Journal: JCI Insight

Article Title: Erythromycin inhibits neutrophilic inflammation and mucosal disease by upregulating DEL-1

doi: 10.1172/jci.insight.136706

Figure Lengend Snippet: (A) DEL1 mRNA expression determined by qPCR and DEL-1 protein levels determined by ELISA in control or GHSR siRNA-transfected HMVECs treated with ERM (10 μg/mL) or ghrelin (5 μg/mL) 3 hours (mRNA) or 6 hours (protein) (n = 6 culture sets/group). Data normalized against GAPDH mRNA are expressed as fold induction relative to ethanol (set as 1). (B) HMVECs, pretreated for 24 hours with control or GHSR siRNA (20 nM), were incubated with ERM and assayed for phosphorylation at indicated points. (C) After 1-hour pretreatment with AG490 (10 μM), LY294002 (20 μM), or SB203580 (10 μM), HMVECs were incubated 3 hours with ERM and assayed for DEL1 expression (n = 6 culture sets/group). Data normalized against GAPDH mRNA were expressed as fold induction relative to ethanol control (set as 1). (D) HMVECs were transiently transfected with hEDIL3-promoter-Luc reporter plasmid, pretreated 1 hour with inhibitors, and subsequently incubated 8 hours with ERM or control followed by luciferase assay. Data are presented as fold change relative to ethanol control, set as 1 (n = 6 culture sets/group). (E) HMVECs, pretreated as above with inhibitors, were incubated 4 hours with ERM and subjected to ChIP analysis of C/EBPβ occupancy at the EDIL3 promoter (n = 4 culture sets/group). (F) After 30-minute pretreatment with ERM or RvD1 (100 nM), HMVECs were stimulated (3 hours), or not, with IL-17 (5 ng/mL). DEL1 mRNA expression was assayed and presented as above (n = 6 HMVEC culture sets/group). (G) HMVECs were transiently transfected with hEDIL3-promoter-Luc reporter plasmid and pretreated with inhibitors. After 1 hour, the cells were treated with ERM, RvD1, or control for 30 minutes, followed by 8-hour stimulation with IL-17 and luciferase activity assay (n = 6 culture sets/group). Data are presented as fold change relative to ethanol (set as 1). (H) After 1-hour pretreatment with inhibitors, HMVECs were treated with ERM, RvD1, or ethanol for 30 minutes, followed by 4-hour stimulation with IL-17. Chromatin was immunoprecipitated with anti–C/EBPβ IgG and subjected to qPCR of the DEL1 promoter. Nonimmunoprecipitated cell extracts served as input samples. (I) After 1-hour pretreatment with inhibitors, HMVECs were incubated with RvD1 or ERM for 30 minutes and assayed for phosphorylation. In experiments shown in A–I, sequential treatments were performed without intermediate washing steps. Each compound was used at the same concentration in all experiments. Data are shown as means ± SD. **P < 0.001, ***P < 0.0001 by 1-way ANOVA with Tukey’s multiple comparisons test (A and C–H).

Article Snippet: TaqMan probes, sense primers, and antisense primers for the expression of a housekeeping gene ( GAPDH ; Mm99999915_g1, Gapdh ; Hs02786624_g1), as well as investigated genes Edil3 ( Del1 ) ( Del1 ; Mm01291247_m1, DEL1 ; Hs00964112_m1), Il6 (Mm00446190_m1), Il1b (Mm00434228_m1), Tnf (Mm00443258_m1), Il10 (Mm01288386_m1), and Il17a (Mm00439618_m1), were purchased from Thermo Fisher Scientific.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control, Transfection, Incubation, Phospho-proteomics, Plasmid Preparation, Luciferase, Activity Assay, Immunoprecipitation, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Thrombospondin-1 Partly Mediates the Cartilage Protective Effect of Adipose-Derived Mesenchymal Stem Cells in Osteoarthritis

doi: 10.3389/fimmu.2017.01638

Figure Lengend Snippet: List of primers and assays for PCR analysis and transfection experiments.

Article Snippet: EDIL3 , Hs00174781_m1 , .

