EMBO/SourceData huggingface dataset:
Figure 1Quantitative RT-PCR shows that Lpin1 expression is depleted in the skeletal muscles Gastrocnemius (GC) and Tibialis Anterior (TA), but not in White Adipose Tissue (WAT) from 3-month-old HSACre/+/Lpin1fEx2-3/fEx2-3 mice (HSA-Cre) compared to control mice. Expression levels are corrected for expression of the control gene Pinin and presented in the graph as fold change relative to control mice.PAP1 and PAP2 activities were respectively substantially decreased and increased in TA, of HSA-Cre mice as compared to control mice, respectively (n = 6; *P<0.05).Measurements of muscle/body weight ratio of GC and TA of 3-month-old HSA-Cre and control mice.Hematoxylin and Eosin (HE) staining of representative transverse sections of GC muscles from 3-month-old HSA-Cre and control mice. The black arrows indicate fibers with centrally located nuclei and yellow arrows indicate mononuclear cell infiltration. Scale bars: 40 μmRelative force measurements after normalization of absolute tetanic force to GC muscle weight from 3-month-old HSA-Cre and control male mice.
Figure 2Phosphatidic Acid (PA) levels in HSA-Cre and control GC muscles.Phospholipidome in HSA-Cre and control GC muscles. Proportions of indicated phospholipid classes in total phospholipids are represented. PC; Phosphatidylcholine, PE; Phosphatidylethanolamine, PI; Phosphatidylinositol, PS; Phoshpatidylserine, PA; Phosphatidic Acid, PG; Phosphatidylglycerol.Amounts of indicated phospholipid classes normalized by protein content in HSA-Cre and control GC muscles. (D) Total phospholipid (PL) amount normalized by protein content in HSA-Cre and control GC muscles.Distribution of the total carbon number in acyl chains of PC in HSA-Cre and control GC muscles.Distribution of the number of double bonds in acyl chains of PC in HSA-Cre and control GC muscles.Distribution of the total carbon number in acyl chains of PE in HSA-Cre and control GC muscles.Distribution of the number of double bonds in acyl chains of PE in HSA-Cre and control GC muscles.
Figure 3HE, Oil Red O (ORO), Cytochrome c Oxidase (COX) and Succinate Deshydrogenase (SDH) staining of representative frozen sections of TA muscle from 3-month-old HSA-Cre and control mice.Quantification of triacylglycerol (TAG) levels in GC muscles from 3-month-old HSA-Cre and and control mice.Content of TAG assessed by MS in HSA-Cre and control GC muscles.Cholesterol, (E) Cholesteryl ester (CE), (F) DAG was assessed by MS in HSA-Cre and control GC muscles.
Figure 4Quantitative RT-PCR detection of the level of expression of Fatty acid synthase (Fasn), ATP citrate lyase (Acly), Thyroid hormone-inducible hepatic protein 1 (Thrsp1), Angiopoietin-like protein 4/ fast-induced adipose factor (ANGPTL4/Fiaf), Insulin-induced gene 1 (Insig1) and Peroxisome proliferator-activated receptor gamma (PPARγ) in TA from 3-month-old HSA-Cre and control mice. Expression levels are corrected for expression of the control gene Pinin and presented in the graph as fold change relative to control mice.Immunoblot analysis of Fasn in TA muscle of 3-month-old HSA-Cre and control mice.Quantitative RT-PCR measurement of Carnitine palmitoyltransferase 1B (Cpt1b) and Medium-chain acyl-CoA dehydrogenase (Mcad) in TA from 3-month-old HSA-Cre and control mice.Immunoblot analysis of full length and N-terminal SREBP1c and SREBP2 protein levels in GC muscle of 4-month-old HSA-Cre and control mice (n=7-4). The ratio of the densitometric assay of the N-term and Full length forms is presented.
Figure 5Metabolomics profile of GC muscle of 4-month-old HSA-Cre and control mice presented as heatmap visualization and hierarchical clustering analysis of compoundsFold change of phospholipid precursors in GC muscles of 4-month-old HSA-Cre and control mice measured by liquid-chromatography (LC) tandem mass spectrometry (MS/MS) (n=7-4, *P<0.05).Fold change of Lactate/Pyruvate, Pyruvate/AcetylCoA, Pyruvate/Citrate, ATP/AMP, and ATP/ADP ratios in GC muscles of 4 month-old HSA-Cre and control mice (n=7-4, *P<0.05).
