Journal: Annals of Clinical and Translational Neurology

Article Title: Exosome Proteomics of SOD1 D90A Mutation Suggest Early Disease Mechanisms, and FN1 as a Biomarker

doi: 10.1002/acn3.70208

Figure Lengend Snippet: Fibronectin protein becomes increasingly abundant in the exosomes of patients after symptom onset. (A) Schematic representation of Fibronectin (FN1) gene and its isoforms, which are the result of alternative splicing. The isoforms detected in this study are labeled with red box. (B) The FN1 protein levels (as detected by peptide ID P02751 ) in the Son (T2), Father, and Patient #2. Regression analyses of protein levels with respect to ALSFRS scores. R 2 = 0.9712. (C) Isoform 2 of FN1 protein levels (as detected by peptide ID P02751 ‐2) in the Son (T2), Father, and Patient #2. Regression analyses of protein levels with respect to ALSFRS scores. R 2 = 0.8658. (D) Isoform 4, 5, and 6 of FN1 protein levels (as detected by peptide ID P02751 ‐4. P02751 ‐5, P02751 ‐6, respectively) in the Son (T2), Father, and Patient #2. Regression analyses of protein levels with respect to ALSFRS scores. R 2 = 0.9916. (E) Isoform 7, 8, and 9 of FN1 protein levels (as detected by peptide ID P02751 ‐7, P02751 ‐8, P02751 ‐9, respectively) in the Son (T2), Father, and Patient #2. Regression analyses of protein levels with respect to ALSFRS scores. R 2 = 0.9712. Blue dot = Son; red dot = Father; black dot = Patient #2.

Article Snippet: ELISA was used to quantify levels of FN1 protein in the serum (Cat. # DFBN10, R&D Systems).

Techniques: Alternative Splicing, Labeling

Journal: Annals of Clinical and Translational Neurology

Article Title: Exosome Proteomics of SOD1 D90A Mutation Suggest Early Disease Mechanisms, and FN1 as a Biomarker

doi: 10.1002/acn3.70208

Figure Lengend Snippet: ELISA confirmation of FN1 levels in the serum. (A) FN1 protein was also detected in the serum of the Son, Father and Patient #2, with increasing concentrations. (B) FN1 protein was increasingly present in the serum of an ALS patient, who had TDP‐43 G348C mutation (Time between T1 and T2 = 10 months). (C) FN1 protein was increasingly present in the serum of an ALS patient, with PABPN1 Ala11dub intron expansion mutation (Time between T1 and T2 = 6 months). Red dot depicts the age and sex‐matched control for each patient.

Article Snippet: ELISA was used to quantify levels of FN1 protein in the serum (Cat. # DFBN10, R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Mutagenesis, Control

Journal: Chinese Medical Journal

Article Title: Exosomal miR-485-3p derived from pancreatic ductal epithelial cells inhibits pancreatic cancer metastasis through targeting PAK1

doi: 10.1097/CM9.0000000000002154

Figure Lengend Snippet: Suppressive effects of Exo hTERT-HPNE on PC cells and PSCs through delivering mature miR-485-3p into the corresponding cells. (A) Transwell migration and invasion assays of BxPC-3 and PANC-1 cells treated with or without Exo hTERT-HPNE (bar: 100 μm). Cells were imaged at 200 × magnification in five random fields and counted using ImageJ software. (B) Transwell migration assay of PSCs treated with or without Exo hTERT-HPNE (bar: 100 μm). (C) FN1, COLIA1, and FAP levels in cell supernatant of PSCs treated with or without Exo hTERT-HPNE assessed by ELISA. (D) Detection of miR-485-3p in exosomes of hTERT-HPNE, PSCs, and PC cells, as well as in the corresponding parental cells using RT-PCR. MiR-485-3p was not detected in Exo PSC , Exo BxPC-3 , and Exo MIA PaCa-2 . U6 served as endogenous reference. (E) Mature miR-485-3p and its precursors (pre-miR-485-3p, pri-miR-485-3p) of PSCs and PC cells were detected by RT-PCR. U6 and GAPDH, respectively, served as endogenous reference of miRNA and mRNA. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. COLIA1: Collagen type alpha 1 chain; ELISA: Enzyme linked immunosorbent assay; FAP: Fibroblast activation protein alpha; FN1: Fibronectin 1; miRNAs: microRNAs; PC: Pancreatic cancer; PSCs: Pancreatic stellate cells; RT-PCR: Real-time PCR; WT: Wild type.

