Journal: Bone & Joint Research

Article Title: The role of EDIL3 in maintaining cartilage extracellular matrix and inhibiting osteoarthritis development

doi: 10.1302/2046-3758.1212.BJR-2023-0087.R1

Figure Lengend Snippet: The expression of EGF-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) is higher in human osteoarthritis (OA) cartilage compared with normal cartilage. Paraffin tissue sections were established from both pathological and normal cartilage. Safranin O and immunofluorescence staining were performed for the human articular cartilage sections. a) and b) Compared with normal cartilage, OA cartilage demonstrated chondrocyte cell clustering, empty lacunae morphology, and increased EDIL3 fluorescence signal. c) and d) The chondrocyte cluster and EDIL3-positive cells in the articular cartilage were quantified. Data are presented as means and standard errors. **p < 0.05, ***p < 0.001; independent-samples t -test. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: The next morning, plugs were treated with either vehicle PBS or recombinant EDIL3 protein (2 μg/ml, 6046-ED; R&D Systems, USA).

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence

Journal: Bone & Joint Research

Article Title: The role of EDIL3 in maintaining cartilage extracellular matrix and inhibiting osteoarthritis development

doi: 10.1302/2046-3758.1212.BJR-2023-0087.R1

Figure Lengend Snippet: EGF-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) prevents chondrocyte clustering and chondrocyte loss, and maintains enhanced SOX9 expression in human osteoarthritis (OA) cartilage. a) Osteochondral plugs obtained from patients were cultured ex vivo and treated with phosphate-buffered saline (PBS) (vehicle) or recombinant EDIL3 proteins for six days. Representative images of Safranin O staining and magnified images of regions include the superficial zone (SZ), middle zone (MZ), and deep zone (DZ). Compared with the vehicle control group, the EDIL3 protein-treated group demonstrated prevention of chondrocyte clustering, cell loss, and lacunae morphology in SZ, MZ, and DZ regions. b) The percentage of clustered chondrocytes and chondrocyte number in the cartilage were quantified. The EDIL3 protein treatment reduces the severity of chondrocyte clustering and chondrocyte loss. c) and d) SOX9 protein is displayed in green, and 4′,6-diamidino-2-phenylindole-stained nuclei are in blue. The EDIL3 protein treatment increased the expression level of SOX9. Data are presented as means and standard errors. *p < 0.05, **p < 0.01, ***p < 0.001; two-way analysis of variance, followed by Bonferroni's multiple comparison test for selected pairs of groups for multiple comparisons.

Article Snippet: The next morning, plugs were treated with either vehicle PBS or recombinant EDIL3 protein (2 μg/ml, 6046-ED; R&D Systems, USA).

Techniques: Expressing, Cell Culture, Ex Vivo, Saline, Recombinant, Staining, Control, Comparison

Journal: Bone & Joint Research

Article Title: The role of EDIL3 in maintaining cartilage extracellular matrix and inhibiting osteoarthritis development