Figure 6UPR activation was analysed by immunoblotting with indicated antibodies in GC muscles of 4-month-old HSA-Cre and control mice (n=7-4). Quantification by densitometric analyses of Fgf21, Bip, sXbp1 Atf6 cleaved form, p-Eif2a and Chop protein levels is presented as a graph. Data are normalized to Gapdh and expressed as a fold change relative to the control mice.Quantitative RT-PCR measurement of Fgf21, Gdf15, Bip/Grp78, hsp90b1, and Edem1 in GC muscles from 4-month-old HSA-Cre and control mice.EM analysis of EDL fibers of HSA-Cre mice revealed the presence of areas in which the non-junctional SR is more vesiculated and fragmented compared to control (black arrows). Remodeling of the SR is also accompanied by modification in the positioning of mitochondria (white arrows). A representative Calcium Release Unit (CRU) is indicated (empty arrow). Scale bars: 0.50 μm.
Figure 7The relative mtDNA copy number was determined by the ratio of mitochondrial DNA-encoded Cytochrome B (CytB) and nuclear-encoded NADH dehydrogenase (ubiquinone) flavoprotein 1 (Ndufv1) genes in GC and TA from HSA-Cre and control mice.Mitochondria-encoded gene and Nuclear-encoded gene expression levels by quantitative RT-PCR in GC muscles from 4-month-old HSA-Cre and control mice.Electron micrographs of HSA-Cre EDL muscles of 3-month-old mice showed that mitochondria are almost exclusively positioned at the two sides of Z lines in EDL fibers from control mice (black arrows). On the other hand, in Lipin1 deficient EDL fibers mitochondria are more often clustered and display electron-dense contact sites (white arrows). Scale bars: 0.20 μm.Ultrastructural analysis of HSA-Cre EDL muscles revealed the presence of numerous autophagic vacuoles of different morphologies (autophagosomes and lysosomes, black arrows; multilamellar bodies, white arrow) containing amorphous material and possibly degraded mitochondria (M). Scale bars: 0.50 μm
Figure 8Representative immunoblots from protein extract of GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice, with the indicated antibodies.Quantitative RT-PCR measurement of Fgf21 and GDF15 expression in GC muscle of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice.Histological analysis of TA muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice by Oil Red O staining to detect the neutral lipids. Scale bars: 40 μm.TAG level measurement in GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice.In vivo force measurements performed on GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice.Representative staining of IgG positive fibers on frozen sections of TA of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice. The sections were stained with a secondary fluorescent anti-mouse IgG antibody. Quantification of the number of necrotic fibers over the total number of fibers.
Figure 9Representative immunoblots from protein extract of GC muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice, with the indicated antibodies.Quantitative RT-PCR measurement of Fgf21 and GDF15 expression in GC muscle of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice.Histological analysis of TA muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice by Oil Red O staining to detect the neutral lipids.TAG level measurement in GC muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice.In vivo force measurements performed on GC muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice.Representative staining of IgG positive fibers on frozen sections of TA of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice. The sections were stained with a secondary fluorescent anti-mouse IgG antibody. Quantification of the number of necrotic fibers over the total number of fibers.
Figure 1Hoxb8-FL cells generated from bone marrow of mice of the indicated genotypes were treated for 24h with graded doses of the CHK1-inhibitors PF-477736 (PF) or CHIR-124 (CHIR). Representative histograms of CHK1i-treated (grey) or DMSO-treated (black) cells, analyzed for cell cycle distribution and cell death using Nicoletti staining and flow cytometry are shown.WT Hoxb8-FL cells were treated with CHIR-124 in the presence or absence of the pan-caspase inhibitor QVD. Cell death was assessed after 24h using flow cytometry as in (A).Western blot of CHIR-treated Hoxb8-FL WT and Bax-/-Bak-/- cells using the indicated antibodies.WT and Bax-/-Bak-/- Hoxb8-FL cells were treated for up to 8h with 500 nM CHIR and p21 mRNA levels were compared to Hprt in response to CHK1-inhibition by real-time RT-qPCR. Bars represent means ± S.D. of independent experiments (n=4-6), performed in duplicates.50.000 Hoxb8-FL cells of the indicated genotypes were seeded to assess cell growth in response to CHK1-inhibition. Cells were counted 48h post seeding.
