Journal: Scientific Reports

Article Title: Epidermal Growth Factor Like-domain 7 and miR-126 are abnormally expressed in diffuse Systemic Sclerosis fibroblasts

doi: 10.1038/s41598-019-39485-8

Figure Lengend Snippet: EGFL7 immunostaining in HC and dcSSc skin. ( A , D ) In HC skin, strong EGFL7 expression was detected in ECs and pericytes of vessels (black arrows), in fibroblasts of papillary and reticular dermis (black open arrows), and cells of the basal layer of the epidermis and keratinocytes. ( B , E ) In EOS dcSSc skin, strong EGFL7 expression was detected in ECs and pericytes of vessels (black arrows), in fibroblasts of papillary and reticular dermis and epidermal (black open arrows), cells of the basal layer of the epidermis and keratinocytes as in the HC skin. ECs of the most vessels were strong immunostained for EGFL7. However, ECs of a few vessels displayed a weaker immunostaining intensity for EGFL7 (blu arrows). Inset shows EGFL7 positive fibroblast ( C and F ) In LSS dcSSc skin, the remaining microvessels displayed a weak (or absent) immunostaining for EGFL7. Furthermore, fibroblasts of papillary and reticular dermis and cells of the basal layer of the epidermis and keratinocytes displayed a weak (or absent) immunostaining for EGFL7. Inset shows EGFL7 weak positive fibroblast. Semiquantitative scoring was performed independently by 2 blinded observers, based on the observation of immunostained skin sections. Scores are defined as follows: +++ intense staining; +++/− intense staining but not homogeneous positive staining; ++ moderate staining; + weak staining; +/− weak staining but not homogeneous positive staining.

Article Snippet: Non specific antibody binding was blocked with 10% casein in PBS for 20 min. All cells were incubated for 1 hr with a rabbit polyclonal anti-EGFL7 (Bioss) and mouse monoclonal α-SMA antibody (abcam).

Techniques: Immunostaining, Expressing, Staining

Journal: Scientific Reports

Article Title: Epidermal Growth Factor Like-domain 7 and miR-126 are abnormally expressed in diffuse Systemic Sclerosis fibroblasts

doi: 10.1038/s41598-019-39485-8

Figure Lengend Snippet: Abnormal expression of EGFL7 mRNA and protein levels in FBs of SSc patients ( A–C ). FBs isolated from the skin of EOS SSc patients, express a statistically significant higher levels of EGFL7 mRNA, when compared to HC and LSS SSc-FBs ( *** p = 0.0001). The experiments were performed in triplicate for each patient and HC. Bars represent mean values ± SEM (N = 10 for each group) ( A ). 18 s gene served as the control. WB analysis of EGFL7 ( B ). These results confirm at protein level the results of qRT-PCR analysis. Blots were stripped using Re-Blot Plus Western Blot recycling kit (Chemicon International, USA) and re-probed with anti-mouse IgG β-actin antibody (Sigma-Aldrich, USA) to confirm similar loading of the gels and efficiency in electrophoretic transfer. The experiments were performed in triplicate for each patient and HC. Full length blot is represented in Supplementary Fig. . Immunoreactive bands were acquired by chemidoc (ImageLab). Densitometric analysis of the bands was performed using ImageJ software (NIH; online at http://rsbweb.nih.gov/ij ) ( C ). IF on cultured FBs at 60% of confluence. Negative controls were obtained by omitting the primary antibody.