doi: 10.1302/2046-3758.1212.BJR-2023-0087.R1

Figure Lengend Snippet: EGF-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) has beneficial effects on cartilage maintenance in mice with osteoarthritis (OA). a) Schematic representation of the timeline of the antibody drug or recombinant protein (EDIL3 or CD98) treatment during 16 to 21 weeks of age. STR/ort mice were injected weekly through the tail vein with phosphate-buffered saline (PBS, vehicle) combined with either the antibody or the protein for six consecutive weeks. Mice were killed at 22 weeks. This was followed by paraffin tissue sections and Safranin O and immunofluorescence (IF) staining in knee articular cartilage. b) Representative images include articular cartilage in the tibial plateau knee joints. Arrows indicate representative matrix-producing (red) and matrix-non-producing (black) chondrocytes. c) The total chondrocyte number in the articular cartilage was quantified. EDIL3 protein treatment increased the number of chondrocytes. d) The number and percentage of matrix-non-producing chondrocytes (MNCs) in the articular cartilage were quantified. EDIL3 antibody treatment increased the number of MNCs. e) EDIL3 protein treatments decreased the Osteoarthritis Research Society International (OARSI) score in the LFC. f) Representative images of IF staining (green) in whole articular cartilage obtained from STR/ort mice for the EDIL3; 4',6-diamidino-2-phenylindole (DAPI) (blue) stained nuclei; orange dashed lines define the cartilage region. EDIL3 antibody treatments decreased the EDIL3 contents in the cartilage region. g) The fluorescence of EDIL3 was quantified in the whole articular cartilage region. EDIL3 antibody significantly decreased EDIL3 expression. h) Representative images of IF staining (green) in whole articular cartilage obtained from STR/ort mice for the indicated OA markers; DAPI (blue) stained nuclei; orange dashed lines define the cartilage region. i) Fluorescence was quantified in the whole articular cartilage region. Data are presented as means and standard errors in . Data are presented as mean and maximum with 95% confidence intervals in . CD98 protein significantly increased SOX9 expression, and EDIL3 protein significantly reduced aggrecan fragments. LTP, lateral tibial plateau; MFC, medial femoral condyle; MMP, matrix metalloproteinase; MTP, medial tibial plateau. Data presented in Figures 3c to 3e were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test for selected pairs of groups for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001. Data in Figures 3g to 3i were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test. Isotype Ab, immunoglobulin G (IgG)1 antibody.

Article Snippet: The next morning, plugs were treated with either vehicle PBS or recombinant EDIL3 protein (2 μg/ml, 6046-ED; R&D Systems, USA).

Techniques: Recombinant, Injection, Saline, Immunofluorescence, Staining, Fluorescence, Expressing, Comparison

Journal: Bone & Joint Research

Article Title: The role of EDIL3 in maintaining cartilage extracellular matrix and inhibiting osteoarthritis development

doi: 10.1302/2046-3758.1212.BJR-2023-0087.R1

Figure Lengend Snippet: EGF-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) treatment prevents subchondral bone plate thickness (PI.Th) and epiphyseal trabecular mineralization. a) Representative cross and horizontal sections of knee joints obtained from STR/ort mice. The 3D reconstruction of a proximal tibia with a plane indicates the location from which the cross and horizontal sections were obtained. The vehicle control mice exhibited uneven trabecular bone distribution; moreover, EDIL3 antibody treatment was observed to worsen this phenomenon. However, EDIL3 and CD98 protein prevented subchondral bone mineralization. b) Trabecular bone morphometric parameters were calculated using micro-CT analysis. The medial and lateral subchondral bone plate and underlying epiphyseal trabecular bone were analyzed separately. Epiphysis was manually selected as representative of subchondral bone. The epiphyseal trabeculae were split from the subchondral bone plate, and trabecular bone morphometric parameters were calculated. EDIL3 protein treatment prevented osteoarthritis (OA)-associated reduction in subchondral bone total porosity (Po(tot)). EDIL3 protein treatment also decreased the subchondral PI.Th, bone volume (BV/TV), and trabecular parameters (trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular spacing (Tb.Sp)). Analyses were conducted with two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. Data are presented as means and standard deviations. *p < 0.05, **p < 0.01; analyzed using a two-way analysis of variance followed by Tukey’s multiple comparison test.

Article Snippet: The next morning, plugs were treated with either vehicle PBS or recombinant EDIL3 protein (2 μg/ml, 6046-ED; R&D Systems, USA).

Techniques: Control, Micro-CT, Comparison

Journal: Bone & Joint Research

Article Title: The role of EDIL3 in maintaining cartilage extracellular matrix and inhibiting osteoarthritis development