Figure 2LSK (Lin- Sca1+ cKit+) cells of the indicated genotypes were isolated by cell sorting from the fetal liver at embryonic day E13.5 or the bone marrow of adult mice and treated for 48h with PF-477736 [1.5 μM]. Survival was assessed using AnnexinV-staining and flow cytometry. Bars represent means ± S.E.M. from n=6 wild type and n=3 Vav-BCL2 fetal liver LSK cells (left) and n=3 bone marrow LSK cells (right).WT and Vav-BCL2 derived E14.5 total fetal liver cells (B) were treated for 72h (B) with different doses of CHK1i. Survival was assessed using Nicoletti staining and flow cytometry. Bars represent means ± S.E.M. (n=3 biological replicates per genotype).WT and Vav-BCL2 derived E14.5 total bone marrow cells (C) were treated for up to 48h with different doses of CHK1i. Survival was assessed using Nicoletti staining and flow cytometry. Bars represent means ± S.E.M. (n=3 biological replicates per genotype).
Figure 3MACS-purified CD34+ human cord-blood derived HSPC were treated with PF-477736 or CHIR-124 ± the caspase-inhibitor QVD (50 μM). Cell death was assessed using AnnexinV/7-AAD staining and flow cytometry.CD34+ cells were transduced with a BCL2-encoding lentivirus or a control virus, along with IRES-GFP with a MOI of 10 for two consecutive days, followed by CHK1i application for 48 h. Survival was assessed using Nicoletti staining and flow cytometry.Percentage of living GFP+ transduced human CD34+ HSPCs, cultured in the absence or presence of CHK1i for 48 hours.Colony formation potential of human CD34+ HSPCs transduced with empty control or BCL2 encoding virus. Colonies were counted 10 days post seeding of 1000 CD34+ cells per plate/sample. "Myeloid colonies" include granulocyte/monocyte, monocyte, granulocyte and GEMM (granulocyte, erythroid, monocyte/macrophage and megakaryocyte) colonies.
Figure 4Representative pictures of E13.5 and E18.5 embryos of the indicated genotypes. Scale bar E13.5 = 1 mm, E18.5 = 5 mm. Quantification of fetal liver cell number at E13.5. Bars represent means ± S.E.M. (N=4 for Chk1fl/+, N=3 for Chk1fl/+ Vav-Cre and N=4 for Chk1fl/- Vav-Cre).Representative pictures of isolated fetal livers at E13.5. Scale bar = 1 mm.Representative H&E-staining of fetal liver sections from E13.5 embryos. Black arrows indicate lack of erythroid cells in the blood vessels of Chk1fl/- Vav-Cre fetal livers. Scale bar = 500 µM.Single cell suspensions of fetal livers from Chk1fl/+, Chk1fl/+ Vav-Cre and Chk1fl/- Vav-Cre embryos were stained with antibodies recognizing ckit, CD71, Ter119 or CD45 to assess primitive and definitive erythropoiesis by flow cytometry. Large 'EryA' erythroblasts (CD71high Ter119high FSChigh), smaller, more mature 'EryB' erythroblasts (CD71high Ter119high FSClow); most mature erythroblast subset is EryC (CD71low Ter119high FSClow). Bars represent means ± S.E.M. ctrl N=5 (pooled N=4 Chk1fl/+ and N=1 Chk1fl/+ Vav-Cre), N=3 for Chk1fl/- Vav-Cre.
Figure 5Representative dot-plots of the LSK cell phenotype of Chk1fl/+ and Chk1fl/- Vav-Cre embryos observed in E13.5 fetal livers and relative distribution / absolute numbers of LKhi and LSKhi cells, as well as LKlow and LSKlow cells, found in the fetal liver on E13.5. Bars represent means ± S.E.M. N=4 for Chk1fl/+ and N=4 for Chk1fl/- Vav-Cre.Colony formation potential of FACS-sorted LSK cells (left panel, N=4 for Chk1fl/+ and N=5 for Chk1fl/- Vav-Cre) and total fetal liver cells (right panel, N=3 for Chk1fl/+ and N=6 for Chk1fl/- Vav-Cre) in methyl-cellulose assays. Bars represent means ± S.E.M.Flow cytometric analysis of LSKhi or LSKlow cells stained with antibodies specific for CD34 and Flt3 (N=4 for Chk1fl/+ and N=4 for Chk1fl/- Vav-Cre), to discriminate LT- and ST-HSC from MPP within the LSK-subset. Bars represent means ± S.E.M.
