Values are expressed as mean ± SD (n=3); ****P < 0.0001, **P < 0.01 vs control; and #### P < 0.0001, ## P < 0.01, # P< 0.05 vs rotenone.

Article Snippet: Immunostaining was performed by incubating the cells overnight at 4 °C with primary antibodies targeting PGC-1α (1:50, sc-518038, Santa Cruz), Mitofusin-1 (1:100, ab126575, Abcam), and TOMM20 (1:100, ab186735, Abcam).

Techniques: Immunofluorescence, Functional Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Control

Journal: bioRxiv

Article Title: Human Platelet-derived Lysates and Extracellular Vesicles Restore Mitochondrial Function and Redox Balance in Neuronal Models

doi: 10.1101/2025.09.11.675564

Figure Lengend Snippet: (A) changes in ZF embryo brain tissue mitochondria and cristae observed under TEM, representative images captured at 15X magnification, scale bar: 1 μm. Blue arrows represent organized dense mitochondrial cristae, red arrows show collapsed mitochondrial matrix with abnormal cristae, black arrow defines mitochondrial membrane integrity, yellow arrow shows damaged mitochondrial membranes. (B) Quantification of key mitochondrial characteristics included a mitochondrial area (um 2 ) (C) Swelling index determined by circulatory which measures mitochondrial shape (<0.6 elongated/tubular and 1 round/bloated) (D) Measurement of the observed cristae quality. Each dot represents one mitochondrion, with a total number of 50 mitochondria in each condition from atleast three biological replicates. Values are expressed as mean ± SD (n=3); ***P < 0.001, ****P < 0.0001 vs control; # P< 0.05, ### P< 0.001, #### P< 0.0001 vs rotenone group.

Article Snippet: Immunostaining was performed by incubating the cells overnight at 4 °C with primary antibodies targeting PGC-1α (1:50, sc-518038, Santa Cruz), Mitofusin-1 (1:100, ab126575, Abcam), and TOMM20 (1:100, ab186735, Abcam).

Techniques: Membrane, Control

Journal: Biology Direct

Article Title: Enhanced itaconic acid secretion from macrophages mediates the protection of mesenchymal stem cell-derived exosomes on lipopolysaccharide-induced acute lung injury mice

doi: 10.1186/s13062-024-00586-8

Figure Lengend Snippet: AMs aggravate LPS-induced injury in PMVECs via M1 polarization-mediated inflammatory response. (A) PMVECs were cultured in the presence of AMs or alone, followed by 100 ng/mL LPS treatment. Equivalent PBS was used as the control. After 24 h of culture, (B) PMVEC permeability was assessed using HRP leakage assays. (C) Western blot analysis revealed the relative expressions of both Claudin-5 and ZO-1 proteins, with GAPDH as the internal reference. (D) Annexin V-FITC/PI staining and (E) TUNEL assays demonstrated the apoptosis rate of PMVECs. (E) ELISA was used for inflammatory cytokines (F) IL-6 and (G) TNF-α concentration determination. (H) The western blot analysis for iNOS and Arg-1 represented the level of M1 polarization. Data are presented as mean ± SEM. Statistical significance was determined using appropriate methods, with * p < 0.05, *** p < 0.001. PMVECs: pulmonary microvascular endothelial cells; AMs: alveolar macrophages; HRP: horseradish peroxidase

Article Snippet: The levels of IL-6 and TNF-α in the supernatant of cultured cells or BALF of mice were assessed by Mouse IL-6 and TNF-α ELISA kit (SEKM-0007 and SEKM-0034; Solarbio, Beijing, China), respectively.

