Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: The Nucleus Bypasses Obstacles by Deforming Like a Drop with Surface Tension Mediated by Lamin A/C.

doi: 10.1002/advs.202201248

Figure Lengend Snippet: Figure 1. Nuclear invaginations around microposts permit unhindered forward motion of the nucleus. A) Schematic illustrates micropost geometry relative to cell shape. B) SEM image shows the circular pattern of fabricated microposts (left, scale bar is 20 μm) and micropost geometry (right). C) SEM of an MDCK-II cell on microposts, yellow arrowheads indicate internalized microposts (Scale bar is 5 μm). D) Time-lapse confocal images of an NIH 3T3 fibroblast stably expressing GFP-BAF deforming around 5 μm tall rhodamine-fibronectin coated PDMS microposts (red) and forming transient deep, local invaginations in the nuclear surface (Scale bar is 5 μm). Nuclear outlines relative to the position of microposts are shown on the right. E) Plot shows the percentage of cells with nuclei that formed deep invaginations around the microposts in different cell types, including fibroblasts (NIH 3T3 and MEF), epithelial cells (MDCK-II), and breast cancer cells (MDA-MB-231); n = 50 cells per condition from 3 independent experiments. F) Nuclear trajectories of NIH 3T3 fibroblasts migrating on a flat substrate (left) and microposts (right); n = 30 cells imaged for 2 hours for each condition from 3 independent experiments. G) Bar graph shows the mean nuclear speed in cells migrating against a varying number of microposts. Error bars represent SEM. (ns p > 0.05; Brown–Forsythe and Welch ANOVA test). H) Time-lapse confocal images of an NIH 3T3 fibroblast stably expressing GFP-BAF deforming around 11 μm tall Si microposts (red), forming deep invaginations in the x-y plane, separated by lobes of nearly constant curvature (Scale bar is 5 μm). Nuclear outlines relative to the position of microposts are shown on the right. I) Nuclear envelope rupture in fibroblasts caused by micropost indentation. White arrowhead points to the local enrichment of GFP-BAF indicative of rupture (Scale bar is 5 μm).

Article Snippet: Fluorescent Labeling: The micropost pattern was coated with rhodamine-conjugated fibronectin (FNR01, Cytoskeleton Inc., Denver, CO) at a concentration of 5 μg mL−1 to promote cell adhesion while fluorescently-labeling the microposts.

Techniques: Stable Transfection, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: The Nucleus Bypasses Obstacles by Deforming Like a Drop with Surface Tension Mediated by Lamin A/C.

doi: 10.1002/advs.202201248

Figure Lengend Snippet: Figure 2. Microposts in nuclear invaginations are not exposed to cellular or nuclear contents. A) Confocal images of a fibroblast cultured on microposts, with plasma membrane labeled with PlasMem bright red, and expressing GFP-lamin A (green). The apical, middle and basal planes are shown along with the corresponding x–z view situated along the white-dashed line (Scale bar is 5 μm). B) Confocal images of F-actin stained with phalloidin (white) and GFP-BAF (green) expressing fibroblast with nucleus deformed around the microposts coated with Rhodamine-fibronectin (red); corresponding x–z views are also shown. (Scale bar is 5 μm). C) Confocal image of F-actin stress fibers (green) in the basal plane of a fibroblast cultured on rhodamine- fibronectin-coated microposts (red) (Scale bar is 5 μm). D) Images of an MCF-10A cell stably expressing GFP-N144 (green) and mCherry-nes2-KASH (red) cultured on microposts. White arrowheads point to the ring of the inner and outer nuclear membrane proteins around a single micropost present in a nuclear invagination (Scale bar is 5 μm). E) Time-lapse image sequence showing the development of nuclear invaginations (yellow arrowheads) in a GFP-lamin A expressing fibroblast (Scale bar is 5 μm).

Article Snippet: Fluorescent Labeling: The micropost pattern was coated with rhodamine-conjugated fibronectin (FNR01, Cytoskeleton Inc., Denver, CO) at a concentration of 5 μg mL−1 to promote cell adhesion while fluorescently-labeling the microposts.

