Journal: Cancers

Article Title: Proteomic Analyses of Fibroblast- and Serum-Derived Exosomes Identify QSOX1 as a Marker for Non-invasive Detection of Colorectal Cancer

doi: 10.3390/cancers13061351

Figure Lengend Snippet: Primary fibroblast-derived EXOs display an activation status dependent protein cargo. The established fibroblast cell lines were subjected to cellular protein isolation after 48 h incubation of equal cell numbers in normal growth medium. EXOs were isolated using combined differential centrifugation and ultrafiltration approach following 48 h of fibroblast incubation in starvation medium. Exosomal protein was isolated in biological triplicates and subjected to Mass Spectrometry. Statistical Analysis was performed in Perseus. ( A ) Immunoblot analysis of vimentin (VIM), α-smooth-muscle actin (αSMA), fibroblast activation protein α (FAPα), caveolin 1 (CAV1), cluster of differentiation 90 (CD90)/Thy1 and fibroblast-specific protein 1 (FSP1) in primary fibroblasts, including GAPDH as loading control. ( B ) Particle size distribution in the isolated EXOs (mean of n = 4–10) as measured by nanoparticle tracking analysis (NTA) using ZetaView ® . ( C ) Representative transmission electron microscopy (TEM) image of fibroblast-derived EXOs. ( D ) Immunoblot analysis of EXO markers CD9, CD63, CD81, Flotillin 1 and Tumor susceptibility 101 (TSG101), including calreticulin as negative control. ( E ) Heat map of mass spectrometry data illustrating significantly deregulated vesicular proteins. Proteins with more than 4 undefined values in total or more than 3 undefined values in the NF/CAF subgroups are excluded. Paired t test: q < 0.05, diff. > |1.0|. LFQ: label-free quantification; nda: no data acquired. ( F ) Immunoblot analysis of quiescin sulfhydryl oxidase 1 (QSOX1), actinin α4 (ACTN4), thrombospondin 1 (THBS1) and EGF-like repeats and discoidin domains 3 (EDIL3) in EXOs. ( G ) Immunoblot analysis of QSOX1, ACTN4, THBS1 and EDIL3 in primary fibroblasts whole cell lysate, including GAPDH as loading control. The images of uncropped western blot figures are shown in .

Article Snippet: The following primary antibodies were used: anti-Actin, α-smooth muscle antibody, mouse monoclonal (A5228, Sigma-Aldrich, 1:1000), human EDIL3 antibody (MAB6046, R&D Systems, 1:200), recombinant anti-quiescin Q6 antibody [EPR21866] (ab235444, Abcam, 1:200), mouse (E5Y6Q) mAb IgG2a (61656, Cell Signaling Technology, 1:200) and rabbit (DA1E) mAb IgG XP ® isotype control (3900, Cell Signaling Technology, 1:200).

Techniques: Derivative Assay, Activation Assay, Isolation, Incubation, Centrifugation, Mass Spectrometry, Western Blot, Control, Transmission Assay, Electron Microscopy, Negative Control, Quantitative Proteomics

Journal: Cancers

Article Title: Proteomic Analyses of Fibroblast- and Serum-Derived Exosomes Identify QSOX1 as a Marker for Non-invasive Detection of Colorectal Cancer

doi: 10.3390/cancers13061351

Figure Lengend Snippet: Selected primary fibroblast activation status dependent EXO markers display specificity to blood EXOs in matched CRC patient plasma. ( A ) Particle size distribution of patient-matched plasma EXOs (pEXO) in comparison to whole plasma (wP) and EXO-depleted plasma (edP) as measured by NTA. ( B ) Immunoblot analysis of quiescin sulfhydryl oxidase 1 (QSOX1), thrombospondin 1 (THBS1), EGF-like repeats and discoidin domains 3 (EDIL3), the EXO markers CD9, CD63 and Flotillin 1, including calreticulin as negative control, and albumin in wP, pEXO and edP protein lysates. ( C ) Graphical analysis of immunoblots shown in (B) using ImageJ, comparing signal strength to pEXO mix. Unpaired t -test: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following primary antibodies were used: anti-Actin, α-smooth muscle antibody, mouse monoclonal (A5228, Sigma-Aldrich, 1:1000), human EDIL3 antibody (MAB6046, R&D Systems, 1:200), recombinant anti-quiescin Q6 antibody [EPR21866] (ab235444, Abcam, 1:200), mouse (E5Y6Q) mAb IgG2a (61656, Cell Signaling Technology, 1:200) and rabbit (DA1E) mAb IgG XP ® isotype control (3900, Cell Signaling Technology, 1:200).