doi: 10.1302/2046-3758.1212.BJR-2023-0087.R1

Figure Lengend Snippet: EGF-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) inhibition promotes pro-inflammatory cytokine production in STR/ort mice. a) The variable expressions of 48 cytokines among vehicle control, isotype IgG1, EDIL3 IgG1, recombinant EDIL3 protein, and CD98 protein groups were identified by comparing the expression pattern in the serum of STR/ort mice and those with vehicle control group by heat-map analysis. Arrows indicate representative anti-inflammatory cytokines (red) and pro-inflammatory cytokines (green). b) Quantitation of each cytokine demonstrated serum samples from different groups using Luminex xMAP technology. Six anti-inflammatory cytokines and 20 pro-inflammatory cytokines were identified with the most significant difference between the five groups. c) Notably, many pro-inflammatory cytokines in serum increase with ageing, including monocyte chemotactic protein-3 (MCP-3), RANTES, interleukin (IL)-17A, IL-22, and GRO-alpha. EDIL3 antibody significantly promoted the increase of these pro-inflammatory cytokines. Analyses were conducted with one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test for selected pairs of groups. Data are presented as means and standard errors. *p < 0.05, **p < 0.01, ***p < 0.001. Data presented in Figure 5c were analyzed using one-way analysis of variance followed by Tukey’s multiple comparison test for selected pairs of groups for multiple comparisons. IgG, immunoglobulin G.

Article Snippet: The next morning, plugs were treated with either vehicle PBS or recombinant EDIL3 protein (2 μg/ml, 6046-ED; R&D Systems, USA).

Techniques: Inhibition, Control, Recombinant, Expressing, Quantitation Assay, Luminex, Comparison

Journal: Bone & Joint Research

Article Title: The role of EDIL3 in maintaining cartilage extracellular matrix and inhibiting osteoarthritis development

doi: 10.1302/2046-3758.1212.BJR-2023-0087.R1

Figure Lengend Snippet: EGF-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) prevents chondrocyte loss by inhibiting phosphorylation of glycogen synthase kinase 3 alpha/beta (GSK-3α/β) and phospholipase C gamma 1 (PLC-γ1). a) Chondrocytes were transfected with siEDIL3 or scramble small interfering RNA (siRNA). In comparison with scramble siRNA, siEDIL3 successfully inhibited EDIL3 protein expression in chondrocytes. b) and c) The siRNA-mediated EDIL3 knockdown in chondrocytes attenuated cell viability and increased TUNEL signal. d) Intracellular proteins were collected from chondrocytes and subsequently phosphokinase protein arrays were performed to measure the phosphorylation profiles of the kinases. e) Spots with high-intensity changes were measured by Image J software. EDIL3 attenuated the interleukin (IL)-1β-enhanced phosphokinase protein expression pattern in the chondrocytes, including GSK-3α/β, p38α, PLC-γ1, Src, STAT5ab, and WNK1. f) Hypertrophic chondrocyte-related genes were measured in IL-1β-treated chondrocytes, including SOX9, type II procollagen gene (COL2A1), and COL10A1. EDIL3 restored IL-1β-decreased SOX9 and COL2A1 expression. EDIL3 did not prevent IL-1β-increased type X procollagen gene (COL10A1) expression. Data are presented as means and standard deviations. *p < 0.05, **p < 0.01, ***p < 0.001. Data in c) were analyzed using an independent-samples t -test. Data presented in f) were analyzed using one-way analysis of variance followed by Tukey’s multiple comparison test for selected pairs of groups for multiple comparisons. DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: The next morning, plugs were treated with either vehicle PBS or recombinant EDIL3 protein (2 μg/ml, 6046-ED; R&D Systems, USA).

Techniques: Phospho-proteomics, Transfection, Small Interfering RNA, Comparison, Expressing, Knockdown, TUNEL Assay, Software

Journal: Molecular Cancer

Article Title: Elevated autocrine EDIL3 protects hepatocellular carcinoma from anoikis through RGD-mediated integrin activation