Figure 6Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of fetal liver cryosections of the indicated genotypes at E13.5 and quantification. Scale bar 100x = 200 µM, 400x = 50 µM. The percentage of TUNEL+ area/field was assessed across three random fields from two control (Chk1fl/+) and two knock-out embryos (Chk1fl/- Vav-Cre) using ImageJ, as reported by us before (60). Bars represent means ± S.D.Total fetal liver cell suspensions of Chk1fl/+ (+/+), Chk1fl/+ Vav-Cre (+/-) and Chk1fl/- Vav-Cre (-/-) E13.5 embryos were processed for Western blot analysis using the indicated antibodies. Each lane represents samples isolated from an individual littermate embryo.DAPI-negative viable LK- and LSK-cells of the indicated genotypes (littermates) were sorted from E13.5 fetal livers and immediately fixed in 70% EtOH. Fixed cell suspensions were stained for ɣH2A.X and DAPI intracellularly. Shown here are representative dot-plots and quantification of N=4 embroys per genotype; Bars represent means ± S.E.M.Total E13.5 fetal liver cells were processed for intracellular Ki67 staining in combination with cell surface antibody staining to discriminate LK (top panel) from LSK (lower panel) cells. Shown here are representative dot-plots of Ki67 and DAPI staining in LK and LSK cells, quantified in (E). Bars represent means ± S.E.M. N=4 for Chk1fl/+ and N=5 for Chk1fl/- Vav-Cre.
Figure 7Representative dot-plots and quantification of E13.5 fetal liver LSKhi/LSKlow cell phenotypes observed in embryos of the indicated genotypes. N=21 for Chk1fl/+, N=5 for Chk1fl/- Vav-Cre, N=16 for Chk1fl/+ Vav-BCL2, N=5 for Chk1fl/- Vav-Cre Vav-BCL2, N=2 for p53-/- Chk1fl/+ and N=4 for p53-/- Chk1fl/- Vav-Cre.Flow cytometry-based analysis of LSK cells stained with antibodies specific for CD34 and Flt3, to discriminate LT- and ST-HSC from MPP within the LSK subset in fetal livers of embryos of the indicated genotypes. Left: N=25 for Chk1fl/+, N=7 for Chk1fl/+ Vav-Cre and N=7 for Chk1fl/- Vav-Cre. Right: N=16 for Chk1fl/+ Vav-BCL2, N=6 for Chk1fl/+ Vav-Cre Vav-BCL2 and N=5 for Chk1fl/- Vav-Cre Vav-BCL2.Analysis of the colony formation potential of E14.5 total fetal liver cells of the indicated genotypes in methylcellulose. N=3 for controls (pooled; 1 Chk1fl/+, 2 Chk1fl/+ Vav-Cre), N=3 for Chk1fl/- Vav-Cre and N=3 for Chk1fl/- Vav-Cre Vav-BCL2.Western blot analysis of fetal liver cells of the indicated genotypes (each lane represents an embryo) at E14.5 using the indicated antibodies.
