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