Journal: Frontiers in Endocrinology

Article Title: Integration of Metabolomics and Proteomics in Exploring the Endothelial Dysfunction Mechanism Induced by Serum Exosomes From Diabetic Retinopathy and Diabetic Nephropathy Patients

doi: 10.3389/fendo.2022.830466

Figure Lengend Snippet: Serum exosomes from diabetic retinopathy and nephropathy patients could induce endothelial dysfunction. (A) The typical pathological kidney images and fundus images obtained from healthy people and a diabetic microvascular disease patient. PAS : The DMC biopsy sample showed proliferation and swelling of the endothelial cell (blue arrow), reduplication (double contour appearance) of the glomerular basement membrane (red arrow), mesangial expansion (black arrow), arteriolar hyalinosis (green arrow). Scale bar: 50 μm. Methenamine silver: Renal biopsy samples from the DMC patient show the swelling of the endothelial cells (red arrow) and reduplication (double contour appearance) of the glomerular basement membrane (black arrow). Scale bar: 50 μm. Electron microscopy: The slice from the DMC patient showed mesangial expansion (red arrow). Scale bar: 5 μm. Fundus: The fundus from DMC showed microhemangioma (green arrow), retinal exudates (red arrow), intraretinal hemorrhage (black arrow), and intraretinal microvascular abnormalities (IRMAs; violet arrow). (B) Identification of serum exosomes from CON patients and DRDN patients by transmission electron microscopy (TEM). Scale bar: 200 nm. (C) Western blotting was used to look for the exosomal markers CD9, CD63, and TSG101 in exosome samples. (D) Analysis of the size distribution of exosomes from patients using the NanoSight technology; the average size of serum exosomes was 107.5 ± 55.2 nm. (E) Exosome tracing experiment captured by confocal microscope. Blue for DNA dyed by DAPI and green for exosomes derived from DR+DN patients dyed by PKH67. HGECs were subjected to 6, 12, and 24 h of incubation with exosomes. Scale bar: 25 μm. PAS, Periodic acid–Schiff stain; DMC, diabetic microvascular complications; CON, healthy controls; DRDN, patients diagnosed with both diabetic retinopathy and diabetic nephropathy.

Article Snippet: The primary antibodies were anti-CD9 (Abcam, Hong Kong, China; # ab92726), anti-CD63 (Abcam, ab216130), anti-TSG101 (Sino Biological, Beijing, China; #102286-T38), anti-CD31 (Zen-Bioscience, Chengdu, China; #383815), anti-von Willebrand factor (vWF), (Abcam, #ab154193), anti-intercellular adhesion molecule 1 (ICAM-1) (Abcam, #ab171123), anti-vascular cell adhesion protein 1 (VCAM-1) (Abcam, #ab134047), and anti-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) (Goodhere, Hangzhou, China; #AB-P-R 001).

Techniques: Membrane, Electron Microscopy, Transmission Assay, Western Blot, Microscopy, Derivative Assay, Incubation, Staining

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) Protein expression of precipitated EVs. The serum collected from seven mice of wild-type (WT) and EML4-ALK transgenic mouse (TL group) was subjected to Western blot analysis with anti-CHL1, anti-α2 integrin, anti-β1 integrin, and anti-FGFR3 antibodies, which were cancer cell membrane BiCAT molecules previously reported. ( B ) Western blot analysis for TSG101 antigen detection in Sephacryl S-500 fractions. The fractions were concentrated with Nanosep ® centrifugal unit, and then subjected to Western blot analysis with anti-TSG101 antibody. TSG101-(Ub)n indicates ubiquitinated TSG101.