Techniques: Transfection, Sequencing, Variant Assay, Gene Expression

Journal: Frontiers in Immunology

Article Title: Thrombospondin-1 Partly Mediates the Cartilage Protective Effect of Adipose-Derived Mesenchymal Stem Cells in Osteoarthritis

doi: 10.3389/fimmu.2017.01638

Figure Lengend Snippet: Expression of THBS1 in chondrocyte and adipose stem cell (ASC) mono- or cocultures. (A) Proteins identified in the secretome of chondrocyte/ASC cocultures significantly differentially secreted in chondrocyte/ASC cocultures as compared to mixed monocultures. The white bars represented the fold change of protein expression in coculture and the dot plot represented the number of quantified peptides for each proteins. Abbreviations: PYCARD , PYD and CARD domain containing; CSTA , cystatin A; SRGN , serglycin; XYLT1 , xylosyltransferase 1; THBS1 , thrombospondin-1; PTX3 , pentraxin-3; EDIL3 , EGF-like repeats and discoidin domains 3; UCHL3 , ubiquitin C-terminal hydrolase L3; IGFBP5 , insulin-like growth factor binding protein 5; COL4A2 , collagen type IV alpha 2; NDRG1 , N-Myc downstream regulated 1; ARG1 , arginase 1. (B) THBS1 protein was quantified in the supernatants of chondrocyte monocultures (Chondro), ASC monocultures (ASC) or cocultures by Enzyme-linked ImmunoSorbent Assay. Results are expressed as the mean concentration ± SEM ( n = 26–50 patient cell replicates). (C) THBS1 mRNA level was measured in ASCs and in chondrocytes, cultured alone or in cocultures by RT-qPCR. Results are expressed as relative gene expression (2 −ΔCT ) and represented as mean ± SEM ( n = 5 patient cell replicates). (D) THBS1 mRNA level was measured in BM-MSCs and ASCs by RT-qPCR ( n = 5). Statistics used unpaired t -test (B) or Mann–Whitney test (C,D) : * p ≤ 0.05, *** p ≤ 0.001.

Article Snippet: EDIL3 , Hs00174781_m1 , .

Techniques: Expressing, Ubiquitin Proteomics, Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture, Quantitative RT-PCR, Gene Expression, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Thrombospondin-1 Partly Mediates the Cartilage Protective Effect of Adipose-Derived Mesenchymal Stem Cells in Osteoarthritis

doi: 10.3389/fimmu.2017.01638

Figure Lengend Snippet: Proteins identified in the secretome of chondrocyte/adipose stem cells (ASC) cocultures.

Article Snippet: EDIL3 , Hs00174781_m1 , .

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: 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: Oncotarget

Article Title: Overexpressed EDIL3 predicts poor prognosis and promotes anchorage-independent tumor growth in human pancreatic cancer

doi: 10.18632/oncotarget.6772

Figure Lengend Snippet: ( A ) The mRNA expression of EDIL3 is upregulated in PDAC tissues (T) compared with the normal pancreas tissues (N) revealed using the GSE15471 dataset. ( B ) EDIL3 expression in the normal pancreas and PDAC tissues revealed by the GSE28735 dataset. ( C ) EDIL3 expression analysis in the normal pancreas and PDAC tissues in the GSE16515 dataset. ( D ) The mRNA expression level of EDIL3 in 32 matched tumor (T) and non-tumor tissue (N) derived from Ren Ji cohort was detected by Real-time quantitative PCR. ( E ) Representative photographs of the EDIL3 immunoreactivity in normal pancreas (NP), chronic pancreatitis (CP) and PDAC tissues in TMA1 (scale bar: 100 μm). ( F ) Comparisons of EDIL3 expression in TMA1 revealed by IHC analysis in NP, CP and PDAC tissues. ( G ) Representative photographs of the EDIL3 staining in NP, pancreatic intraepithelial neoplasia-3 (PanIN3) and PDAC tissues in TMA2. The arrows represent positive staining of EDIL3 in the islets (scale bar: 100 μm). ( H ) Comparisons of EDIL3 expression in TMA2 revealed by IHC analysis in NP, PanIN3 and PDAC tissues.

Article Snippet: Briefly, after tissue sections were deparaffinized, rehydrated with graded ethanol, incubated with 0.3% hydrogen peroxide for 30 minutes and blocked with 10% BSA (Sangon, Shanghai, China), slides were first incubated using the antibody for EDIL3 (dilution 1:200, Proteintech, US), PCNA (dilution 1:5000, CST, US), cleaved caspase 3 (dilution 1:2000, CST, US) and Bcl-2 (dilution 1:300, Proteintech, US) at 4°C overnight, labeled by HRP (rabbit) second antibody (Thermo Scientific, US) at room temperature for 1 h. Finally, positive staining was visualized with DAB substrate liquid (Gene Tech, Shanghai), and counterstained by hematoxylin.