Article Snippet: The levels of fibronectin 1 (FN1), Col I, and fibroblast activation protein alpha (FAP) in PSCs cell supernatant were detected by using Human FN ELISA Kit (No. E-EL-H0179c, Elabscience, Wuhan, China), Human Col I ELISA Kit (No. JM-03329H1, JINGMEI, Jiangsu, China), and Human FAPα ELISA Kit (No. EH3037, FineTest, Wuhan, China) according to the manufacturer's instructions.

Techniques: Migration, Software, Transwell Migration Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Activation Assay, Real-time Polymerase Chain Reaction

Journal: Chinese Medical Journal

Article Title: Exosomal miR-485-3p derived from pancreatic ductal epithelial cells inhibits pancreatic cancer metastasis through targeting PAK1

doi: 10.1097/CM9.0000000000002154

Figure Lengend Snippet: MiR-485-3p regulated PC cell migration and Invasion. (A, B) miR-485-3p was overexpressed and silenced In BxPC-3 and PANC-1 cells. (C, D) Transwell migration and Invasion assays in BxPC-3 and PANC-1 cells with overexpression or silencing of miR-485-3p (bar: 100 μm). The migrated or invaded cells were imaged at 200 × magnification in five random fields and counted using ImageJ software. (E) MiR-485-3p was overexpressed and silenced in PSCs. (F) FN1, COLIA1, and FAP levels in cell supernatant of PSCs transfected with miR-485-3p mimic or Inh-miR-485-3p were assessed by ELISA. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. COLIA1: Collagen type alpha 1 chain; ELISA: Enzyme linked immunosorbent assay; FAP: Fibroblast activation protein alpha; FN1: Fibronectin 1; PC: Pancreatic cancer; PSCs: Pancreatic stellate cells.

Article Snippet: The levels of fibronectin 1 (FN1), Col I, and fibroblast activation protein alpha (FAP) in PSCs cell supernatant were detected by using Human FN ELISA Kit (No. E-EL-H0179c, Elabscience, Wuhan, China), Human Col I ELISA Kit (No. JM-03329H1, JINGMEI, Jiangsu, China), and Human FAPα ELISA Kit (No. EH3037, FineTest, Wuhan, China) according to the manufacturer's instructions.

Techniques: Migration, Over Expression, Software, Transfection, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: Cell Reports Medicine

Article Title: Spatial transcriptomic validation of a biomimetic model of fibrosis enables re-evaluation of a therapeutic antibody targeting LOXL2

doi: 10.1016/j.xcrm.2024.101695

Figure Lengend Snippet: The anti-LOXL2 antibody AB0023 does not inhibit collagen-cross linking, tissue stiffness, or catalytic amine oxidase activity in a disease-relevant model of fibrosis (A) LOXL2 protein levels were assayed in the serum of patients with IPF ( n = 12) and control subjects ( n = 13). (B) Expression of LOXL2 within spatially resolved regions of control and IPF lung tissue. Data from Eyres et al. (C) Representative images of mRNA expression of LOXL2 (green chromagen) and PLOD2 (red chromagen) within a fibroblast focus (∗) of IPF lung tissue and the 3D spheroid model using RNAscope RNA in situ hybridization. Scale bars are 20 μm. Arrows indicating cells with co-expression of LOXL2 and PLOD2 . (D–I) Lung fibroblasts from patients with IPF ( n = 3 donors across 2 independent experiments) were used in the 3D spheroid model in the presence of AB0023 or an isotype control antibody at the same concentrations, as well as with PXS-S2A or its vehicle control (0.1% DMSO). (D) Total mature trivalent (PYD + DPD) collagen cross-links determined by ELISA ( n = 3). (E) Tissue stiffness measured from parallel-plate compression testing determined by Young’s modulus and represented as a proportion of control ( n = 6). (F) Total collagen content determined by hydroxyproline assay. (G) LOXL2 catalytic amine oxidase activity within the conditioned media was assessed using an activity-based probe ( n = 3). (H) VEGFA within cell-conditioned media determined by ELISA. (I) Fibronectin within cell-conditioned media determined by ELISA. Data are mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Unpaired two-tailed t test (A) (t = 2.651, degrees of freedom = 23), Wilcoxon test with Benjamini-Hochberg multiple test correction (B), and ANOVA with Šídák’s multiple comparisons test (D–H) were used to evaluate statistical significance (F values: (D) 7.508, (E) 2.862, (F) 0.8966, (G) 33.06, (H) 34.1, and (I) 45.03. Degrees of freedom: (D) 12, (E) 30, (F) 12, (G) 12, (H) 12, and (I) 12). Error bars are standard deviation.