Techniques: Cell Culture, Control, Permeability, Western Blot, Staining, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Biology Direct

Article Title: Enhanced itaconic acid secretion from macrophages mediates the protection of mesenchymal stem cell-derived exosomes on lipopolysaccharide-induced acute lung injury mice

doi: 10.1186/s13062-024-00586-8

Figure Lengend Snippet: MSC-exos attenuate LPS-induced PMVEC injury by promoting M2 polarization of AMs. MSC-exos were added to the LPS-treated co-culture system to evaluate their effects on PMVECs. (A) PMVEC permeability was assessed using HRP leakage assays. (B) Western blot analysis was performed to detect the expression levels of tight junction proteins Claudin-5 and ZO-1. (C) Annexin V-FITC/PI staining and (D) TUNEL assays demonstrated the apoptosis rate of PMVECs to determine the anti-apoptosis effect of MSC-exos. (E-F) ELISA for inflammatory cytokines IL-6 and TNF-α in cell supernatant. (G) Western blot analysis for M1 polarization markers assessed the relative levels of iNOS and Arg-1 proteins. (H) M2 polarization was determined via flow cytometry by calculate the percentage of CD206-positive cells. (I) Gas chromatography-mass spectrometry was used to measure ITA concentration. Data are presented as mean ± SEM. Statistical significance was determined using appropriate methods, with * p < 0.05, ** p < 0.01, and *** p < 0.001. MSC-exos: mesenchymal stem cell-derived exosome; PMVECs: pulmonary microvascular endothelial cells; AMs: alveolar macrophages; HRP: horseradish peroxidase

Article Snippet: The levels of IL-6 and TNF-α in the supernatant of cultured cells or BALF of mice were assessed by Mouse IL-6 and TNF-α ELISA kit (SEKM-0007 and SEKM-0034; Solarbio, Beijing, China), respectively.

Techniques: Co-Culture Assay, Permeability, Western Blot, Expressing, Staining, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Gas Chromatography, Mass Spectrometry, Concentration Assay, Derivative Assay

Journal: Biology Direct

Article Title: Enhanced itaconic acid secretion from macrophages mediates the protection of mesenchymal stem cell-derived exosomes on lipopolysaccharide-induced acute lung injury mice

doi: 10.1186/s13062-024-00586-8

Figure Lengend Snippet: ITA mediates the effect of MSC-exos on AMs. To investigate the role of ITA, IRG1 was knocked down in MH-S cells using three siRNA-IRG1 with different sequences. si-RNAs were subsequently transfected to the AMs. (A) The knockdown efficiency was validated by PCR and Western blot analysis, with the most efficient siRNA selected for further assays. After that, (B) the horseradish peroxidase (HRP) permeation assay was conducted and (C) western blot analysis revealed the relative levels of tight junction proteins Claudin-5 and ZO-1. Apoptosis rates were assessed using (D) Annexin V-FITC/PI staining and (E) TUNEL assays. (F) ELISA was performed for (F) IL-6 and (G) TNF-α concentrations. (H) For M1 polarization evaluation, iNOS and Arg-1 proteins were determined by Western blot analysis. (I) For M2 polarization assessment, the proportion of CD206-positive cells were calculated by flow cytometry. Data are presented as mean ± SEM. Statistical significance was determined using appropriate methods, with * p < 0.05, ** p < 0.01, and *** p < 0.001. ITA: itaconic acid; MSC-exos: mesenchymal stem cell-derived exosome; AMs: alveolar macrophages; HRP: horseradish peroxidase

Article Snippet: The levels of IL-6 and TNF-α in the supernatant of cultured cells or BALF of mice were assessed by Mouse IL-6 and TNF-α ELISA kit (SEKM-0007 and SEKM-0034; Solarbio, Beijing, China), respectively.

Techniques: Transfection, Knockdown, Western Blot, Staining, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Derivative Assay

Journal: Biology Direct

Article Title: Enhanced itaconic acid secretion from macrophages mediates the protection of mesenchymal stem cell-derived exosomes on lipopolysaccharide-induced acute lung injury mice

doi: 10.1186/s13062-024-00586-8

Figure Lengend Snippet: AMs aggravate LPS-induced injury in PMVECs via M1 polarization-mediated inflammatory response. (A) PMVECs were cultured in the presence of AMs or alone, followed by 100 ng/mL LPS treatment. Equivalent PBS was used as the control. After 24 h of culture, (B) PMVEC permeability was assessed using HRP leakage assays. (C) Western blot analysis revealed the relative expressions of both Claudin-5 and ZO-1 proteins, with GAPDH as the internal reference. (D) Annexin V-FITC/PI staining and (E) TUNEL assays demonstrated the apoptosis rate of PMVECs. (E) ELISA was used for inflammatory cytokines (F) IL-6 and (G) TNF-α concentration determination. (H) The western blot analysis for iNOS and Arg-1 represented the level of M1 polarization. Data are presented as mean ± SEM. Statistical significance was determined using appropriate methods, with * p < 0.05, *** p < 0.001. PMVECs: pulmonary microvascular endothelial cells; AMs: alveolar macrophages; HRP: horseradish peroxidase

