mouse anti human fibronectin primary antibody  (Millipore)

Journal: The FASEB Journal

Article Title: Fibronectin rescues aberrant phenotype of endothelial cells lacking either CCM1, CCM2 or CCM3

doi: 10.1096/fj.201902888r

Figure Lengend Snippet: FIGURE 1 Deregulation of extracellular matrix components after long-term CCM3 inactivation. A, Western Blot analyzes verified complete CCM3 inactivation in clonally expanded CCM3−/− CI-huVECs used in this study (clones I-IV). Expression levels normalized to the CCM3+/+ control group are given below the panel. B, RNA-Seq data of CCM3+/+ control (x-axis) and CCM3−/− CI-huVECs (y-axis) are presented as scatter dot plot. FPKM = fragments per kilobase of exon model per million mapped reads. C, Expression levels of FN1, FBLN5, and POSTN were validated by qPCR. D, Western Blot results revealed less fibronectin in cell culture supernatants of CCM3−/− CI-huVECs and reduced DOC-insoluble fibronectin aggregates upon CCM3-inactivation. Expression levels normalized to the CCM3+/+ control group are given below the subpanels. E, Fluorometric cell adhesion assays demonstrated no major cell binding abnormalities of CCM3−/− CI-huVECs to ECM components. F, A reduced fibronectin expression was observed in immunofluorescence imaging of 1 × 104 cells/well cultured on a tissue culture treated 96-well plate after 48 hours. Plasma fibronectin supplementation promoted fibronectin matrix assembly. Scale bars ≙ 100 µm in the left and 50 µm in the right panels. Images were acquired using the same setting for each sample and no changes were implemented. ND = not detected, RFU = relative fluorescence units, ctrl = CCM3+/+ control cells, Col I = type I collagen, Col II = type II collagen, Col IV = type IV collagen, FN = fibronectin, LN = laminin, TN = tenascin, VN = vitronectin, and Neg = bovine serum albumin. Data are presented as mean and single data points (n = 3-4). Multiple t tests were used for statistical analyzes. ****P < .0001

Article Snippet: Primary mouse anti-human fibronectin antibodies (1:160, MAB19182, R&D Systems) and Alexa Fluor 488-conjugated secondary goat anti-mouse IgG antibodies (1:200, A-11029, Life Technology, Carlsbad, CA, USA) were incubated at RT for 1 hour.

Techniques: Western Blot, Clone Assay, Expressing, Control, RNA Sequencing, Cell Culture, Binding Assay, Immunofluorescence, Imaging, Clinical Proteomics, Fluorescence

Journal: The FASEB Journal

Article Title: Fibronectin rescues aberrant phenotype of endothelial cells lacking either CCM1, CCM2 or CCM3

doi: 10.1096/fj.201902888r

Figure Lengend Snippet: FIGURE 2 Restored endothelial function of CCM3−/− CI-huVECs by fibronectin replacement. A, CCM3−/− CI-huVECs cultured on fibronectin coated plates (5 µg/cm2) regained a typical endothelial morphology. Black arrowheads indicate a compact cell shape, while white arrowheads indicate cells that show a spindle-shaped morphology. CCM3+/+ and CCM3−/− CI-huVECs were seeded with 1 × 104 cells/well on a 96-well plate. Scale bar ≙ 200 µm. B, Fibronectin supplementation (32 µg/mL) significantly improved the spheroid organization of CCM3−/− CI-huVECs. The circularity and the cross-sectional area of the spheroids were determined. The manually traced perimeter of the shown spheroid is depicted in the upper right corner. Scale bar ≙ 100 µm. C, CCM3−/− CI-huVECs cultured on fibronectin-coated plates demonstrated a reduced actin stress fiber formation (1 × 104 cells/well; 96-well plate). Confocal microscopy was used for image acquisition. Phalloidin-iFluor 488 and DAPI staining are shown in green and blue, respectively. The brightness was adjusted equally for all images to show the relevant structures of F-actin formation. Original images are shown in Figure S1. Scale bar ≙ 50 µm. D, The reduced ability of CCM3−/− CI-huVECs to form tube- like structures could not be rescued by fibronectin supplementation (32 µg/mL). Scale bar ≙ 1 mm. E, Neither fibronectin coating (5 µg/cm2) nor supplementation to the culture medium (32 µg/mL) had an effect on staurosporine-induced Caspase-3 activity. F, Representative Phospho-Kinase array membranes are shown for CCM3−/− CI-huVECs cultured without and with fibronectin supplementation (5 µg/cm2). Spots showing the detection of phosphorylated forms of Src and FAK are marked in green or red, respectively. G and H, RNA-Seq data of CCM3+/+ control cells without (x-axis) and CCM3−/− CI-huVECs with 5 µg/cm2 fibronectin supplementation (y-axis) (G) or CCM3−/− CI-huVECs without (x-axis) and with (y-axis) fibronectin supplementation (H) are presented as scatter dot plot. FPKM = fragments per kilobase of exon model per million mapped reads. ctrl = CCM3+/+ control cells, FN = fibronectin, Nb = Number. Data are presented as mean and single data points (n = 3-5). Two-way ANOVA with Holm-Šidák's multiple comparisons test, multiple t test or Student's t test were used for statistical analyzes: *P < .05; **P < .01; ****P < .0001