Techniques: Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Staining

Journal: Oncotarget

Article Title: Overexpressed EDIL3 predicts poor prognosis and promotes anchorage-independent tumor growth in human pancreatic cancer

doi: 10.18632/oncotarget.6772

Figure Lengend Snippet: Correlations between EDIL3 expression and clinicopathologic parameters in patients with PDAC in TMA2

Article Snippet: Briefly, after tissue sections were deparaffinized, rehydrated with graded ethanol, incubated with 0.3% hydrogen peroxide for 30 minutes and blocked with 10% BSA (Sangon, Shanghai, China), slides were first incubated using the antibody for EDIL3 (dilution 1:200, Proteintech, US), PCNA (dilution 1:5000, CST, US), cleaved caspase 3 (dilution 1:2000, CST, US) and Bcl-2 (dilution 1:300, Proteintech, US) at 4°C overnight, labeled by HRP (rabbit) second antibody (Thermo Scientific, US) at room temperature for 1 h. Finally, positive staining was visualized with DAB substrate liquid (Gene Tech, Shanghai), and counterstained by hematoxylin.

Techniques: Expressing

Journal: Oncotarget

Article Title: Overexpressed EDIL3 predicts poor prognosis and promotes anchorage-independent tumor growth in human pancreatic cancer

doi: 10.18632/oncotarget.6772

Figure Lengend Snippet: ( A ) The correlation between EDIL3 expression and patient survival was conducted in GSE28735 dataset. ( B ) Overall survival analysis of PDAC patients with different EDIL3 protein expression in TMA1. ( C ) Overall survival analysis of PDAC patients with different EDIL3 protein expression in TMA2. ( D ) Comparison of overall survival in patients with or without lymph node metastasis was conducted based on EDIL3 expression. ( E ) Comparisons of overall survival between lower EDIL3 expression group and higher EDIL3 expression group in early TNM stage (I–II) cohort and in advanced TNM stage (III–IV) cohort. P value was calculated by log-rank test.

Article Snippet: Briefly, after tissue sections were deparaffinized, rehydrated with graded ethanol, incubated with 0.3% hydrogen peroxide for 30 minutes and blocked with 10% BSA (Sangon, Shanghai, China), slides were first incubated using the antibody for EDIL3 (dilution 1:200, Proteintech, US), PCNA (dilution 1:5000, CST, US), cleaved caspase 3 (dilution 1:2000, CST, US) and Bcl-2 (dilution 1:300, Proteintech, US) at 4°C overnight, labeled by HRP (rabbit) second antibody (Thermo Scientific, US) at room temperature for 1 h. Finally, positive staining was visualized with DAB substrate liquid (Gene Tech, Shanghai), and counterstained by hematoxylin.

Techniques: Expressing, Comparison

Journal: Oncotarget

Article Title: Overexpressed EDIL3 predicts poor prognosis and promotes anchorage-independent tumor growth in human pancreatic cancer

doi: 10.18632/oncotarget.6772

Figure Lengend Snippet: Univariate and multivariate analysis of prognostic parameters for survival in patients with PDAC in TMA2

Article Snippet: Briefly, after tissue sections were deparaffinized, rehydrated with graded ethanol, incubated with 0.3% hydrogen peroxide for 30 minutes and blocked with 10% BSA (Sangon, Shanghai, China), slides were first incubated using the antibody for EDIL3 (dilution 1:200, Proteintech, US), PCNA (dilution 1:5000, CST, US), cleaved caspase 3 (dilution 1:2000, CST, US) and Bcl-2 (dilution 1:300, Proteintech, US) at 4°C overnight, labeled by HRP (rabbit) second antibody (Thermo Scientific, US) at room temperature for 1 h. Finally, positive staining was visualized with DAB substrate liquid (Gene Tech, Shanghai), and counterstained by hematoxylin.