Article Snippet: After blocking with 5% skim milk solution, transferred membrane was reacted with first antibodies as follows: anti-mouse and human CHL1 antibody (AF2147 and MAB2126; R&D systems, MN; 1 μg/ml), HRP-conjugated anti-mouse and human CHL1 antibody (described above), anti-α2 integrin antibody (ab133557; Abcam; 1 μg/ml), anti-β1 integrin antibody (610467; BD transduction laboratories, NJ; 0.25 μg/ml), anti-FGFR3 antibody (c-15; Santa cruz, TX; 0.2 μg/ml 5% BSA-PBS), anti-TSG101 antibody (EXOAB-TSG101-1; System Biosciences, CA; 1:500), anti-CD63 antibody (EXOAB-CD63A-1; System Biosciences, CA; 1:500), anti-CD5L antibody (AF2834; R&D systems, MN; 0.4 μg/ml), anti-Pregnancy Zone Protein (PZP) antibody (PAG324Ra01; CLOUD-CLONE, TX; 0.5 μg/ml), anti-SLC4A1 antibody (18566-1-AP; PROTEINTECH, IL; 0.6 μg/ml), anti-Thrombospondin 1 (THBS1) antibody (PAA611Hu01; CLOUD-CLONE, TX; 0.5 μg/ml), and anti-caspase 14 antibody (MAB8215; R&D systems, MN; 0.5 μg/ml).

Techniques: Expressing, Transgenic Assay, Western Blot

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A, B ) Western blot analysis of TSG101 ( A ) and CD63 ( B ) in serum EVs from WT, TL, and TS using anti-TSG101 and CD63 antibody. Arrows indicate the detected band of monomer TSG101 and CD63. The black bar indicates wide range of molecular weight due to an ubiquitination in TSG101 (TSG101-(Ub)n). ( C ) Western blot analysis of EMARS products with anti-CHL1 antibody. The EMARS products were concentrated and purified by immunoprecipitation with the anti-fluorescein antibody Sepharose. The resulting samples were subjected to SDS-PAGE analysis with fluorescence detection. “IP” indicates the immunoprecipitated samples, and “Lys” indicates the lysate samples before immunoprecipitation. Arrow indicates the detected band of CHL1. Asterisk indicates unknown bands (predicted as non-specific or partial fragments).

Article Snippet: After blocking with 5% skim milk solution, transferred membrane was reacted with first antibodies as follows: anti-mouse and human CHL1 antibody (AF2147 and MAB2126; R&D systems, MN; 1 μg/ml), HRP-conjugated anti-mouse and human CHL1 antibody (described above), anti-α2 integrin antibody (ab133557; Abcam; 1 μg/ml), anti-β1 integrin antibody (610467; BD transduction laboratories, NJ; 0.25 μg/ml), anti-FGFR3 antibody (c-15; Santa cruz, TX; 0.2 μg/ml 5% BSA-PBS), anti-TSG101 antibody (EXOAB-TSG101-1; System Biosciences, CA; 1:500), anti-CD63 antibody (EXOAB-CD63A-1; System Biosciences, CA; 1:500), anti-CD5L antibody (AF2834; R&D systems, MN; 0.4 μg/ml), anti-Pregnancy Zone Protein (PZP) antibody (PAG324Ra01; CLOUD-CLONE, TX; 0.5 μg/ml), anti-SLC4A1 antibody (18566-1-AP; PROTEINTECH, IL; 0.6 μg/ml), anti-Thrombospondin 1 (THBS1) antibody (PAA611Hu01; CLOUD-CLONE, TX; 0.5 μg/ml), and anti-caspase 14 antibody (MAB8215; R&D systems, MN; 0.5 μg/ml).

Techniques: Western Blot, Molecular Weight, Purification, Immunoprecipitation, SDS Page, Fluorescence

Journal: Separations

Article Title: Alternative Method for HDL and Exosome Isolation with Small Serum Volumes and Their Characterizations

doi: 10.3390/separations8110204

Figure Lengend Snippet: Figure 6. The protein pattern and biomarker of the exosomes isolated from different sample volumes. A protein ladder reference (Vivantis Technologies, Shah Alam, Selangor Darul Ehsan, Malaysia, cat no. PR0623) had 2 reference bands (25 and 72 kDa) coupled with blue chromophore for easy identification (A). SDS-PAGE stained with Coomassie blue G250 showed multiple bands with the same patterns (B). The Western blot results showed a single band of TSG101 at about 48.9 kDa exhibiting the exosome biomarker (C). Thirty micrograms of protein were loaded for both SDS-PAGE and the Western blot. All experiments above were run with a reducing agent. M = Marker of protein molecular weight.