Article Snippet: VEGFA and Fibronectin concentrations were assayed in conditioned media from 3D spheroid models using Human VEGF DuoSet ELISA and Human Fibronectin DuoSet ELISA kits (RnD systems).

Techniques: Activity Assay, Control, Expressing, RNAscope, RNA In Situ Hybridization, Enzyme-linked Immunosorbent Assay, Hydroxyproline Assay, Two Tailed Test, Standard Deviation

Journal: Cell Reports Medicine

Article Title: Spatial transcriptomic validation of a biomimetic model of fibrosis enables re-evaluation of a therapeutic antibody targeting LOXL2

doi: 10.1016/j.xcrm.2024.101695

Figure Lengend Snippet:

Article Snippet: VEGFA and Fibronectin concentrations were assayed in conditioned media from 3D spheroid models using Human VEGF DuoSet ELISA and Human Fibronectin DuoSet ELISA kits (RnD systems).

Techniques: Control, Recombinant, Luminex, RNAscope, Enzyme-linked Immunosorbent Assay, Gene Expression, Software

Journal: Biomedicines

Article Title: Niclosamide Attenuates Inflammation-Associated Profibrotic Responses in Human Subepithelial Lung Myofibroblasts.

doi: 10.3390/biomedicines11072032

Figure Lengend Snippet: Figure 1. The effect of niclosamide on the IL-1α and TNF-α-inducible mRNA expression of collagen type I, collagen type III, and fibronectin. Collagen type I (A), collagen type III (B), and fibronectin (C) mRNA expression levels in SELMs after stimulation with IL-1α, TNF-α or their combination, treated or not with niclosamide at 30 nM and 100 nM concentrations. Mean values of data from experiments per- formed in triplicates on SELMs from 4 individuals are shown. 2C: IL-1α + TNF-α, N30: niclosamide 30 nM, N100: niclosamide 100 nM.

Article Snippet: Human fibronectin was measured in SELM culture supernatants using a commercially available kit (human fibronectin enzyme-linked immunosorbent assay [ELISA] kit, Origene, Biomedicines 2023, 11, 2032 5 of 14 Rockville, MD, USA) as previously described [27] and according to the manufacturer’s instructions.

Techniques: Expressing

Journal: Biomedicines

Article Title: Niclosamide Attenuates Inflammation-Associated Profibrotic Responses in Human Subepithelial Lung Myofibroblasts.

doi: 10.3390/biomedicines11072032

Figure Lengend Snippet: Figure 2. The effect of niclosamide on the TGF-β1-inducible expression of collagen type I, collagen type III, and fibronectin. Collagen type I (A), collagen type III (B), and fibronectin (D) mRNA expression levels in SELMs after stimulation with TGF-β1 with or without treatment with niclosamide at 100 nM concentration. Total secreted collagen from SELMs stimulated with TGF-β1 with or without treatment with niclosamide at 100 nM concentration (C). Mean values of data from experiments performed in triplicate on SELMs from 4 individuals (A,B,D) and 3 individuals (C) are shown. N100: niclosamide 100 nM.

Article Snippet: Human fibronectin was measured in SELM culture supernatants using a commercially available kit (human fibronectin enzyme-linked immunosorbent assay [ELISA] kit, Origene, Biomedicines 2023, 11, 2032 5 of 14 Rockville, MD, USA) as previously described [27] and according to the manufacturer’s instructions.

Techniques: Expressing, Concentration Assay

Journal: Journal of Translational Medicine

Article Title: LTBP1 promotes esophageal squamous cell carcinoma progression through epithelial-mesenchymal transition and cancer-associated fibroblasts transformation

doi: 10.1186/s12967-020-02310-2

Figure Lengend Snippet: LTBP1 contributed to CAFs transformation and expression of FN1 induced by ESCC cells. a The expression of LTBP1 and FN1 were positively correlated in the GEPIA database (Spearman correlation analysis, r = 0.6, p < 0.001). b , c The expression of LTBP1 and FN1 were detected in various cells by western blot and ELISA. d , f Western blot and immunofluorescent analysis of α-SMA and FN1 expression in fibroblasts which were con-cultured with si-NC, si-LTBP1 ESCC cells or negative for 96 h. e CCK8 assays comparing the effect of (± siLTBP1 ESCC cells or negative) conditioned medium on the activity of the fibroblasts. Each experiment was performed in triplicate. Paired-samples t-test, *p < 0.05

Article Snippet: The levels of FN1 in conditioned medium of various cells were measured using Human Fibronectin ELISA Kit (CSB-E04551h; CUSABIO) according to the manufacturer’s instructions.

Techniques: Transformation Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture, Activity Assay