Journal: PLoS ONE

Article Title: The extracellular matrix protein Edil3 stimulates osteoblast differentiation through the integrin α5β1/ERK/Runx2 pathway

doi: 10.1371/journal.pone.0188749

Figure Lengend Snippet: (A) Edil3 mRNA levels in several tissues of mice at post-natal day (PN) 0 were assessed by qRT-PCR and normalized to β-actin levels. Edil3 level in the heart at PN 0 was adjusted to 1. All data are shown as mean ± S.E. (n = 3). (B) Hematoxylin and eosin (H&E) staining of coronal sections from newborn mouse heads. Scale bar indicates 1 mm. (C-G) High-magnification images for boxed area of B. Scale bar indicates 250 μm. (C'-G') Higher magnifications of boxed periosteal areas in the frontal bone of C-G. Scale bar indicates 50 μm. Specimens were stained with hematoxylin and eosin (B, C, and C’) or antibodies against Edil3 (D, D'), Col1a1 (E, E'), and Runx2 (F, F'). Anti-IgG antibody was used as a negative control (G, G'). Arrows indicate positive immunoreactivities against antibodies. Note that signals for Edil3, Col1a1, and Runx2 were strong in the periosteum. Br, Brain; Fb, frontal bone; Ca, Cartilages; Dm, Dura mater.

Article Snippet: Recombinant human Edil3 protein was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Quantitative RT-PCR, Staining, Negative Control

Journal: PLoS ONE

Article Title: The extracellular matrix protein Edil3 stimulates osteoblast differentiation through the integrin α5β1/ERK/Runx2 pathway

doi: 10.1371/journal.pone.0188749

Figure Lengend Snippet: (A) Endogenous expression of Edil3 mRNA in various progenitor cells. Total RNAs were isolated from cells that had been cultured for 3 days and used for RT-PCR with mouse Edil3 and β–actin primers. (B) Expression of Edil3 mRNA during BMP2-induced osteoblast differentiation. MC3T3-E1 cells were cultured with BMP2 (200 ng/ml) for up to 8 days. (C, D) MC3T3-E1 cells were maintained for up to 6 days in osteogenic medium (OM: α-MEM containing 10% FBS, 50 μg/ml ascorbic acid, and 5 mM β-glycerophosphate) and harvested at the indicated time points. RT-PCR was carried out using specific primers after total RNA isolation (B, C). Western blot analysis was performed with Edil3 and β-actin antibodies to evaluate Edil3 protein expression (D).

Article Snippet: Recombinant human Edil3 protein was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Isolation, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: The extracellular matrix protein Edil3 stimulates osteoblast differentiation through the integrin α5β1/ERK/Runx2 pathway

doi: 10.1371/journal.pone.0188749

Figure Lengend Snippet: (A, B) MC3T3-E1 cells were treated for indicated days with 200 ng/ml of Edil3 protein in the presence or absence of osteogenic medium. Cells were then harvested and RT-PCR (left panel) and qRT-PCR (middle and right panels) were performed using specific primers. The number indicates fold ratio between both groups. *, P < 0 . 05; **, P < 0 . 01 . (C) Cells were treated with indicated concentration of Edil3 protein in growth medium (GM). After 4 days of treatment, cells were harvested and RT-PCR was performed. (D) Cells were cultured with indicated concentrations of Edil3 protein for 14 days in the presence of osteogenic medium and stained with alizarin red solution. (E) Graph showing quantified staining levels in (D). *, P < 0 . 05 compared to OM-treated group.

Article Snippet: Recombinant human Edil3 protein was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Concentration Assay, Cell Culture, Staining

Journal: PLoS ONE

Article Title: The extracellular matrix protein Edil3 stimulates osteoblast differentiation through the integrin α5β1/ERK/Runx2 pathway

doi: 10.1371/journal.pone.0188749

Figure Lengend Snippet: (A) Integrin expression in MC3T3-E1 cells. Flow cytometry analysis revealed that the distribution of cells stained for integrins (shaded regions) and IgG as background control (open regions). (B) Effects of integrin-blocking antibodies on Edil3-induced osteoblast differentiation. Cells were incubated with 1 μg/ml of specific or control antibodies and 200 ng/ml of Edil3 for 2 days, and then harvested for RT-PCR analysis. (C) MC3T3-E1 cells were treated with Edil3 and indicated antibodies against integrins, and maintained for 14 days in osteogenic medium. These cells were stained with alizarin red solution. (D) Graph showing quantification of staining levels in (C). **, P < 0 . 01 vs. indicated group. mIgG, mouse IgG; rIgG, rat IgG.