Techniques: Expressing

Journal: Oncotarget

Article Title: Overexpressed EDIL3 predicts poor prognosis and promotes anchorage-independent tumor growth in human pancreatic cancer

doi: 10.18632/oncotarget.6772

Figure Lengend Snippet: The mRNA ( A ), protein ( B ), secreted ( C ) levels of EDIL3 were assessed in six pancreatic cancer cell lines as well as a nonmalignant cell line hTERT-HPNE by quantitative real-time PCR, Western blotting and ELISA, respectively. ( D ) Interfere efficacy in SW1990 and BxPC-3 cell was detected by Western blotting. Knockdown of EDIL3 promoted anoikis as revealed by flow cytometry ( E ) and caspase-3/7 activity ( F ), and inhibited the colony formation ability ( G ) of SW1990 and BxPC-3 cells. Scale bar: 5 mm. sh-Ctrl versus sh-1 or sh-2, * P < 0.05, ** P < 0.01.

Article Snippet: Briefly, after tissue sections were deparaffinized, rehydrated with graded ethanol, incubated with 0.3% hydrogen peroxide for 30 minutes and blocked with 10% BSA (Sangon, Shanghai, China), slides were first incubated using the antibody for EDIL3 (dilution 1:200, Proteintech, US), PCNA (dilution 1:5000, CST, US), cleaved caspase 3 (dilution 1:2000, CST, US) and Bcl-2 (dilution 1:300, Proteintech, US) at 4°C overnight, labeled by HRP (rabbit) second antibody (Thermo Scientific, US) at room temperature for 1 h. Finally, positive staining was visualized with DAB substrate liquid (Gene Tech, Shanghai), and counterstained by hematoxylin.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Knockdown, Flow Cytometry, Activity Assay

Journal: Oncotarget

Article Title: Overexpressed EDIL3 predicts poor prognosis and promotes anchorage-independent tumor growth in human pancreatic cancer

doi: 10.18632/oncotarget.6772

Figure Lengend Snippet: ( A ) Detection of purified recombinant human EDIL3 protein by coomassie blue staining. ( B ) PDAC cells derived EDIL3 promoted tumor angiogenesis in vitro . Scale bar: 200 μm. Treatment with recombinant EDIL3 protein inhibited anoikis as revealed by flow cytometry ( C ) and caspase-3/7 activity ( D ), and promoted the colony formation ability ( E ) of PANC1 and AsPC1 cells in a dose-dependent manner. Scale bar: 5 mm. Ctrl versus 10 nM EDIL3 or 100 nM EDIL3, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Briefly, after tissue sections were deparaffinized, rehydrated with graded ethanol, incubated with 0.3% hydrogen peroxide for 30 minutes and blocked with 10% BSA (Sangon, Shanghai, China), slides were first incubated using the antibody for EDIL3 (dilution 1:200, Proteintech, US), PCNA (dilution 1:5000, CST, US), cleaved caspase 3 (dilution 1:2000, CST, US) and Bcl-2 (dilution 1:300, Proteintech, US) at 4°C overnight, labeled by HRP (rabbit) second antibody (Thermo Scientific, US) at room temperature for 1 h. Finally, positive staining was visualized with DAB substrate liquid (Gene Tech, Shanghai), and counterstained by hematoxylin.

Techniques: Purification, Recombinant, Staining, Derivative Assay, In Vitro, Flow Cytometry, Activity Assay

Journal: Oncotarget

Article Title: Overexpressed EDIL3 predicts poor prognosis and promotes anchorage-independent tumor growth in human pancreatic cancer

doi: 10.18632/oncotarget.6772

Figure Lengend Snippet: ( A ) Three weeks later, mice in sh-EDIL3 group showed relatively larger tumors compared with that in control group. ( B ) Tumor volume in sh-EDIL3 group was smaller than that control group ( n = 7). ( C ) Tumor weight in sh-EDIL3 group was reduced compared with control group ( n = 7). ( D ) Representative images of EDIL3, PCNA and cleaved caspase 3 in tissues from sh-EDIL3 and sh-Ctrl mice. Compared with sh-Ctrl mice, decreased expression of PCNA ( E ) and increased expression of cleaved caspase 3 ( F ) was observed in the tissue samples of sh-EDIL3 mice. Scale bar: 100 μm. sh-Ctrl versus sh-1 or sh-2, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Briefly, after tissue sections were deparaffinized, rehydrated with graded ethanol, incubated with 0.3% hydrogen peroxide for 30 minutes and blocked with 10% BSA (Sangon, Shanghai, China), slides were first incubated using the antibody for EDIL3 (dilution 1:200, Proteintech, US), PCNA (dilution 1:5000, CST, US), cleaved caspase 3 (dilution 1:2000, CST, US) and Bcl-2 (dilution 1:300, Proteintech, US) at 4°C overnight, labeled by HRP (rabbit) second antibody (Thermo Scientific, US) at room temperature for 1 h. Finally, positive staining was visualized with DAB substrate liquid (Gene Tech, Shanghai), and counterstained by hematoxylin.