Article Snippet: Non specific antibody binding was blocked with 10% casein in PBS for 20 min. All cells were incubated for 1 hr with a rabbit polyclonal anti-EGFL7 (Bioss) and mouse monoclonal α-SMA antibody (abcam).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Western Blot, Software, Cell Culture

Journal: Scientific Reports

Article Title: Epidermal Growth Factor Like-domain 7 and miR-126 are abnormally expressed in diffuse Systemic Sclerosis fibroblasts

doi: 10.1038/s41598-019-39485-8

Figure Lengend Snippet: EGFL7 is inducible by TGF-β on HC-FBs and decreases the expression of COL1A1 ( A–G ). The experiments were performed in triplicate. Bars represent mean values ± SEM before and after stimulation with 10 ng/ml of TGF-β for 48 hrs. The absence of rh proteins from the medium of stimulation was considered as a negative control. HC-FBs, after stimulation by TGF-β, expressed significantly higher levels of EGFL7 RNA, when compared to untreated HC-FBs ( *** p < 0.0001). On the contrary, TGF-β treated EOS SSc-FBs did not show any difference in the EGFL7-RNA levels, when compared to the untreated EOS SSc-FBs. No effect was observed, on the EGFL7 RNA levels for the TGF-β treated and untreated LSS SSc-FBs ( A ). The levels of TGF-β in untreated HC-FBs were set to 100% and all the results were normalized to this value. After EGFL7 stimulation, we observed a dose-related fashion decrease of the expression of COL1A1 ( * p = 0.018; *** p = 0.0001 for RNA levels), both in RNA and protein levels, in SSc-FBs. On the contrary, when HC-FBs were treated with the same rhEGFL7 concentrations, the expression of COL1A1 protein did not change at any concentration of EGFL7 stimulation ( B,C ). Full length blot is represented in Supplementary Fig. . Densitometric analysis of the bands was performed using ImageJ software (NIH; online at http://rsbweb.nih.gov/ij ) ( D ).

Article Snippet: Non specific antibody binding was blocked with 10% casein in PBS for 20 min. All cells were incubated for 1 hr with a rabbit polyclonal anti-EGFL7 (Bioss) and mouse monoclonal α-SMA antibody (abcam).

Techniques: Expressing, Negative Control, Concentration Assay, Software

Journal: Scientific Reports

Article Title: Epidermal Growth Factor Like-domain 7 and miR-126 are abnormally expressed in diffuse Systemic Sclerosis fibroblasts

doi: 10.1038/s41598-019-39485-8

Figure Lengend Snippet: EGFL7 knockdown in HC- and SSc-FBs up-regulates Col1A1 expression after TGF-β stimulation. SSc-FBs were transfected with specific EGFL7-siRNA (siRNA) or non-targeting siRNA (scr), and EGFL7 mRNA expression was evaluated by qRT-PCR analysis. The cells transfected with EGFL7-siRNA showed a decreased expression of EGFL7 mRNA levels when compared with cells transfected with scr siRNA ( A ). After TGF-β stimulation, the silenced cells show an overexpression of COL1A1, suggesting the anti-fibrotic role of EGFL7, confirming the effects observed in Fig. . Each experimental condition was performed in triplicate. Bars represent mean values ± SEM.

Article Snippet: Non specific antibody binding was blocked with 10% casein in PBS for 20 min. All cells were incubated for 1 hr with a rabbit polyclonal anti-EGFL7 (Bioss) and mouse monoclonal α-SMA antibody (abcam).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Over Expression

Journal: Scientific Reports

Article Title: Epidermal Growth Factor Like-domain 7 and miR-126 are abnormally expressed in diffuse Systemic Sclerosis fibroblasts

doi: 10.1038/s41598-019-39485-8

Figure Lengend Snippet: EGFL7 promotes migration and invasion but not proliferation of EOS dcSSc-FBs. At basal state EOS SSc-FBs, expressed a significantly lower ki67 transcript, when compared to that of HC. EGFL7 did not modulates the proliferation of treated HC- and EOS-FBs ( A ). Furthermore, we assessed the migratory ability and invasion of our cells and we found that HC- and EOS dcSSc-FBs migration and invasion were significantly induced by EGFL7 in a concentration dependent-manner (1–100 ng/ml) (p = 0.013 and p = 0.0044 and p = 0.0020) ( B ). All assays were independently performed in triplicate and repeated at least thrice. Bars represent mean values ± SEM. Representative photomicrographs show SSc-FBs migration at basal state ( C ), following stimulation with EGFL7 1 ng/ml ( D ) and EGFL7 100 ng/ml ( E ). Original magnification X20.