Article Snippet: The primary antibodies, human ApoA-1 antibody (cat no. MAB 36641-SP) and human TSG101 antibody (cat no. orb337267), were supplied by R&D System, Inc., Minneapolis, MN, USA and Biorbyt, Cambridge, Cambridgeshire, UK, respectively.

Techniques: Biomarker Discovery, Isolation, SDS Page, Staining, Western Blot, Marker, Molecular Weight

Journal: eLife

Article Title: TCF7L1 promotes skin tumorigenesis independently of β-catenin through induction of LCN2

doi: 10.7554/eLife.23242

Figure Lengend Snippet: ( A ) Real time PCR analysis of expression of LCN2 in SCC13 and SRB12 cells that were transduced to express Tcf7l1 and its various mutants. ( B ) Real time PCR analysis of Lcn2 in enriched epithelial cells from mouse normal skin, DMBA/TPA-induced papilloma and SCC. ( C ) Real time PCR analysis of LCN2 in SRB12 cells that were transduced to express shRNA against nonspecific sequence (shNS) or two different sets of shRNAs against LCN2 . ( D ) Western analysis of LCN2 in SRB12 cells that were transduced to express empty vector control or Tcf7l1 with shRNA against nonspecific sequence or two different sets of shRNAs against LCN2 . Drug selected transduced cells expressing shRNAs were grafted onto NSG mice and tumors were isolated at the end of eight weeks. ( E ) Quantification of tumor mass. Data are presented as box and whisker plots where boxes span first and third quartiles, bars as the median values, and whiskers as minimum and maximum of all data. *p<0.05, **p<0.01, ***p<0.001 (One-way ANOVA with Dunnett’s post-hoc test). n = 5 (shNS+vector), n = 4 (sh LCN2 #1+vector), n = 2 (sh LCN2 #2+vector), n = 3 (shNS+ Tcf7l1 ), n = 3 (sh LCN2 #1+ Tcf7l1 ), n = 3 (sh LCN2 #2+ Tcf7l1 ). ( F ) Representative images of tumors. ( G ) Quantification of cell migration in transwell assay. Human SCC (SRB12) cells with tet-inducible Tcf7l1 were treated with doxycycline for 48 hr and their migratory ability was measured using the Boyden chamber-based cell migration assay. Cells were allowed to migrate toward the lower compartment containing media with 10% FBS as chemoattractant, supplemented with neutralizing antibody against LCN2 or mouse IgG isotype control. After 30 hr, migrated cells were fixed by 4%PFA, counterstained with crystal violet, and quantified. Data are presented as mean ± s.d. **p<0.01 (One-way ANOVA with Dunnett’s post-hoc test). DOI: http://dx.doi.org/10.7554/eLife.23242.018

Article Snippet: Rabbit anti-Myc epitope (1:1,000, Cell Signaling Technology #2278 RRID: AB_490778 ), guinea pig anti-TCF7L1 (1:1,000, lab-generated), rabbit anti-TCF7L2 (1:1,000, Cell Signaling #2569S RRID: AB_2199816 ), rabbit anti-human LCN2 (1:1,000, Abcam #ab125075 RRID: AB_10978084 ), mouse anti-β-actin (1:5,000, Sigma #A5441 RRID: AB_476744 ).