Techniques: Activation Assay, Clinical Proteomics, Comparison, Western Blot, Negative Control

Journal: Cancers

Article Title: Proteomic Analyses of Fibroblast- and Serum-Derived Exosomes Identify QSOX1 as a Marker for Non-invasive Detection of Colorectal Cancer

doi: 10.3390/cancers13061351

Figure Lengend Snippet: In vivo marker expression in patient-matched healthy and malignant colon tissue. ( A ) Representative images of paraffin embedded tissue slides of healthy (hC) and malignant (CRC) colon tissue derived from patients 1–3, hematoxylin and eosin (H&E) or immunohistochemically stained for the proteins quiescin sulfhydryl oxidase 1 (QSOX1), thrombospondin 1 (THBS1), EGF-like repeats and discoidin domains 3 (EDIL3), α-smooth-muscle actin (αSMA) and IgG control (Ctrl.). Scale bars equal 250 µm. ( B ) Graphical IHC staining analysis performed in QuPath. From each patient and tissue, a minimum of three representative areas were subjected to graphical and statistical analysis. Mann-Whitney-U test: * p < 0.05, ** p < 0.01. ns = not significant.

Article Snippet: The following primary antibodies were used: anti-Actin, α-smooth muscle antibody, mouse monoclonal (A5228, Sigma-Aldrich, 1:1000), human EDIL3 antibody (MAB6046, R&D Systems, 1:200), recombinant anti-quiescin Q6 antibody [EPR21866] (ab235444, Abcam, 1:200), mouse (E5Y6Q) mAb IgG2a (61656, Cell Signaling Technology, 1:200) and rabbit (DA1E) mAb IgG XP ® isotype control (3900, Cell Signaling Technology, 1:200).

Techniques: In Vivo, Marker, Expressing, Derivative Assay, Staining, Control, Immunohistochemistry, MANN-WHITNEY

Journal: Cancers

Article Title: Proteomic Analyses of Fibroblast- and Serum-Derived Exosomes Identify QSOX1 as a Marker for Non-invasive Detection of Colorectal Cancer

doi: 10.3390/cancers13061351

Figure Lengend Snippet: Exosomal fibroblast activity marker validation in an independent validation cohort. Twenty additional fibroblast cell lines derived from 10 CRC patients were subjected to cellular protein and EXO isolation. Exosomal protein was isolated and subjected to immunoblot. ( A ) Immunoblot analysis of quiescin sulfhydryl oxidase 1 (QSOX1), thrombospondin 1 (THBS1), EGF-like repeats and discoidin domains 3 (EDIL3), in primary fibroblasts-derived EXOs. ( B ) Immunoblot analysis of QSOX1, THBS1 and EDIL3 in primary fibroblasts whole cell lysate, including GAPDH as loading control. ( C ) Graphical analysis of EXO protein immunoblots shown in ( A ) using ImageJ, relative to GAPDH. Mann-Whitney-U test: * p < 0.05. ( D ) Graphical analysis of cellular protein immunoblots shown in ( B ) using ImageJ, relative to GAPDH. Mann-Whitney-U test: * p < 0.05, *** p < 0.001. ns = not significant.

Article Snippet: The following primary antibodies were used: anti-Actin, α-smooth muscle antibody, mouse monoclonal (A5228, Sigma-Aldrich, 1:1000), human EDIL3 antibody (MAB6046, R&D Systems, 1:200), recombinant anti-quiescin Q6 antibody [EPR21866] (ab235444, Abcam, 1:200), mouse (E5Y6Q) mAb IgG2a (61656, Cell Signaling Technology, 1:200) and rabbit (DA1E) mAb IgG XP ® isotype control (3900, Cell Signaling Technology, 1:200).

Techniques: Activity Assay, Marker, Biomarker Discovery, Derivative Assay, Isolation, Western Blot, Control, MANN-WHITNEY

Journal: Current Pharmaceutical Biotechnology

Article Title: Evaluation of Changes in the Expression Pattern of EDIL3 in Different Grades of Endometrial Cancer

doi: 10.2174/1389201020666190408112822

Figure Lengend Snippet: Optical density of the reaction product for EDIL3 in different grades of endometrial cancer and control.

Article Snippet: Rabbit polyclonal anti-EDIL3 antibody (Novus Biological, USA) was used to perform immunohistochemical staining of the prepared slides.

Techniques: Control

Journal: Molecular Medicine Reports

Article Title: EDIL3 knockdown inhibits retinal angiogenesis through the induction of cell cycle arrest in vitro

doi: 10.3892/mmr.2017.7122

Figure Lengend Snippet: Silencing EDIL3 expression using RNA interference inhibits HREC proliferation in vitro . HRECs were transfected with siEDIL3 or scramble siRNA. (A) EDIL3 mRNA expression levels were assessed using reverse transcription-quantitative polymerase chain reaction. (B) EDIL3 protein expression levels were detected using western blot analysis. (C) Proliferation of HRECs was examined using a Cell Counting kit 8 assay. **P<0.01, ***P<0.001 vs. control and scramble groups. EDIL3, epidermal growth factor-like repeat and discoidin I-like domain-containing protein 3; HRECs, human retinal endothelial cells; si, small interfering.

Article Snippet: Antibodies against EDIL3 (Catalog: 12580-1-AP), cyclin B (Catalog: 12580-1-AP, cyclin D (Catalog: 60186-1-Ig), cyclin E (Catalog: 11554-1-AP) and GAPDH (Catalog: 10494-1-AP) were purchased from Wuhan Sanying Biotechnology (Wuhan, China).