Article Snippet: Apoptosis of PMVECs was assessed using the Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime) according to manufacturer’s protocols.

Techniques: Cell Culture, Control, Permeability, Western Blot, Staining, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Biology Direct

Article Title: Enhanced itaconic acid secretion from macrophages mediates the protection of mesenchymal stem cell-derived exosomes on lipopolysaccharide-induced acute lung injury mice

doi: 10.1186/s13062-024-00586-8

Figure Lengend Snippet: MSC-exos attenuate LPS-induced PMVEC injury by promoting M2 polarization of AMs. MSC-exos were added to the LPS-treated co-culture system to evaluate their effects on PMVECs. (A) PMVEC permeability was assessed using HRP leakage assays. (B) Western blot analysis was performed to detect the expression levels of tight junction proteins Claudin-5 and ZO-1. (C) Annexin V-FITC/PI staining and (D) TUNEL assays demonstrated the apoptosis rate of PMVECs to determine the anti-apoptosis effect of MSC-exos. (E-F) ELISA for inflammatory cytokines IL-6 and TNF-α in cell supernatant. (G) Western blot analysis for M1 polarization markers assessed the relative levels of iNOS and Arg-1 proteins. (H) M2 polarization was determined via flow cytometry by calculate the percentage of CD206-positive cells. (I) Gas chromatography-mass spectrometry was used to measure ITA concentration. Data are presented as mean ± SEM. Statistical significance was determined using appropriate methods, with * p < 0.05, ** p < 0.01, and *** p < 0.001. MSC-exos: mesenchymal stem cell-derived exosome; PMVECs: pulmonary microvascular endothelial cells; AMs: alveolar macrophages; HRP: horseradish peroxidase

Article Snippet: Apoptosis of PMVECs was assessed using the Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime) according to manufacturer’s protocols.

Techniques: Co-Culture Assay, Permeability, Western Blot, Expressing, Staining, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Gas Chromatography, Mass Spectrometry, Concentration Assay, Derivative Assay

Journal: Biology Direct

Article Title: Enhanced itaconic acid secretion from macrophages mediates the protection of mesenchymal stem cell-derived exosomes on lipopolysaccharide-induced acute lung injury mice

doi: 10.1186/s13062-024-00586-8

Figure Lengend Snippet: ITA mediates the effect of MSC-exos on AMs. To investigate the role of ITA, IRG1 was knocked down in MH-S cells using three siRNA-IRG1 with different sequences. si-RNAs were subsequently transfected to the AMs. (A) The knockdown efficiency was validated by PCR and Western blot analysis, with the most efficient siRNA selected for further assays. After that, (B) the horseradish peroxidase (HRP) permeation assay was conducted and (C) western blot analysis revealed the relative levels of tight junction proteins Claudin-5 and ZO-1. Apoptosis rates were assessed using (D) Annexin V-FITC/PI staining and (E) TUNEL assays. (F) ELISA was performed for (F) IL-6 and (G) TNF-α concentrations. (H) For M1 polarization evaluation, iNOS and Arg-1 proteins were determined by Western blot analysis. (I) For M2 polarization assessment, the proportion of CD206-positive cells were calculated by flow cytometry. Data are presented as mean ± SEM. Statistical significance was determined using appropriate methods, with * p < 0.05, ** p < 0.01, and *** p < 0.001. ITA: itaconic acid; MSC-exos: mesenchymal stem cell-derived exosome; AMs: alveolar macrophages; HRP: horseradish peroxidase

Article Snippet: Apoptosis of PMVECs was assessed using the Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime) according to manufacturer’s protocols.