doi: 10.1186/1476-4598-13-226

Figure Lengend Snippet: EDIL3 is significantly up-regulated in HCC and PVTT compared with normal liver (NL) and cirrhotic livers (CL) and is closely related to the prognosis of HCC. A , The transcriptional level of EDIL3 is measured via qRT-PCR in 5 NLs, 10 CLs and 49 HCCs. The relative mRNA level is normalized to β-actin and is presented as Δ–ΔCq. The mRNA of EDIL3 in HCC is significantly higher than both NL and CL; B , The protein level of EDIL3 in NL, CL, HCC and PVTT was examined by western blot with β-actin as a loading control. The EDIL3/β-actin densitometry is performed and shown as density value below. EDIL3 is mildly expressed In NL and CL while significantly elevated in HCC and PVTT; C , Representative pictures demonstrating EDIL3 staining in different liver samples including NL, CL, HCC, PVTT and microscopic thrombi by IHC. Scale bars, 50 μm or 150 μm; Arrow heads indicate that high EDIL3 expression is largely localized to cancer cells. D , Confocal microscopic observation of immunofluorescence staining of CD31 (green), an endothelium marker, and EDIL3 (red) in HCC samples show EDIL3 is not only localized with endothelium, but also widely and diffusively located in cancer cells clusters. White arrows indicates the endothelium; Grey arrows indicates the cancer cell cluster. E , Kaplan-Meier analysis of overall survival between EDIL3-negative or moderately positive patients and highly positive patients shows a significant survival advantage in EDIL3 low express group. **: P < 0.01.

Article Snippet: Human EDIL3 (NM_005711 Origene) with the signal peptide truncated were cloned into the episomal expression vector pCEP-Pu-Strep II-tag (N-terminal) in-frame with the sequence of the BM-40 (SPARC/osteonectin) signal peptide downstream of the CMV promoter, which has been described elsewhere [ ].

Techniques: Quantitative RT-PCR, Western Blot, Staining, Expressing, Immunofluorescence, Marker

Journal: Molecular Cancer

Article Title: Elevated autocrine EDIL3 protects hepatocellular carcinoma from anoikis through RGD-mediated integrin activation

doi: 10.1186/1476-4598-13-226

Figure Lengend Snippet: EDIL3 exhibits a unique expression pattern in cell lines. A , detailed analysis of EDIL3 expression in mRNA level, cell lysates and CMs of 7 HCC and 2 non-HCC cell lines demonstrated an approximate correlation between mRNA and secreted EDIL3 in CMs, whereas EDIL3 in cell lysates exhibited almost same intensity. Notably, Huh-7 and CSQT-2 exhibited a much higher level of secreted EDIL3. B , ELISA assay testing the EDIL3 in CMs of 9 cell lines validates the secreted EDIL3 level is approximately correlated with mRNA level. C , Immunofluorescence staining of EDIL3, F-actin and DAPI in confocal microscope shows EDIL3 is localized within cells and at almost the same intensity in all the 9 cell lines under test, despite their varied mRNA level.

Article Snippet: Human EDIL3 (NM_005711 Origene) with the signal peptide truncated were cloned into the episomal expression vector pCEP-Pu-Strep II-tag (N-terminal) in-frame with the sequence of the BM-40 (SPARC/osteonectin) signal peptide downstream of the CMV promoter, which has been described elsewhere [ ].

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Microscopy

Journal: Molecular Cancer

Article Title: Elevated autocrine EDIL3 protects hepatocellular carcinoma from anoikis through RGD-mediated integrin activation

doi: 10.1186/1476-4598-13-226

Figure Lengend Snippet: Treatment of EDIL3 contributes to anoikis resistance and anchorage-independent growth advantage in HCC. A , Different concentration of recombinant EDIL3 is used to treat SMMC-7721 suspended in poly-hema coated dishes. It sustains the viability of SMMC-7721 as demonstrated by caspase3WSt-8 assay in a time and dose dependent manner compared with control protein. B , Recombinant EDIL3 ameliorate the anoikis compared with control protein in SMMC-7721 as demonstrated by caspase3/7 intensity assay in a time and dose dependent manner. C-D , MHCC-97H is subjected to the assays same as SMMC-7721 and obtain consistent result. E , Administration of recombinant EDIL3 increases the anchorage-independent growth of SMMC-7721 and MHCC-97H cells in soft agar compared with control protein in a dose dependent manner. The assays last for 28 days. *: P < 0.05; **: P < 0.01.

Article Snippet: Human EDIL3 (NM_005711 Origene) with the signal peptide truncated were cloned into the episomal expression vector pCEP-Pu-Strep II-tag (N-terminal) in-frame with the sequence of the BM-40 (SPARC/osteonectin) signal peptide downstream of the CMV promoter, which has been described elsewhere [ ].