Figure 8Three independent Chk1fl/fl mT/mG (coloured) and mT/mG (grey) Hoxb8-FL progenitor cell lines were transduced with a retroviral construct carrying Cre (pMIP-CRE-IRES-PURO) and monitored over time for GFP-expression. Shown here are three technical replicates per genotype. Additionally, Chk1fl/fl mT/mG Hoxb8-FL progenitor cells were FACS-sorted for GFP+ and dsRED+ cells 48h after transduction with the Cre-construct. Sorted cells were processed for western blotting using the indicated antibodies.Chk1fl/fl Hoxb8-FL progenitor cells were transduced with a retroviral construct carrying Cre and GFP (pMIG-CRE-IRES-GFP). Cells were FACS-sorted 72h after transduction with the retroviral construct and processed for western blotting using the indicated antibodies.Percentage of GFP+ cells indicative for CRE expression and recombination in the peripheral blood after tamoxifen administration by gavage (5 x 2 mg, 200 μl/mouse; daily). Bars represent means ± S.E.M. (Day 0 N=9/10, WT/Chk1fl/fl, Day 7 N=8/7, Day 14 N=3/3).Quantification of Chk1-del PCR-product enrichment in DNA from GFP+ vs. Tomato+ bone marrow cells sorted on day 8 after first TAM administration. Transgenic Cre levels were quantified in parallel for reference. Bars represent means ± S.E.M. from n=5 mT/mG Vav-CreERT2 Chk1fl/fl mice.Percentage of living LK or LSK-cells and (F) percentage of GFP+ LK or LSK-cells found in the bone marrow of mT/mG Vav-CreERT2 (WT) and mT/mG Vav-CreERT2 Chk1fl/fl (Chk1fl/fl) mice on day 8 or 15 post first TAM administration. Bars in (E)/(F) represent means ± S.E.M. (Day 8 N=8/8, WT/Chk1fl/fl, Day 15 N=7/8).GFP+ (CRE) and Tomato+ (CRE-) total bone marrow cells were sorted on day 8 after first TAM-administration and processed for Western Blot using the indicated antibodies. Sorted cells from three animals - mT/mG Vav-CreERT2 (WT) and mT/mG Vav-CreERT2 Chk1fl/fl (Chk1fl/fl) - were pooled per lane.Thymocytes of the indicated genotypes were sorted based on GFP+ and Tomato+ on day 15 after the first TAM administration for western analysis using the indicated antibodies.
Figure 1Quantification of human EGFL7 by qRT-PCR in primary GBM biopsies and corresponding PDX xenografts upon implantation of GBM-derived organotypic spheroids (n = 3; Mann-Whitney U-test; mean ± SEM); B - GBM biopsy; G - generation.Alignment of EGFL7 expression along with stromal (VWF, VEGFR1, VEGFR2, ACTA2, CD248, ITGAX, AIF1, CD68, CD4, and PTPRC) or tumor markers (EGFR, PDGFRA, VEGFA, VIM, and NES) in primary GBM biopsies or GBM-derived organotypic spheroids (PDX xenografts).Immunohisto­chemical (IHC) analyses revealed EGFL7 expression in blood vessels (arrow) and neurons (arrowheads) of healthy gray matter. Expression in malignant brain tumor specimens was restricted to blood vessels, in particular with a large diameter (arrows). Scale bar represents 50 µm. Quantitative scoring of EGFL7 staining in glioma specimens yielded about 25-40% EGFL7-positive blood vessels (n = 13 astrocytoma WHO° grade II, n = 25 astrocytoma WHO° grade III, n = 24 GBMs WHO° grade IV).
Figure 2Overexpression of human EGFL7 (hE7) or murine EGFL7 (mE7) increased tumor growth (n = 9; one-way ANOVA).Immunocompetent mice bearing GL261 tumors. Representative MRI images confirmed tumor implantation in all experimental groups (left). Kaplan-Meier curves revealed decreased survival of mice bearing EGFL7 expressing tumors (right; 34 d (control) vs. 29.5 d (hE7) vs. 31.5 d (mE7); n = 8; log-rank test). Immunodeficient mice bearing U87 tumors. Representative magnetic resonance images confirmed tumor implantation in all experimental groups (left). Kaplan-Meier curves revealed decreased survival of mice bearing EGFL7 expressing tumors (right; 70 d (control) vs. 42.5 d (hE7) vs. 40 d (mE7); n = 8; log-rank test).miR-126 KO did not affect overall survival of GL261 tumor-bearing mice (n = 8).EGFL7 KO prolonged overall survival of GL261 tumor-bearing mice as compared to wild-type (WT) littermates (44 d in KO vs. 35.5 d in WT; n = 8 each; log-rank test).Residual EGFL7 expression in BTPC11 glioma cells was reduced by a lentivirus-based approach. Knock-down of EGFL7 (shE7_1 or shE7_2) prolonged the median survival time of glioma-bearing mice (68 d or 69 d) as compared to scrambled control (shScr; 56 d; n = 6; log-rank test).The blood vessel-specific and miR126-independent KO of EGFL7 in EGFL7fl/fl;Cdh5-CreERT2 mice prolonged the overall survival of GL261 tumor-bearing mice as compared to WT littermates (38 d in KO vs. 33 d in WT; n = 7 for KO; n = 5 for WT; log-rank test). All data are presented as mean ± SEM. Scale bars represent 5 mm.