Techniques: Transfection, Knockdown, Western Blot, Staining, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Derivative Assay

Journal: Endocrinology

Article Title: The presence of adenosine A2a receptor in thyrocytes and its involvement in Graves' IgG-induced VEGF expression.

doi: 10.1210/en.2012-2258

Figure Lengend Snippet: Figure 2. The A2aR agonist and GD IgG increase VEGF transcription in thyrocytes. (A) After treating FRTL-5 cells with various concentrations of CGS21680 (CGS) (A2aR-specific agonist) for 12 (a1), 24 (a2), and 48 (a3) h or with increasing concentrations of GD IgG for 48 hours (a4), the mRNA expression of the VEGF splice forms was determined by RT-PCR. The PCR products were separated by agarose gel electrophoresis. From top to bottom, the bands corresponding to the 4 VEGF splice isoforms, VEGF188 (619 bp), VEGF164 (547 bp), VEGF144 (487 bp), and VEGF120 (415 bp), are depicted. The data are normalized to -actin and are presented as an expression ratio compared with the control group. Representative graphs of 3 independent experiments are presented. The results are expressed as the mean SD (*, P .05 compared with the corresponding control). VEGF protein in supernatants and cell lysates was measured (a5 and a6). The supernatant from CGS21680 and GD IgG-stimulated FRTL-5 cells was collected, and the volume was measured. The cells were collected in cell lysate buffer. The total concentration of VEGF protein (the sum of all VEGF isoforms) in the lysates and media were determined with an ELISA kit according to the manufacturer’s instructions. Data are presented as the ratio of the VEGF concentration to the total protein content or an expression ratio compared with the control group. The panels are representative of 3 independent experiments, and the results are expressed as the mean SD (*, P .05 compared with control). (B) PHTs were incubated with CGS21680, normal IgG, GD IgG, normal serum, or GD serum (containing an equivalent amount of IgG as the previous groups) for 48 hours. The mRNA expression of the VEGF splice forms in PHT was determined by RT-PCR, and the PCR products were separated by agarose gel electrophoresis. From top to bottom, the bands corresponding to the 4 VEGF splice isoforms, VEGF189 (648 bp), VEGF165 (576 bp), VEGF145 (516 bp), and VEGF121 (444 bp), are depicted. The data are normalized to -actin and are presented as an expression ratio compared with the control group. The panels are representative of 5 independent experiments, and the results are expressed as the mean SD (*, P .05 compared with control).

Article Snippet: Primary antibodies The following antibodies were used: rat A2aR, VEGF, and peroxisome proliferator-activated receptor coactivator-1 (PGC-1 ) (Santa Cruz Biotechnology, Inc); human A2aR and -actin (Abcam, Inc); phosphorylated (p)-cAMP-response element-binding protein (CREB) and CREB (Cell Signaling Technology); and hypoxia-inducible factor-1 (HIF-1 ) (Novus Biologicals).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Endocrinology

Article Title: The presence of adenosine A2a receptor in thyrocytes and its involvement in Graves' IgG-induced VEGF expression.

doi: 10.1210/en.2012-2258

Figure Lengend Snippet: Figure 3. Adenosine A2aR stimulation and GD IgG augment VEGF expression via CREB phosphorylation and increased nuclear PGC-1 in thyrocytes. (A, a1) After treatment with various concentrations of CGS21680, FRTL-5 cell total protein and nuclear extracts were prepared and separated by SDS-PAGE. Specific antibodies against active (Ser133 phosphorylated) CREB (p-CREB), total CREB, PGC-1, and HIF-1 were used. Sequential incubation of the membranes with an anti--actin antibody confirmed equal protein loading. (a2) After a 48-hour incubation with various concentrations of forskolin (an adenylyl cyclase [AC] activator), FRTL-5 cell cytoplasmic and nuclear extracts were separated by SDS-PAGE and analyzed by Western blotting using antibodies against VEGF, PGC-1, and HIF-1. (a3) FRTL-5 cells were incubated in the presence or absence of CGS21680 or GD IgG with or without a 1-hour preincubation with H89 (10M). Nuclear proteins were analyzed by Western blotting using antibodies against p-CREB and PGC-1. The panels are representative of 3 independent experiments. (B) The presumed pathway is illustrated. (C) Example goiters were obtained from the Ad-TSHR-289 mouse model (n 20 in each group). Paraffin sections were randomly chosen from 8 mice in each group and were incubated with specific antibodies to VEGF, PGC-1, p-CREB, and HIF-1. The brown staining represents the labeled proteins, and the blue staining depicts nuclei (scale bars, 20 m).