Techniques: Concentration Assay, Recombinant

Journal: Molecular Cancer

Article Title: Elevated autocrine EDIL3 protects hepatocellular carcinoma from anoikis through RGD-mediated integrin activation

doi: 10.1186/1476-4598-13-226

Figure Lengend Snippet: EDIL3 promotes HCC tumorigenesis in vivo. A total of 1.0 × 10 6 of EDIL3-overexpressing or control SMMC-7721 cells are subcutaneously implanted into the right flank of 5 nude mice of each groups. The mice were observed and tumors formed are measured every week and resected after 6 weeks. A , After 6 weeks, EDIL3-overexpressing group shows relatively larger tumors compare with control group. B , qRT-PCR of 5 tumors in each group demonstrates a significant elevation in mRNA level. C , Tumor growth curve reveals a shorter latency in the EDIL3-overexpressing group (2 weeks) vs. the control group (4 weeks) and tumors in the EDIL3-overexpressing group are significantly larger than control group from 2 nd week to 4 th week. D , IHC stain in the same region of the tumor confirms the overexpression of EDIL3 and suggests more active proliferation (PCNA) and lower apoptosis (tunel) upon EDIL3 overexpression. **: P < 0.01.

Article Snippet: Human EDIL3 (NM_005711 Origene) with the signal peptide truncated were cloned into the episomal expression vector pCEP-Pu-Strep II-tag (N-terminal) in-frame with the sequence of the BM-40 (SPARC/osteonectin) signal peptide downstream of the CMV promoter, which has been described elsewhere [ ].

Techniques: In Vivo, Quantitative RT-PCR, Staining, Over Expression, TUNEL Assay

Journal: Molecular Cancer

Article Title: Elevated autocrine EDIL3 protects hepatocellular carcinoma from anoikis through RGD-mediated integrin activation

doi: 10.1186/1476-4598-13-226

Figure Lengend Snippet: Sustained activation of FAK-Src by EDIL3 through RGD recognition. A , Western blot and densimetric analysis suggests EDIL3 overexpression sustains the signal intensity of FAK-Src and results in higher AKT phosphorylation within suspended SMMC-7721 cells over 48 hours. The elevated 397 p-FAK, 416 p-Src and 473 p-AKT axis in overexpressing cells compare with control cells exists at most of the time points. B , Cilengitide (10 μM) reversed the activation of the FAK-Src-AKT axis induced by EDIL3. The signal intensity was examined after 24 h of suspension.

Article Snippet: Human EDIL3 (NM_005711 Origene) with the signal peptide truncated were cloned into the episomal expression vector pCEP-Pu-Strep II-tag (N-terminal) in-frame with the sequence of the BM-40 (SPARC/osteonectin) signal peptide downstream of the CMV promoter, which has been described elsewhere [ ].

Techniques: Activation Assay, Western Blot, Over Expression

Journal: Molecular Cancer

Article Title: Elevated autocrine EDIL3 protects hepatocellular carcinoma from anoikis through RGD-mediated integrin activation

doi: 10.1186/1476-4598-13-226

Figure Lengend Snippet: Disrupting integrin-EDIL3 ligation deprives HCC of anoikis resistance induced by EDIL3 in SMMC-7721 and MHCC-LM3. A , Cilengitide (10 μM) reduces the WST-8 value and increased casepase3/7 of the EDIL3 overexpressing group back to levels of control group, suggesting a blockage of EDIL3’s effect. There was not any effect observed in the control group in WST-8 and caspase3/7 assay. Both the two cell lines show consistent results. B , when integrin αV was knocked down to a very low level in both cell lines by two siRNAs (Si2 and Si3), the alteration in WST-8 and casepase3/7 value of EDIL3-overexpressing cells compared with control cells disappear. Interestingly, the WST-8 value and casepase3/7 intensity results suggest the overall anoikis significantly declined upon integrin αV silencing in both siRNA knock-down groups compare with si-control group. **: P < 0.01.

Article Snippet: Human EDIL3 (NM_005711 Origene) with the signal peptide truncated were cloned into the episomal expression vector pCEP-Pu-Strep II-tag (N-terminal) in-frame with the sequence of the BM-40 (SPARC/osteonectin) signal peptide downstream of the CMV promoter, which has been described elsewhere [ ].