Techniques: Control, Expressing

Journal: Oncotarget

Article Title: Overexpressed EDIL3 predicts poor prognosis and promotes anchorage-independent tumor growth in human pancreatic cancer

doi: 10.18632/oncotarget.6772

Figure Lengend Snippet: Altered protein expression level of Bcl-2, Bcl-xL and Bax was detected upon knockdown of EDIL3 ( A ) or treatment with recombinant EDIL3 protein ( B ) ( C ) IHC analysis showed representative positive (up) and negative (down) staining of EDIL3 and Bcl-2 in consecutive sections. Indicated areas were marked by a square. Scale bar: 100 μm. ( D ) Statistical analysis of the correlation between EDIL3 and Bcl-2 expression in TMA1. P values were calculated by the Spearman rank correlation test. In the presence of 1 μM ABT-199, the effects of recombinant EDIL3 protein (100 nM) on caspase-3/7 activity ( E ) and colony formation ability ( F ) was measured.

Article Snippet: Briefly, after tissue sections were deparaffinized, rehydrated with graded ethanol, incubated with 0.3% hydrogen peroxide for 30 minutes and blocked with 10% BSA (Sangon, Shanghai, China), slides were first incubated using the antibody for EDIL3 (dilution 1:200, Proteintech, US), PCNA (dilution 1:5000, CST, US), cleaved caspase 3 (dilution 1:2000, CST, US) and Bcl-2 (dilution 1:300, Proteintech, US) at 4°C overnight, labeled by HRP (rabbit) second antibody (Thermo Scientific, US) at room temperature for 1 h. Finally, positive staining was visualized with DAB substrate liquid (Gene Tech, Shanghai), and counterstained by hematoxylin.

Techniques: Expressing, Knockdown, Recombinant, Staining, Activity Assay

Journal: Journal of Dental Sciences

Article Title: Laser-modified titanium surfaces induce sex-dimorphic secretion of angiogenic factors by gingiva-derived mesenchymal stromal cells

doi: 10.1016/j.jds.2025.09.013

Figure Lengend Snippet: Laser-modified titanium upregulates angiogenesis-related genes in human gingiva-derived mesenchymal stromal cells (GMSCs). Assay for transposase-accessible chromatin sequencing (ATAC-seq) and RNA-sequencing (RNA-seq) were performed on GMSCs cultured on machined, lasered, and SLA (sand-blasted, large-grit, acid-etched) titanium discs for 72 h (n = 2 per group; age-matched 50-year-old female and male). (A) Venn diagrams show differentially upregulated (UP) and downregulated (DOWN) genes in the lasered group and SLA group compared to the machined group, respectively. (B) Volcano plot highlights differentially expressed angiogenesis-related genes. (C) Gene Ontology (GO) enrichment analysis of genes upregulated in the lasered group versus the machined group. (D) Thirty-three genes were identified at the intersection of GO:0016477 (cell migration) and genes upregulated in both female and male GMSCs on laser-modified versus machined surfaces. The heatmap shows shared migration-related genes upregulated in both sexes. (E) Chromatin accessibility and RNA expression levels of CCN1 and EDIL3. F, female. M, male. CCN1, cellular communication network factor 1. EDIL3, EGF like repeats and discoidin domains 3.

Article Snippet: Subsequently, the medium was replaced with fresh exosome-free α-MEM and cells were cultured for an additional 48 h. Conditioned medium was collected for the tube formation assay, and GMSCs were harvested for RNA extraction. mRNA expression was validated using TaqMan Fast Advanced Master Mix (4444556, Invitrogen) with TaqMan Gene Expression Assays (Invitrogen) for CYR61 (Hs00155479_m1), EDIL3 (Hs00964112_m1), and GAPDH (Hs02786624_g1), according to the manufacturer's protocol.