Article Snippet: Primary antibodies The following antibodies were used: rat A2aR, VEGF, and peroxisome proliferator-activated receptor coactivator-1 (PGC-1 ) (Santa Cruz Biotechnology, Inc); human A2aR and -actin (Abcam, Inc); phosphorylated (p)-cAMP-response element-binding protein (CREB) and CREB (Cell Signaling Technology); and hypoxia-inducible factor-1 (HIF-1 ) (Novus Biologicals).

Techniques: Expressing, Phospho-proteomics, SDS Page, Incubation, Western Blot, Staining, Labeling

Journal: Endocrinology

Article Title: The presence of adenosine A2a receptor in thyrocytes and its involvement in Graves' IgG-induced VEGF expression.

doi: 10.1210/en.2012-2258

Figure Lengend Snippet: Figure 4. An A2aR-specific antagonist blocks the increase in nuclear PGC-1 and p-CREB and the GD IgG-mediated induction of VEGF. (A) FRTL-5 cells were incubated in the absence or presence of CGS21680 with or without a 30-minute preincubation with ZM241385 (ZM). Nuclear proteins were extracted, separated by SDS-PAGE, and analyzed by Western blotting using antibodies against PGC-1 and active (Ser133 phosphorylated) CREB (p-CREB). The panels are representative of 3 independent experiments. (B) FRTL-5 cells were incubated with normal IgG or GD IgG for 48 hours with or without a 30-minute preincubation with ZM. The mRNA levels of the VEGF splice forms were determined by RT-PCR and were separated by agarose gel electrophoresis. Data are normalized to -actin and are presented as the ratio to the control group. The panels are representative of 5 independent experiments, and the results are expressed as the mean SD (*, P .05 compared with control, &, P .05 compared with the 10M ZM group).

Article Snippet: Primary antibodies The following antibodies were used: rat A2aR, VEGF, and peroxisome proliferator-activated receptor coactivator-1 (PGC-1 ) (Santa Cruz Biotechnology, Inc); human A2aR and -actin (Abcam, Inc); phosphorylated (p)-cAMP-response element-binding protein (CREB) and CREB (Cell Signaling Technology); and hypoxia-inducible factor-1 (HIF-1 ) (Novus Biologicals).

Techniques: Incubation, SDS Page, Western Blot, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Control

Journal: Endocrinology

Article Title: The presence of adenosine A2a receptor in thyrocytes and its involvement in Graves' IgG-induced VEGF expression.

doi: 10.1210/en.2012-2258

Figure Lengend Snippet: Figure 5. Both A2aR-specific antagonist and A2aR silence block the GD IgG-mediated induction of VEGF. (A) FRTL-5 cells were incubated with GD IgG for 48 hours with or without a 30-minute preincubation with ZM241385 (ZM). The supernatant from cells was collected, and the volume was measured. The cells were collected in cell lysate buffer. The total concentration of VEGF protein (the sum of all VEGF isoforms) in the lysates and media were determined with an ELISA kit according to the manufacturer’s instructions. Data are presented as an expression ratio compared with the control group. The panels are representative of 3 independent experiments, and the results are expressed as the mean SD (*, P .05 compared with control). (B) FRTL-5 cells were incubated with GD IgG for 48 hours with or without nontargeting or A2aR siRNA. The supernatant from cells was collected, and the volume was measured. The cells were collected in cell lysate buffer. The total concentration of VEGF protein (the sum of all VEGF isoforms) in the lysates and media were determined with an ELISA kit according to the manufacturer’s instructions. Data are presented as an expression ratio compared with the control group. The panels are representative of 3 independent experiments, and the results are expressed as the mean SD (*, P .05 compared with control). NC Si, Nontargeting SiRNA treated group; Si, silenced.