Techniques: Real-time Polymerase Chain Reaction, Expressing, shRNA, Sequencing, Western Blot, Plasmid Preparation, Control, Isolation, Whisker Assay, Migration, Transwell Assay, Cell Migration Assay

Journal: eLife

Article Title: TCF7L1 promotes skin tumorigenesis independently of β-catenin through induction of LCN2

doi: 10.7554/eLife.23242

Figure Lengend Snippet: ( A ) Immunofluorescence images of LCN2 staining in control and TCF7L1-induced skin. Control or tet-inducible Tcf7l1 mice were on a doxycycline containing diet for five days and their skins were immunostained for LCN2 (green) and basement membrane marker β4-integrin (red). ( B ) Images of immunohistochemical staining of LCN2 in normal mouse skin, and DMBA/TPA-induced papilloma and skin SCC. Bar denotes 100 µm. DOI: http://dx.doi.org/10.7554/eLife.23242.019

Article Snippet: Rabbit anti-Myc epitope (1:1,000, Cell Signaling Technology #2278 RRID: AB_490778 ), guinea pig anti-TCF7L1 (1:1,000, lab-generated), rabbit anti-TCF7L2 (1:1,000, Cell Signaling #2569S RRID: AB_2199816 ), rabbit anti-human LCN2 (1:1,000, Abcam #ab125075 RRID: AB_10978084 ), mouse anti-β-actin (1:5,000, Sigma #A5441 RRID: AB_476744 ).

Techniques: Immunofluorescence, Staining, Control, Membrane, Marker, Immunohistochemical staining

Journal: eLife

Article Title: TCF7L1 promotes skin tumorigenesis independently of β-catenin through induction of LCN2

doi: 10.7554/eLife.23242

Figure Lengend Snippet: ( A ) Representative images of negative and positive immunohistochemical staining of TCF7L1 and LCN2 in human skin SCC (middle and bottom panels) and adjacent normal skin (top). Bar denotes 100 µm. ( B ) Graph summarizing the expression of TCF7L1 and LCN2 in human skin SCC samples. ( C ) Graph summarizing the expression of LCN2 in TCF7L1-subgrouped human skin SCC samples (n = 16). DOI: http://dx.doi.org/10.7554/eLife.23242.020

Article Snippet: Rabbit anti-Myc epitope (1:1,000, Cell Signaling Technology #2278 RRID: AB_490778 ), guinea pig anti-TCF7L1 (1:1,000, lab-generated), rabbit anti-TCF7L2 (1:1,000, Cell Signaling #2569S RRID: AB_2199816 ), rabbit anti-human LCN2 (1:1,000, Abcam #ab125075 RRID: AB_10978084 ), mouse anti-β-actin (1:5,000, Sigma #A5441 RRID: AB_476744 ).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: eLife

Article Title: TCF7L1 promotes skin tumorigenesis independently of β-catenin through induction of LCN2

doi: 10.7554/eLife.23242

Figure Lengend Snippet: Xenografted tumors (n = 3) expressing empty vector or vector expressing tet-inducible Tcf7l1 with or without sh LCN2#1 were dissociated and analyzed by flow cytometry for CD11b+Ly6G+Ly6C+ (neutrophils) and CD11b +F4/80+ (macrophages). Representative plots showing the gating of ( A ) CD11b+Ly6G+Ly6C+ and ( B ) CD11b +F4/80+. Graph quantifying the percentage of total cells that express ( C ) CD11+Ly6G+Ly6C+ and ( D ) CD11b +F4/80+. Data are presented as mean ± s.d. **p<0.05 (One-way ANOVA with Dunnett’s post-hoc test). DOI: http://dx.doi.org/10.7554/eLife.23242.021

Article Snippet: Rabbit anti-Myc epitope (1:1,000, Cell Signaling Technology #2278 RRID: AB_490778 ), guinea pig anti-TCF7L1 (1:1,000, lab-generated), rabbit anti-TCF7L2 (1:1,000, Cell Signaling #2569S RRID: AB_2199816 ), rabbit anti-human LCN2 (1:1,000, Abcam #ab125075 RRID: AB_10978084 ), mouse anti-β-actin (1:5,000, Sigma #A5441 RRID: AB_476744 ).

Techniques: Expressing, Plasmid Preparation, Flow Cytometry