Techniques: Modification, Derivative Assay, Sequencing, RNA Sequencing, Cell Culture, Migration, RNA Expression

Journal: Journal of Dental Sciences

Article Title: Laser-modified titanium surfaces induce sex-dimorphic secretion of angiogenic factors by gingiva-derived mesenchymal stromal cells

doi: 10.1016/j.jds.2025.09.013

Figure Lengend Snippet: Laser-modified titanium surfaces upregulate CCN1 expression in human gingiva-derived mesenchymal stromal cells (GMSCs). Relative gene expression levels of ADM2, CCN1, EDIL3, and β-actin in GMSCs cultured on three different titanium disc surfaces: machined, lasered, and SLA (sand-blasted, large-grit, acid-etched). (A) CCN1 mRNA levels were significantly upregulated in the laser-treated and SLA groups compared to the machined group. (B) and (C) No significant differences in ADM2 and EDIL3 expression were observed among the three groups. (D) β-actin expression was significantly increased in the laser-treated and SLA groups compared to the machined group. Data are means ± standard deviation (n = 8 per group, 4 females and 4 males). Paired t-test. ∗ P < 0.05, ∗∗∗∗ P < 0.0001. mRNA, messenger ribonucleic acid. CCN1, cellular communication network factor 1. ADM2, adrenomedullin 2. EDIL3, EGF like repeats and discoidin domains 3. RPS18, ribosomal protein S18.

Article Snippet: Subsequently, the medium was replaced with fresh exosome-free α-MEM and cells were cultured for an additional 48 h. Conditioned medium was collected for the tube formation assay, and GMSCs were harvested for RNA extraction. mRNA expression was validated using TaqMan Fast Advanced Master Mix (4444556, Invitrogen) with TaqMan Gene Expression Assays (Invitrogen) for CYR61 (Hs00155479_m1), EDIL3 (Hs00964112_m1), and GAPDH (Hs02786624_g1), according to the manufacturer's protocol.

Techniques: Modification, Expressing, Derivative Assay, Gene Expression, Cell Culture, Standard Deviation

Journal: Journal of Dental Sciences

Article Title: Laser-modified titanium surfaces induce sex-dimorphic secretion of angiogenic factors by gingiva-derived mesenchymal stromal cells

doi: 10.1016/j.jds.2025.09.013

Figure Lengend Snippet: Sex-dimorphic secretion of EDIL3 and CCN1 by human gingiva-derived mesenchymal stromal cells (GMSCs). ELISA assays were performed to quantify CCN1 and EDIL3 levels in conditioned medium collected from GMSCs cultured on machined, lasered, and SLA (sand-blasted, large-grit, acid-etched) titanium discs for 72 h. (A) ELISA results from conditioned medium without Triton X treatment. Data are mean ± standard deviation (n = 8; four females and four males). Dotted line: paired t-test. Solid line: unpaired t-test. ∗ P < 0.05. (B) ELISA results from conditioned medium treated with Triton X. Data are mean ± standard deviation (n = 8; four females and four males). Dotted line: paired t-test. Solid line: unpaired t-test. ∗ P < 0.05, ∗∗ P < 0.01. F, female. M, male. CCN1, cellular communication network factor 1. EDIL3, EGF like repeats and discoidin domains 3.

Article Snippet: Subsequently, the medium was replaced with fresh exosome-free α-MEM and cells were cultured for an additional 48 h. Conditioned medium was collected for the tube formation assay, and GMSCs were harvested for RNA extraction. mRNA expression was validated using TaqMan Fast Advanced Master Mix (4444556, Invitrogen) with TaqMan Gene Expression Assays (Invitrogen) for CYR61 (Hs00155479_m1), EDIL3 (Hs00964112_m1), and GAPDH (Hs02786624_g1), according to the manufacturer's protocol.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Standard Deviation

Journal: Journal of Dental Sciences

Article Title: Laser-modified titanium surfaces induce sex-dimorphic secretion of angiogenic factors by gingiva-derived mesenchymal stromal cells