Article Snippet: Recombinant human Edil3 protein was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Flow Cytometry, Staining, Control, Blocking Assay, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: The extracellular matrix protein Edil3 stimulates osteoblast differentiation through the integrin α5β1/ERK/Runx2 pathway

doi: 10.1371/journal.pone.0188749

Figure Lengend Snippet: (A) MC3T3-E1 cells were treated with 200 ng/ml of Edil3 for the indicated time period. Immunoblot analysis was performed with specific antibodies against Akt, ERK, p38, and Runx2 in order to identify its signaling pathway. (B) Cells were cultured with specific inhibitors (MEK/ERK inhibitor U0126, 10 μM; p38 inhibitor SB203580, 10 μM; or Akt inhibitor LY294002, 10 μM) for 1 h, separately, and then treated with 200 ng/ml of Edil3. These cells were harvested after 4 days of treatment and subjected to RT-PCR. (C) Cells were treated with U0126, SB203580, or LY294002 for 1 h. After removing inhibitor-containing medium, cells were incubated with 200 ng/ml Edil3 in osteogenic medium. After 14 days, cells were harvested for AR-staining. (D) Graph showing quantification of staining levels in (C). *, P < 0 . 05 vs. indicated group.

Article Snippet: Recombinant human Edil3 protein was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Incubation, Staining

Journal: PLoS ONE

Article Title: The extracellular matrix protein Edil3 stimulates osteoblast differentiation through the integrin α5β1/ERK/Runx2 pathway

doi: 10.1371/journal.pone.0188749

Figure Lengend Snippet: (A) Runx2 protein expression. MC3T3-E1 cells were treated with or without 200 ng/ml of Edil3 for indicated time periods and then subjected to Western blot analysis. Upper panel is a representative image. Lower graph shows quantitative and relative level of Runx2 protein (n = 3). *, P < 0 . 05 ; **, P < 0 . 01 vs. before treatment. # , P < 0 . 05 ; ## , P < 0 . 01 vs. time control group. (B) Cells were cultured with specific inhibitors (MEK/ERK inhibitor U0126, 10 μM; p38 inhibitor SB203580, 10 μM; or Akt inhibitor LY294002, 10 μM) for 1 h followed by treatment with 200 ng/ml of Edil3 for 4 days. Cells were then harvested for RT-PCR. (C) Cells were treated with Edil3 in the presence or absence of U0126 (+, 5 μM; ++, 10 μM) for 1 h. After changing into growth medium containing 200 ng/ml of Edil3, cells were cultured for 48 h. Western blot analysis was performed with indicated antibodies to evaluate Runx2 expression. Upper panel is a representative image and lower graph shows quantitative level of Runx2 protein (n = 3). *, P < 0 . 05 vs. indicated group. (D) Effects of Edil3 on luciferase activity using a Runx2 luciferase reporter plasmid containing six copies of Runx2-binding osteoblast specific element (6x OSE-Luc). MC3T3-E1 cells were co-transfected with indicated plasmids, including 6x OSE-luc (500 ng), pYX-Asc-Edil3 (+, 300 ng; ++, 600 ng), pCS-Myc-Runx2 (600 ng), and a control mock plasmid (600 ng) with pCMV-β-galactosidase (500 ng). Luciferase activity was measured and normalized to β-galactosidase activity. *, P < 0 . 05 ; **, P < 0 . 01 vs. Mock control.