Article Snippet: Primary mouse anti-human fibronectin antibodies (1:160, MAB19182, R&D Systems) and Alexa Fluor 488-conjugated secondary goat anti-mouse IgG antibodies (1:200, A-11029, Life Technology, Carlsbad, CA, USA) were incubated at RT for 1 hour.

Techniques: Cell Culture, Confocal Microscopy, Staining, Activity Assay, RNA Sequencing, Control

Journal: The FASEB Journal

Article Title: Fibronectin rescues aberrant phenotype of endothelial cells lacking either CCM1, CCM2 or CCM3

doi: 10.1096/fj.201902888r

Figure Lengend Snippet: FIGURE 3 Fibronectin replacement improves spheroid organization of CCM3−/− hCMEC/D3 cells. A, Western Blot analyzes verified complete CCM3 inactivation in clonally expanded hCMEC/D3 used in this study (clones I-III). Expression levels normalized to the CCM3+/+ control group are given below the panel. B, A reduced fibronectin expression was observed in immunofluorescence imaging of cells cultured on a tissue culture treated 96-well plate (1 × 104 cells/well). Scale bars ≙ 200 µm in the left and 50 µm in the right panels. C, Plasma fibronectin supplementation (32 µg/mL) significantly improved spheroid organization of CCM3−/− hCMEC/D3 cells. Shown are the circularity and the area of the spheroids. Scale bar ≙ 100 µm. ctrl = CCM3+/+ hCMEC/D3 cells, FN = fibronectin, ND = not detected. Data are presented as mean and single data points (n = 3). Student's t test was used for statistical analyzes: ***P < .001

Article Snippet: Primary mouse anti-human fibronectin antibodies (1:160, MAB19182, R&D Systems) and Alexa Fluor 488-conjugated secondary goat anti-mouse IgG antibodies (1:200, A-11029, Life Technology, Carlsbad, CA, USA) were incubated at RT for 1 hour.

Techniques: Western Blot, Clone Assay, Expressing, Control, Immunofluorescence, Imaging, Cell Culture, Clinical Proteomics

Journal: The FASEB Journal

Article Title: Fibronectin rescues aberrant phenotype of endothelial cells lacking either CCM1, CCM2 or CCM3

doi: 10.1096/fj.201902888r

Figure Lengend Snippet: FIGURE 4 The 120 kD fragment of fibronectin is sufficient to rescue the cytoskeletal changes and spheroid organization of CCM3−/− CI-huVECs. A, Phalloidin-iFluor 488-(green) and DAPI-(blue) co-staining demonstrated that the actin stress fiber content was significantly decreased in CCM3−/− CI-huVECs that had been cultured on plates coated with cFN, 70 kD or 120 kD fibronectin fragments (5 µg/cm2; 1 × 104 cells/well). Scale bar ≙ 50 µm. B, Only the supplementation of a 120 kD fibronectin fragment (32 µg/mL) and type IV collagen (60 µg/mL) but not of cellular fibronectin (cFN) or a 70 kD fibronectin fragment (32 µg/mL) significantly rescued circularity and proper spheroid organization of CCM3−/− CI-huVECs. Scale bar ≙ 100 µm. ctrl = CCM3+/+ control cells, cFN = cellular fibronectin, 70 kD FN = 70 kD fibronectin fragment, 120 kD FN = 120 kD fibronectin fragment, Col IV = type IV collagen. Data are presented as mean and single data points (n = 3). Two-way ANOVA with Holm-Šidák's multiple comparisons test was used for statistical analyzes: ***P < .001, ****P < .0001