doi: 10.1016/j.jds.2025.09.013

Figure Lengend Snippet: Laser-modified titanium promotes the secretion of extracellular vesicles enriched with CCN1 and EDIL3 from human gingiva-derived mesenchymal stromal cells (GMSCs). (A) Primary GMSCs from one female and one male donor were cultured on machined, lasered, and SLA discs for 72 h, followed by ATAC-seq and RNA-seq analysis. Heatmaps show exosome-related genes. M, machined. L, lasered. S, SLA (sand-blasted, large-grit, acid-etched). (B) Primary GMSCs were cultured on machined, laser-treated, and SLA (sand-blasted, large-grit, acid-etched) titanium discs for 72 h. The conditioned medium was collected and centrifuged at 300× g for 10 min. The supernatant was then filtered through a 0.22 μm membrane, and particle size was analyzed using nanoparticle tracking analysis (NTA). Data are presented as mean values (n = 8 per group; 4 females and 4 males). (C) Western blot analysis was performed to evaluate CCN1, EDIL3 and ADM2 protein levels in cells, extracellular vehicles (EVs), and non-extracellular vesicle (non-EV) fractions derived from GMSCs (51-year-old male, 51M, and 63-year-old female, 63F) were cultured on machined (M), lasered (L), and SLA (sand-blasted, large-grit, acid-etched, S) titanium discs for 72 h. Laser-modified surfaces increased CCN1 in male GMSC EVs and EDIL3 in female EVs compared to machined surfaces. Lower panels show Ponceau S staining of the corresponding PVDF membranes. Results shown are representative of at least three independent experiments. CCN1, cellular communication network factor 1. ADM2, adrenomedullin 2. EDIL3, EGF like repeats and discoidin domains 3. TSG101, tumor susceptibility gene 101.

Article Snippet: Subsequently, the medium was replaced with fresh exosome-free α-MEM and cells were cultured for an additional 48 h. Conditioned medium was collected for the tube formation assay, and GMSCs were harvested for RNA extraction. mRNA expression was validated using TaqMan Fast Advanced Master Mix (4444556, Invitrogen) with TaqMan Gene Expression Assays (Invitrogen) for CYR61 (Hs00155479_m1), EDIL3 (Hs00964112_m1), and GAPDH (Hs02786624_g1), according to the manufacturer's protocol.

Techniques: Modification, Derivative Assay, Cell Culture, RNA Sequencing, Membrane, Western Blot, Staining

Journal: Journal of Dental Sciences

Article Title: Laser-modified titanium surfaces induce sex-dimorphic secretion of angiogenic factors by gingiva-derived mesenchymal stromal cells

doi: 10.1016/j.jds.2025.09.013

Figure Lengend Snippet: Silencing CCN1 or EDIL3 in gingiva-derived mesenchymal stromal cells (GMSCs) impairs HUVEC angiogenesis. GMSCs were seeded on machined, lasered, or SLA (sand-blasted, large-grit, acid-etched) titanium discs and transfected with siRNAs targeting CCN1 or EDIL3 for 24 h. (A) Relative mRNA expression of CCN1 and EDIL3 after siRNA transfection. NC, negative control siRNA. (B) Representative images of HUVEC tube formation in response to conditioned medium (CM) collected from female (F) and male (M) GMSCs. Images were captured using the ImageXpress Pico system. Scale bars = 500 μm. (C) Quantification of tube formation (branch points, total length, segments, and nodes) using ImageJ. Data are mean ± standard deviation (n = 2, one female and one male). One-way ANOVA analysis with Tukey's multiple comparison test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. ns, not significant.

Article Snippet: Subsequently, the medium was replaced with fresh exosome-free α-MEM and cells were cultured for an additional 48 h. Conditioned medium was collected for the tube formation assay, and GMSCs were harvested for RNA extraction. mRNA expression was validated using TaqMan Fast Advanced Master Mix (4444556, Invitrogen) with TaqMan Gene Expression Assays (Invitrogen) for CYR61 (Hs00155479_m1), EDIL3 (Hs00964112_m1), and GAPDH (Hs02786624_g1), according to the manufacturer's protocol.

Techniques: Derivative Assay, Transfection, Expressing, Negative Control, Standard Deviation, Comparison

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human recombinant EDIL3 (DEL1) , Abnova , Cat. #H00010085-P01.

Techniques: Virus, Generated, Control, Isolation, Recombinant, Purification, Staining, Diagnostic Assay, Cell Isolation, Enzyme-linked Immunosorbent Assay, Labeling, Gene Expression, Reverse Transcription, Synthesized, Software, Transfection, Imaging, Flow Cytometry, Microscopy, Western Blot

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Recombinant human CD99 (H00004267-P01) and EDIL3/DEL1 (H00010085-P01) proteins were from Abnova Corporation (Taipei, Taiwan).

Techniques: Generated, Isolation, Recombinant, Purification, Staining, Diagnostic Assay, Cell Isolation, Enzyme-linked Immunosorbent Assay, Labeling, Expressing, Synthesized, Software, Transfection, Imaging, Flow Cytometry, Microscopy, Western Blot