Article Snippet: Non specific antibody binding was blocked with 10% casein in PBS for 20 min. All cells were incubated for 1 hr with a rabbit polyclonal anti-EGFL7 (Bioss) and mouse monoclonal α-SMA antibody (abcam).

Techniques: Migration, Concentration Assay

Journal: Scientific Reports

Article Title: Epidermal Growth Factor Like-domain 7 and miR-126 are abnormally expressed in diffuse Systemic Sclerosis fibroblasts

doi: 10.1038/s41598-019-39485-8

Figure Lengend Snippet: SSc-FBs are responsible for impaired angiogenesis in an organotypic co-culture assay system in vitro but EGFL7 restores angiogenesis. Angiogenesis was not affected when primary HC-FBs were co-cultured with HUVECS ( A ). Interestingly, when primary SSc-FBs were co-cultured with HUVECs, angiogenesis was impaired when compared with the tubule formation obtained from co-cultured HC-FBs with HUVECs ( B–C ). EGFL7 increases the tubule formation when added to the cultured medium (100 ng/ml) thus adding EGFL7 to the list of the pro-angiogenic molecules that rescue angiogenesis defects in SSc patients ( E–G ). VEGF (25 ng/ml) was used as positive control ( F–K ). Representative microscopic fields at 4X magnification from triplicate wells. Vascular junction and tubule number and total tubule length were analysed by Angiosys System (TCS CellWorks, USA) ( D – H – L ). Bars represent mean values ± SEM (measurment of 12 microscopic fields from triplicate wells). Statistical analysis was performed by unpaired and paired t test, two tailed. * p < 0.05; ** p < 0.001; *** p < 0.0001.

Article Snippet: Non specific antibody binding was blocked with 10% casein in PBS for 20 min. All cells were incubated for 1 hr with a rabbit polyclonal anti-EGFL7 (Bioss) and mouse monoclonal α-SMA antibody (abcam).

Techniques: Co-culture Assay, In Vitro, Cell Culture, Positive Control, Two Tailed Test

Journal: Molecular Cancer

Article Title: A positive feed-forward loop between LncRNA-URRCC and EGFL7/P-AKT/FOXO3 signaling promotes proliferation and metastasis of clear cell renal cell carcinoma

doi: 10.1186/s12943-019-0998-y

Figure Lengend Snippet: URRCC enhances EGFL7 level by mediating histone H3 acetylation of EGFL7 promoter. a : URRCC subnetwork according to the result of Target mRNA PCR Array. Genes colored in blue are down-regulated genes after that cells were transfected with sh-URRCC. Genes colored in red are up-regulated genes after that cells were transfected with sh-URRCC. The size of the gene round represents the fold change. b : The mRNA and protein levels of EGFL7 were detected by qRT-PCR and WB assays in A498 and OSRC-2 cells after transfection with sh-control or sh-URRCC. c : The mRNA and protein levels of EGFL7 were detected by qRT-PCR and WB assays in A498 and OSRC-2 cells after transfection with mock or oe-URRCC. d : EGFL7 expression in ccRCC and normal samples from TCGA KIRC dataset. e : Representative EGFL7 IHC staining of ccRCC tissues compared to paired normal renal tissues (200×, 400×). Black scale bar represents 100 μm (200×) or 50 μm (400×). f and g : Representative EGFL7 IHC staining of xenograft tumors from sh-control, sh-URRCC, mock, and oe-URRCC groups (200×, 400×). Black scale bar represents 100 μm (200×) or 50 μm (400×). h : qRT-PCR and WB analysis of EGFL7 in A498 and OSRC-2 cells treated with DMSO or trichostatin A (TSA) (50 nM or 100 nM) for 72 h ( n = 3). i : ChIP analyses of A498 cells transfected with sh-control or sh-URRCC were conducted on the EGFL7 promoter regions using anti-acetyl-histone H3 and anti-acetyl-histone H4. Enrichment was determined relative to input controls. The data are the means ± standard deviations of three independent biological replicates. j : ChIP analyses of A498 cells transfected with sh-control or sh-URRCC were conducted on the GAPDH promoter regions using anti-acetyl-histone H3 and anti-acetyl-histone H4. Enrichment was determined relative to input controls. k : ChIP analyses of OSRC-2 cells transfected with mock or oe-URRCC were conducted on the EGFL7 promoter regions using anti-acetyl-histone H3 and anti-acetyl-histone H4. Enrichment was determined relative to input controls. The data are the means ± standard deviations of three independent biological replicates. l : EGFL7 promoter region was enriched with H3K27Ac histone mark presented with UCSC data. m : WB analysis of H3K27ac and total H3 in A498 and OSRC-2 cells treated with DMSO or TSA, β-actin was used as a loading control