Techniques: Expressing, In Vitro, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Cell Counting, Control

Journal: Molecular Medicine Reports

Article Title: EDIL3 knockdown inhibits retinal angiogenesis through the induction of cell cycle arrest in vitro

doi: 10.3892/mmr.2017.7122

Figure Lengend Snippet: Silencing EDIL3 expression inhibits HREC migration in vitro . HRECs were transfected with siEDIL3 or scramble siRNA and cell migration was examined using a Transwell assay. Representative photomicrographs of migrated cells in (A) control (B) scramble and (C) siEDIL3 groups. Scale bars, 100 µm. (D) Silencing EDIL3 expression significantly reduced HREC migration. ***P<0.001 vs. control and scramble groups. EDIL3, epidermal growth factor-like repeat and discoidin I-like domain-containing protein 3; HRECs, human retinal endothelial cells; si, small interfering.

Article Snippet: Antibodies against EDIL3 (Catalog: 12580-1-AP), cyclin B (Catalog: 12580-1-AP, cyclin D (Catalog: 60186-1-Ig), cyclin E (Catalog: 11554-1-AP) and GAPDH (Catalog: 10494-1-AP) were purchased from Wuhan Sanying Biotechnology (Wuhan, China).

Techniques: Expressing, Migration, In Vitro, Transfection, Transwell Assay, Control

Journal: Molecular Medicine Reports

Article Title: EDIL3 knockdown inhibits retinal angiogenesis through the induction of cell cycle arrest in vitro

doi: 10.3892/mmr.2017.7122

Figure Lengend Snippet: Silencing EDIL3 expression inhibits HREC tube formation in vitro . HRECs were transfected with siEDIL3 or scramble siRNA and a tube formation assay was performed. Representative photomicrographs of formed capillary-like structures in (A) control (B) scramble and (C) siEDIL3 groups. Scale bars, 200 µm. (D) Silencing EDIL3 expression significantly impaired tube formation in HRECs. *P<0.05 vs. control and scramble groups. EDIL3, epidermal growth factor-like repeat and discoidin I-like domain-containing protein 3; HRECs, human retinal endothelial cells; si, small interfering.

Article Snippet: Antibodies against EDIL3 (Catalog: 12580-1-AP), cyclin B (Catalog: 12580-1-AP, cyclin D (Catalog: 60186-1-Ig), cyclin E (Catalog: 11554-1-AP) and GAPDH (Catalog: 10494-1-AP) were purchased from Wuhan Sanying Biotechnology (Wuhan, China).

Techniques: Expressing, In Vitro, Transfection, Tube Formation Assay, Control

Journal: Molecular Medicine Reports

Article Title: EDIL3 knockdown inhibits retinal angiogenesis through the induction of cell cycle arrest in vitro

doi: 10.3892/mmr.2017.7122

Figure Lengend Snippet: Silencing EDIL3 expression induces cell cycle arrest at the G 1 phase. HRECs were transfected with siEDIL3 or scramble siRNA and cell cycle distribution was analyzed using flow cytometry. Representative images of cell cycle distribution in (A) control (B) scramble and (C) siEDIL3 groups. (D) Silencing EDIL3 expression significantly increased the population of HRECs in the G 1 phase. *P<0.05, **P<0.01 vs. control and scramble groups. EDIL3, epidermal growth factor-like repeat and discoidin I-like domain-containing protein 3; HRECs, human retinal endothelial cells; si, small interfering.

Article Snippet: Antibodies against EDIL3 (Catalog: 12580-1-AP), cyclin B (Catalog: 12580-1-AP, cyclin D (Catalog: 60186-1-Ig), cyclin E (Catalog: 11554-1-AP) and GAPDH (Catalog: 10494-1-AP) were purchased from Wuhan Sanying Biotechnology (Wuhan, China).

Techniques: Expressing, Transfection, Flow Cytometry, Control

Journal: Molecular Medicine Reports

Article Title: EDIL3 knockdown inhibits retinal angiogenesis through the induction of cell cycle arrest in vitro

doi: 10.3892/mmr.2017.7122

Figure Lengend Snippet: Silencing EDIL3 expression interferes with the expression of cell cycle-regulatory proteins and EGFR signaling. HRECs were transfected with siEDIL3 or scramble siRNA and the expression of proteins of interest was detected using western blot analysis. (A) Silencing EDIL3 expression suppressed the protein expression of cyclin D1 and cyclin E1, whereas it had no effect on cyclin B1 expression. (B) Silencing EDIL3 expression appeared to inhibit the phosphorylation of EGFR, Src and ERK in HRECs in vitro . EDIL3, epidermal growth factor-like repeat and discoidin I-like domain-containing protein 3; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; HRECs, human retinal endothelial cells; p-, phosphorylated; si, small interfering.

Article Snippet: Antibodies against EDIL3 (Catalog: 12580-1-AP), cyclin B (Catalog: 12580-1-AP, cyclin D (Catalog: 60186-1-Ig), cyclin E (Catalog: 11554-1-AP) and GAPDH (Catalog: 10494-1-AP) were purchased from Wuhan Sanying Biotechnology (Wuhan, China).

Techniques: Expressing, Transfection, Western Blot, Phospho-proteomics, In Vitro