Article Snippet: Primary mouse anti-human fibronectin antibodies (1:160, MAB19182, R&D Systems) and Alexa Fluor 488-conjugated secondary goat anti-mouse IgG antibodies (1:200, A-11029, Life Technology, Carlsbad, CA, USA) were incubated at RT for 1 hour.

Techniques: Staining, Cell Culture, Control

Journal: The FASEB Journal

Article Title: Fibronectin rescues aberrant phenotype of endothelial cells lacking either CCM1, CCM2 or CCM3

doi: 10.1096/fj.201902888r

Figure Lengend Snippet: FIGURE 6 Deregulation of fibronectin expression upon CCM1 or CCM2 inactivation in CI-huVECs. A, High proportion of CCM1 and CCM2 loss-of-function alleles were found in crRNA:tracrRNA:Cas9 RNP-treated CI-huVEC mixtures. B, Immunofluorescence staining in crRNA:tracrRNA:Cas9 RNP-treated CI-huVECs indicated reduced fibronectin expression (1 × 104 cell/well, 96-well plate). Scale bars ≙ 200 µm in the left and 50 µm in the right panels. C, T7EI analyzes revealed no alterations in predicted off-target regions. Three independent replicates are shown (I-III). Expected length of uncleaved (black) and cleaved (gray) amplicons are depicted at the upper left of each subpanel. crRNA CCM1 = CCM1-targeting RNP, crRNA CCM2 = CCM2-targeting RNP, nc crRNA = non-targeting control RNP, FN = fibronectin. Data are presented as mean and single data points (n = 3)

Article Snippet: Primary mouse anti-human fibronectin antibodies (1:160, MAB19182, R&D Systems) and Alexa Fluor 488-conjugated secondary goat anti-mouse IgG antibodies (1:200, A-11029, Life Technology, Carlsbad, CA, USA) were incubated at RT for 1 hour.

Techniques: Expressing, Immunofluorescence, Staining, Control

Journal: The FASEB Journal

Article Title: Fibronectin rescues aberrant phenotype of endothelial cells lacking either CCM1, CCM2 or CCM3

doi: 10.1096/fj.201902888r

Figure Lengend Snippet: FIGURE 7 Fibronectin replacement attenuates endothelial dysfunction upon CCM1 or CCM2 inactivation in CI-huVECs. A, crRNA:tracrRNA:Cas9 RNP-treated CI-huVECs demonstrated a high proportion of cells with a compact morphology, which was significantly decreased by fibronectin supplementation (1 × 104 cell/well; 96-well plate). Black arrowheads indicate the compact cell shape while white arrowheads indicate cells that regained a spindle-shaped morphology after fibronectin supplementation. Scale bar ≙ 200 µm. B and C, CCM1 and CCM2 inactivation in crRNA:tracrRNA:Cas9 RNP-treated cell mixtures led to an increased stress fiber formation (1 × 104 cells/well; 96-well plate, (B) and an impaired spheroid organization (C), which both were attenuated by fibronectin supplementation. Scale bars ≙ 50 µm (B) and 100 µm (C). crRNA CCM1 = CCM1-targeting RNP, crRNA CCM2 = CCM2-targeting RNP, nc crRNA = non-targeting control RNP. Data are presented as mean and single data points (n = 3). Two-way ANOVA with Holm-Šidák's multiple comparisons test or Student's t test were used for statistical analysis: *P < .05; **P < .01; ***P < .001; ****P < .0001

Article Snippet: Primary mouse anti-human fibronectin antibodies (1:160, MAB19182, R&D Systems) and Alexa Fluor 488-conjugated secondary goat anti-mouse IgG antibodies (1:200, A-11029, Life Technology, Carlsbad, CA, USA) were incubated at RT for 1 hour.

Techniques: Control