Article Snippet: Immunohistochemistry for the target molecules was performed on paraffin sections using a primary antibody against EGFL7 (1:50, proteintech, 19,291–1-AP), FOXO3 (1:500, Abcam, ab12162), P-AKT (1:50, Abcam, ab131443), Ki67 (1:500, Abcam, ab92742) and the proteins in situ were visualized with 3, 3-diaminobenzidine.

Techniques: Transfection, Quantitative RT-PCR, Control, Expressing, Immunohistochemistry

Journal: Molecular Cancer

Article Title: A positive feed-forward loop between LncRNA-URRCC and EGFL7/P-AKT/FOXO3 signaling promotes proliferation and metastasis of clear cell renal cell carcinoma

doi: 10.1186/s12943-019-0998-y

Figure Lengend Snippet: URRCC is associated with EGFL7/P-AKT/FOXO3 signaling pathway. a and b : WB analysis of T-AKT, P-AKT, and FOXO3 in URRCC-overexpressing or URRCC-inhibited A498 and OSRC-2 cells compared with sh-control or mock group, β-actin was used as a loading control. c : CCK8 assays of A498 cells transfected with mock/oe-URRCC and si-NC/si-EGFL7. d : Representative images and the numbers of invasive cells per high-power field reduced after the transfection with mock/oe-URRCC and si-NC/si-EGFL7 in A498 cells. e : WB analysis for EGFL7, T-AKT, P-AKT, and FOXO3 protein levels of A498 cells transfected with mock/oe-URRCC and si-NC/si-EGFL7. β-actin was used as a loading control

Article Snippet: Immunohistochemistry for the target molecules was performed on paraffin sections using a primary antibody against EGFL7 (1:50, proteintech, 19,291–1-AP), FOXO3 (1:500, Abcam, ab12162), P-AKT (1:50, Abcam, ab131443), Ki67 (1:500, Abcam, ab92742) and the proteins in situ were visualized with 3, 3-diaminobenzidine.

Techniques: Control, Transfection

Journal: Molecular Cancer

Article Title: A positive feed-forward loop between LncRNA-URRCC and EGFL7/P-AKT/FOXO3 signaling promotes proliferation and metastasis of clear cell renal cell carcinoma

doi: 10.1186/s12943-019-0998-y

Figure Lengend Snippet: A Schematic Diagram of LncRNA-URRCC-Based Signaling Pathway in ccRCC cells proliferation and invasion. LncRNA-URRCC upregulates AKT signaling by directly targeting EGFL7 via mediating histone H3 acetylation of EGFL7 promoter, and then inducing cell growth and invasion. AKT signaling downstream FOXO3 in turn downregulates URRCC by directly binding to its promoter

Article Snippet: Immunohistochemistry for the target molecules was performed on paraffin sections using a primary antibody against EGFL7 (1:50, proteintech, 19,291–1-AP), FOXO3 (1:500, Abcam, ab12162), P-AKT (1:50, Abcam, ab131443), Ki67 (1:500, Abcam, ab92742) and the proteins in situ were visualized with 3, 3-diaminobenzidine.

Techniques: Binding Assay