Article Snippet: Primary antibodies The following antibodies were used: rat A2aR, VEGF, and peroxisome proliferator-activated receptor coactivator-1 (PGC-1 ) (Santa Cruz Biotechnology, Inc); human A2aR and -actin (Abcam, Inc); phosphorylated (p)-cAMP-response element-binding protein (CREB) and CREB (Cell Signaling Technology); and hypoxia-inducible factor-1 (HIF-1 ) (Novus Biologicals).

Techniques: Blocking Assay, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Control

Journal: Endocrinology

Article Title: The presence of adenosine A2a receptor in thyrocytes and its involvement in Graves' IgG-induced VEGF expression.

doi: 10.1210/en.2012-2258

Figure Lengend Snippet: Figure 6. Monoclonal TSH-stimulating antibody induces nuclear p-CREB and PGC-1 as well as VEGF splicing isoforms expression via A2aR. (A) FRTL-5 cells were incubated in the absence or presence of M22 with or without a 30-minute preincubation with ZM241385 (ZM). Nuclear proteins were extracted, separated by SDS-PAGE, and analyzed by Western blotting using antibodies against PGC-1 and active (Ser133 phosphorylated) CREB (p-CREB). The panels are representative of 3 independent experiments. (B) FRTL-5 cells were incubated in the absence or presence of M22 for 48 hours with or without a 30-minute preincubation with ZM. The mRNA levels of the VEGF splice forms were determined by RT-PCR and were separated by agarose gel electrophoresis. Data are normalized to -actin and are presented as the ratio to the control group. The panels are representative of 3 independent experiments, and the results are expressed as the mean SD (*, P .05 compared with control, &, P .05 compared with the 10M ZM group).

Article Snippet: Primary antibodies The following antibodies were used: rat A2aR, VEGF, and peroxisome proliferator-activated receptor coactivator-1 (PGC-1 ) (Santa Cruz Biotechnology, Inc); human A2aR and -actin (Abcam, Inc); phosphorylated (p)-cAMP-response element-binding protein (CREB) and CREB (Cell Signaling Technology); and hypoxia-inducible factor-1 (HIF-1 ) (Novus Biologicals).

Techniques: Expressing, Incubation, SDS Page, Western Blot, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Control

Journal: Endocrinology

Article Title: The presence of adenosine A2a receptor in thyrocytes and its involvement in Graves' IgG-induced VEGF expression.

doi: 10.1210/en.2012-2258

Figure Lengend Snippet: Figure 7. Silencing A2aR and TSHR blocks the GD IgG-induced increase in VEGF and nuclear PGC-1 and p-CREB. FRTL-5 cells were incubated with GD IgG for 48 hours with or without nontargeting or A2aR siRNA. The efficiency of the A2aR siRNA was determined by immunofluorescence. Immunolabeling of VEGF, p-CREB, PGC-1, and HIF-1 was performed to determine how A2aR silencing modulated the effects of GD IgG. The blue color depicts the DAPI- stained nuclei, red represents A2aR and HIF-1 (tetramethyl rhodamin isothiocyanate [TRITC] conjugated), and green represents VEGF, p-CREB, and PGC-1 (FITC conjugated). The merged images were obtained by superimposing the blue and green/red channels (scale bars, 10 m). The panels are representative of 3 independent experiments. NC Si, Nontargeting SiRNA treated group; Si, silenced.

Article Snippet: Primary antibodies The following antibodies were used: rat A2aR, VEGF, and peroxisome proliferator-activated receptor coactivator-1 (PGC-1 ) (Santa Cruz Biotechnology, Inc); human A2aR and -actin (Abcam, Inc); phosphorylated (p)-cAMP-response element-binding protein (CREB) and CREB (Cell Signaling Technology); and hypoxia-inducible factor-1 (HIF-1 ) (Novus Biologicals).

Techniques: Incubation, Immunofluorescence, Immunolabeling, Staining