Figure 3CD31 staining for endothelial cells revealed increased tumor vascularization in mice bearing human EGFL7 (hE7)- or murine EGFL7 (mE7)-positive tumors (n = 3; one-way ANOVA).T2-weighted magnetic resonance imaging (MRI) analysis of these brains confirmed that all mice developed tumors of similar size. T1-weighted MRI images showed decreased vessel permeability as measured by Gadovist extravasation in tumors expressing hE7 or mE7 (red arrows; n = 6; one-way ANOVA). Scale bar represents 2.5 mm.T2-weighted magnetic resonance imaging (MRI) analysis of these brains confirmed that all mice developed tumors of similar size. T1-weighted MRI images showed decreased vessel permeability as measured by Gadovist extravasation in tumors expressing hE7 or mE7 (red arrows; n = 6; one-way ANOVA). Scale bar represents 2.5 mm.EGFL7 promoted density and maturation state of glioma vessels Enhanced maturation of glioma vessels in the presence of hE7 or mE7 was verified by increased co-localization of PDGFRβ (pericytes) with CD31 (n = 3; one-way ANOVA; quantifications normalized to CD31). Data presented as mean ± SEM, AU-arbitrary units. Scale bars represent 60 µm.Enhanced maturation of glioma vessels in the presence of hE7 or mE7 was verified by increased co-localization of SMA (smooth muscle cells), with CD31 (n = 3; one-way ANOVA; quantifications normalized to CD31). Data presented as mean ± SEM, AU-arbitrary units. Scale bars represent 60 µm.Enhanced maturation of glioma vessels in the presence of hE7 or mE7 was verified by increased co-localization of (I+J) Col IV (basement membrane) with CD31 (n = 3; one-way ANOVA; quantifications normalized to CD31). Data presented as mean ± SEM, AU-arbitrary units. Scale bars represent 60 µm.
Figure 4Structural analysis of recombinant EGFL7 by transmission electron microscopy (TEM) showed aggregates with a fibrillar structure of varying sizes. Black scale bar left represents 50 nm. Higher magnifications depict rod-like EGFL7 particles. Black scale bar right represents 15 nm. White scale bar represents 5 nm.Angiogenic sprouting of primary human umbilical vein endothelial cell (HUVEC) spheroids embedded in a collagen-based matrix was induced by the application of EGFL7 (E7), fibronectin (Fn), or a combination of both (upper row). Co-application of an integrin α5β1 blocking antibody (anti-α5β1) reduced sprouting in all cases but control (lower row). Scale bar represents 100 µm.E7, Fn and a combination of both significantly increased the mean cumulative sprout length per spheroid, an effect blocked by anti-α5β1 (n = 3; one-way ANOVA (inter-group differences) and Mann-Whitney U-test (intra-group differences); mean ± SEM).Measurement of Rho GTPase signaling downstream of integrins. Quantification of (G) Cdc42, (H) Rac1 and (I) RhoA activation by immunoblotting subsequent to seeding HUVECs on E7, Fn or a combination of both. Preferentially, E7 activated Cdc42 and Fn activated Rac1, but both proteins antagonized each other. None of the treatments affected RhoA signaling (n = 3; one-way ANOVA). IP-immunoprecipitation, AU-arbitrary units.
Figure 5Surface biotinylation assays verified increased surface expression of αV and β3 upon appli­cation of E7 but not Fn (n = 3; one-way ANOVA).Surface expression of α5 and β1 was found increased upon treatment with E7 or Fn and was further enhanced by combination of both (n = 3; one-way ANOVA).Ectopic expression of murine EGFL7 (mE7) in GL261 mouse glioma cells followed by intracranial implantation into the striatum of immune-competent mice reduced overall survival but was rescued upon treatment with an integrin α5β1-inhibiting antibody (n = 6; log-rank test). Data presented as mean ± SEM. TCL-total cell lysate, AU-arbitrary units.