Techniques: Ligation

Journal: Molecular Cancer

Article Title: Elevated autocrine EDIL3 protects hepatocellular carcinoma from anoikis through RGD-mediated integrin activation

doi: 10.1186/1476-4598-13-226

Figure Lengend Snippet: Correlation between EDIL3 and key clinicopathological parameters

Article Snippet: Human EDIL3 (NM_005711 Origene) with the signal peptide truncated were cloned into the episomal expression vector pCEP-Pu-Strep II-tag (N-terminal) in-frame with the sequence of the BM-40 (SPARC/osteonectin) signal peptide downstream of the CMV promoter, which has been described elsewhere [ ].

Techniques:

Journal:

Article Title: Del-1 Gene Transfer Induces Cerebral Angiogenesis in Mice

doi: 10.1016/j.brainres.2008.05.003

Figure Lengend Snippet: A. Structure of the 2 AAV vectors with Del-1 or lacZ (as a control). CMV promoter was used to control gene expression in this vector. B. The cDNA encoding human Del-1 was PCR-amplified by using the primers 5′ CG GAA TTC ATG AAG CGC TCG GTA GCC GT 3′ and 5′ CCC AAG CTT TC ATT CCT CCT CTG TGC AGC 3′. The fragment was cloned into the plasmid pAAV-MC with EcoR I/Hind III, and tested by double-enzyme cutting and sequencing. Gel image shows electrophoresis of recombinant pAAV-Del-1after EcoR I/Hind III digestion. Lane 1: molecular size marker. Lanes 2 and 3: Del-1 fragment at 1.2k base pairs.

Article Snippet: We first inserted the human Del-1 cDNA (Origene, Rockville, MD) between two ITRs of pAAV-MC plasmid (Strategene, La Jolla, CA) to generate the pAAV-Del-1.

Techniques: Expressing, Plasmid Preparation, Amplification, Clone Assay, Sequencing, Electrophoresis, Recombinant, Marker

Journal:

Article Title: Del-1 Gene Transfer Induces Cerebral Angiogenesis in Mice

doi: 10.1016/j.brainres.2008.05.003

Figure Lengend Snippet: A. Photomicrograph shows AAV-Del-1 titers using dot blot hybridization. Del-1 gene fragment was amplified by PCR and used as standards. Upper panel in A shows the intensities of dot blot increases with the loading doses of the standards. Low panel in A is a representative blot image of AAV-Del-1 with different loading doses. After hybridization and exposure, the real pixels of dots were measured using software Image J. B. Standard curve, X-axis is log scale of gene copy, and Y-axis is linear scale of pixels. We obtained AAV-Del- 1 of 1.4×1013 /ml.

Article Snippet: We first inserted the human Del-1 cDNA (Origene, Rockville, MD) between two ITRs of pAAV-MC plasmid (Strategene, La Jolla, CA) to generate the pAAV-Del-1.

Techniques: Dot Blot, Hybridization, Amplification, Software

Journal:

Article Title: Del-1 Gene Transfer Induces Cerebral Angiogenesis in Mice

doi: 10.1016/j.brainres.2008.05.003

Figure Lengend Snippet: A. Distribution of X-gal positive staining (blue color) at 5 days following the injection of 5.6 × 1010 particles of AAV-lacZ. The brain section was counter-stained with H&E. B. Representative images of immunofluorescent staining of lectin positive blood vessels demonstrate that AAV-Del-1 transduction increased vascular density in the needle tract region compared to AAV-lacZ transduction. Size bar = 50µm. C. Quantification of microvessel numbers. *, p< 0.05 vs AAV-lacZ, n=8 for each group. The data are representative of 3 separate experiments.

Article Snippet: We first inserted the human Del-1 cDNA (Origene, Rockville, MD) between two ITRs of pAAV-MC plasmid (Strategene, La Jolla, CA) to generate the pAAV-Del-1.