Article Snippet: Recombinant human Edil3 protein was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Control, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Luciferase, Activity Assay, Plasmid Preparation, Binding Assay, Transfection

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: For the detection of proteins of interest, the following primary antibodies were used: recombinant anti-CD9 antibody [EPR2949] (ab92726, Abcam, 1:200), anti-CD63 antibody (ab68418, Abcam, 1:500), CD81 antibody (NBP2-20564, Novus Biologicals, Centennial, CO, USA, 1:400), anti-TSG101 antibody [4A10] (ab83, Abcam, 1:500), Flotillin-1 antibody (3253, Cell Signaling Technology, 1:500), calreticulin antibody (2891, Cell Signaling Technology, 1:1000), recombinant anti-Quiescin Q6 antibody [EPR21866] (ab235444, Abcam, 1:1000), α-actinin 4 (D7U5A) rabbit mAB (15145, Cell Signaling Technology, 1:1000), thrombospondin-1 (D7E5F) rabbit mAb (37879, Cell Signaling Technology, 1:200) and human EDIL3 antibody (MAB6046, R&D Systems, 1:500).

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: For the detection of proteins of interest, the following primary antibodies were used: recombinant anti-CD9 antibody [EPR2949] (ab92726, Abcam, 1:200), anti-CD63 antibody (ab68418, Abcam, 1:500), CD81 antibody (NBP2-20564, Novus Biologicals, Centennial, CO, USA, 1:400), anti-TSG101 antibody [4A10] (ab83, Abcam, 1:500), Flotillin-1 antibody (3253, Cell Signaling Technology, 1:500), calreticulin antibody (2891, Cell Signaling Technology, 1:1000), recombinant anti-Quiescin Q6 antibody [EPR21866] (ab235444, Abcam, 1:1000), α-actinin 4 (D7U5A) rabbit mAB (15145, Cell Signaling Technology, 1:1000), thrombospondin-1 (D7E5F) rabbit mAb (37879, Cell Signaling Technology, 1:200) and human EDIL3 antibody (MAB6046, R&D Systems, 1:500).

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: For the detection of proteins of interest, the following primary antibodies were used: recombinant anti-CD9 antibody [EPR2949] (ab92726, Abcam, 1:200), anti-CD63 antibody (ab68418, Abcam, 1:500), CD81 antibody (NBP2-20564, Novus Biologicals, Centennial, CO, USA, 1:400), anti-TSG101 antibody [4A10] (ab83, Abcam, 1:500), Flotillin-1 antibody (3253, Cell Signaling Technology, 1:500), calreticulin antibody (2891, Cell Signaling Technology, 1:1000), recombinant anti-Quiescin Q6 antibody [EPR21866] (ab235444, Abcam, 1:1000), α-actinin 4 (D7U5A) rabbit mAB (15145, Cell Signaling Technology, 1:1000), thrombospondin-1 (D7E5F) rabbit mAb (37879, Cell Signaling Technology, 1:200) and human EDIL3 antibody (MAB6046, R&D Systems, 1:500).

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: For the detection of proteins of interest, the following primary antibodies were used: recombinant anti-CD9 antibody [EPR2949] (ab92726, Abcam, 1:200), anti-CD63 antibody (ab68418, Abcam, 1:500), CD81 antibody (NBP2-20564, Novus Biologicals, Centennial, CO, USA, 1:400), anti-TSG101 antibody [4A10] (ab83, Abcam, 1:500), Flotillin-1 antibody (3253, Cell Signaling Technology, 1:500), calreticulin antibody (2891, Cell Signaling Technology, 1:1000), recombinant anti-Quiescin Q6 antibody [EPR21866] (ab235444, Abcam, 1:1000), α-actinin 4 (D7U5A) rabbit mAB (15145, Cell Signaling Technology, 1:1000), thrombospondin-1 (D7E5F) rabbit mAb (37879, Cell Signaling Technology, 1:200) and human EDIL3 antibody (MAB6046, R&D Systems, 1:500).

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

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, Control, 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, Control

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, 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: 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, Phospho-proteomics, Control, Suspension

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, Control, Knockdown

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: 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: Recombinant EDIL3 expression, purification and characterization 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 [46].

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: Recombinant EDIL3 expression, purification and characterization 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 [46].

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