Techniques: Staining, Injection, Transduction

Journal:

Article Title: Del-1 Gene Transfer Induces Cerebral Angiogenesis in Mice

doi: 10.1016/j.brainres.2008.05.003

Figure Lengend Snippet: A. Representative images of co-localization of BrdU+ and CD31+ cells showing active endothelial cell proliferation induced by AAV-Del-1 transfection compared to AAV-lacZ treatment. The image is from a section around the needle track. B. Quantification of BrdU and CD31 co-labeled cells. *, p< 0.05 vs AAV-lacZ, n=8 for each group. Scale bar = 50 µm.

Article Snippet: We first inserted the human Del-1 cDNA (Origene, Rockville, MD) between two ITRs of pAAV-MC plasmid (Strategene, La Jolla, CA) to generate the pAAV-Del-1.

Techniques: Transfection, Labeling

Journal:

Article Title: Del-1 Gene Transfer Induces Cerebral Angiogenesis in Mice

doi: 10.1016/j.brainres.2008.05.003

Figure Lengend Snippet: Upper right panel is a representative Nissl-stained brain coronal section showing ischemic infarct at 3 days after MCAO. Lower right panel shows a Nissl-stained normal brain coronal section. Images A and B show representative Del-1 immunostaining at 3 days after ischemic stroke. Arrows indicate positive Del-1 signal. Boxed area indicates location of Del-1 immunostaining in the peri-infarct cortex. Del-1 ποσιτιω̄ε σιγναλσ ωερε νοτ οβσερω̄εδ ιν τηε νον–ισχηεμιχ σηαμ–οπερατεδ μιχε βραινσ (Χ, Δ). Scale bar = 50 µm.

Article Snippet: We first inserted the human Del-1 cDNA (Origene, Rockville, MD) between two ITRs of pAAV-MC plasmid (Strategene, La Jolla, CA) to generate the pAAV-Del-1.

Techniques: Staining, Immunostaining

Journal: Molecular cancer

Article Title: Elevated autocrine EDIL3 protects hepatocellular carcinoma from anoikis through RGD-mediated integrin activation.

doi: 10.1186/1476-4598-13-226

Figure Lengend Snippet: Figure 5 EDIL3 promotes HCC tumorigenesis in vivo. A total of 1.0 × 106 of EDIL3-overexpressing or control SMMC-7721 cells are subcutaneously implanted into the right flank of 5 nude mice of each groups. The mice were observed and tumors formed are measured every week and resected after 6 weeks. A, After 6 weeks, EDIL3-overexpressing group shows relatively larger tumors compare with control group. B, qRT-PCR of 5 tumors in each group demonstrates a significant elevation in mRNA level. C, Tumor growth curve reveals a shorter latency in the EDIL3-overexpressing group (2 weeks) vs. the control group (4 weeks) and tumors in the EDIL3-overexpressing group are significantly larger than control group from 2nd week to 4th week. D, IHC stain in the same region of the tumor confirms the overexpression of EDIL3 and suggests more active proliferation (PCNA) and lower apoptosis (tunel) upon EDIL3 overexpression. **: P < 0.01.

Article Snippet: The secreted EDIL3 in media were quantified using ELISA kits according to the manufacturer’s instructions (CUSABio).

Techniques: In Vivo, Control, Quantitative RT-PCR, Staining, Over Expression, TUNEL Assay

Journal: Molecular cancer

Article Title: Elevated autocrine EDIL3 protects hepatocellular carcinoma from anoikis through RGD-mediated integrin activation.

doi: 10.1186/1476-4598-13-226

Figure Lengend Snippet: Figure 6 Sustained activation of FAK-Src by EDIL3 through RGD recognition. A, Western blot and densimetric analysis suggests EDIL3 overexpression sustains the signal intensity of FAK-Src and results in higher AKT phosphorylation within suspended SMMC-7721 cells over 48 hours. The elevated 397p-FAK, 416p-Src and 473p-AKT axis in overexpressing cells compare with control cells exists at most of the time points. B, Cilengitide (10 μM) reversed the activation of the FAK-Src-AKT axis induced by EDIL3. The signal intensity was examined after 24 h of suspension.

Article Snippet: The secreted EDIL3 in media were quantified using ELISA kits according to the manufacturer’s instructions (CUSABio).

Techniques: Activation Assay, Western Blot, Over Expression, Phospho-proteomics, Control, Suspension

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