Techniques: Cell Culture, Clinical Proteomics, Membrane, Labeling, Expressing, Staining, Stable Transfection, Sequencing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: The Nucleus Bypasses Obstacles by Deforming Like a Drop with Surface Tension Mediated by Lamin A/C.

doi: 10.1002/advs.202201248

Figure Lengend Snippet: Figure 6. Lamin A/C preserves overall nuclear shape during nuclear invagination around microposts. A) Images of fixed Hoechst 33 342 stained MEF WT and MEF Lmna−/−nuclei deformed around microposts. Yellow arrowheads point to the micropost locations, and white arrowheads indicate wispy finger-like nuclear extensions around the microposts (Scale bar is 5 μm). B) Comparison of circularity of MEF WT nuclei and MEF Lmna−/−nuclei, deformed around microposts, or on surfaces devoid of microposts; (n = 31 nuclei for WT, n = 30 nuclei Lmna−/−on flat surface, and n = 32 nuclei for Lmna−/−on microposts from at least three experiments per condition; ns: p > 0.05 and ****: p < 0.0001; Kruskal–Wallis test). C) Top two panels: time- lapse sequences of a MEF WT nucleus and an MEF Lmna−/−nucleus stained with NucSpot Live 650 (a live-nuclear imaging dye) (white) during nuclear invagination around rhodamine-labeled microposts (red). Scale bar is 5 μm. Third panel: time-lapse sequence of an MEF Lmna−/−nucleus expressing WT GFP-Lamin A (green) deforming against a rhodamine-fibronectin labeled micropost (red). Scale bar is 5 μm. Bottom panel: time-lapse sequence of an MEF Lmna−/−nucleus expressing GFP-Lamin A (S22A/S392A mutant) (green) deforming against a rhodamine-fibronectin labeled micropost (red). White arrowhead points to a site of blebbing followed by nuclear envelope rupture (rupture is clear in the corresponding movie 9), and yellow arrows point to regions of near-zero and negative curvatures (Scale bar is 5 μm).

Article Snippet: Fluorescent Labeling: The micropost pattern was coated with rhodamine-conjugated fibronectin (FNR01, Cytoskeleton Inc., Denver, CO) at a concentration of 5 μg mL−1 to promote cell adhesion while fluorescently-labeling the microposts.

Techniques: Staining, Comparison, Imaging, Labeling, Sequencing, Expressing, Mutagenesis

Journal: The American journal of pathology

Article Title: Overexpression of Corin Ameliorates Kidney Fibrosis through Inhibition of Wnt/β-Catenin Signaling in Mice.

doi: 10.1016/j.ajpath.2023.09.008

Figure Lengend Snippet: Figure 1 Wnt/b-catenin signaling is activated and corin expression is decreased along with the increased expression of b-catenin in mouse kidney after unilateral ureteral obstruction (UUO). A and B: Western blot analyses of kidney expression of several proteins, including Wnt1, a-smooth muscle actin (a- SMA), type I collagen, fibronectin, Snail, and plasminogen activator inhibitor-1 (PAI-1), in sham and obstructed kidneys at day 7 after UUO. Representative Western blot analysis (A) and quantitative data relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH; B) are presented. C: Representative mi- crographs using immunohistochemical staining for corin and active b-catenin in kidney tissues at days 4 and 7 after UUO. Boxed areas show magnification of images. Arrows indicate corin- or active b-cateninepositive tubular epithelial cells. Arrowheads in the enlarged boxed areas indicate positive staining. D and E: Western blot analysis of kidney expression of corin and active b-catenin protein at days 4 and 7 after UUO. Representative Western blot analysis (D) and quantitative data (E) are presented. F: Linear regression graph of kidney corin and b-catenin protein expression at days 0, 4, and 7 after UUO. The Spearman correlation coefficient (R2) is shown. G: Representative micrographs using immunohistochemical staining for proprotein convertase subtilisin/kexin-6 (PCSK6) in kidney tissues at days 4 and 7 after UUO. Boxed area shows magnification of image. Arrows indicate PCSK6-positive tubular epithelial cells. Arrowheads in the enlarged boxed area indicate positive staining. H and I: Western blot analysis of PCSK6 protein in kidney at days 4 and 7 after UUO. Representative Western blot analysis (H) and quantitative data (I) are presented. Data are the means SEM (B, E, and I). n Z 6 animals per group (B, E, and I). *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bars: 50 mm (C and G, main images); 200 mm (C and G, enlarged images).

Article Snippet: HKC-8 cells were treated with recombinant human transforming growth factor (TGF)-b1 ajp.amjpathol.org - The American Journal of Pathology Table 1 Used Antibodies in Experiments Antibodies Dilution for IHC/IF Dilution for WB Company Product code Anti-Wnt1 monoclonal antibody e 1:1000 Abcam (Cambridge, UK) ab15251 Anti-corin monoclonal antibody IHC: 1:200 1:1000 Abcam ab274323 Antieb-catenin nonphosphorylated (active) S37/T41 monoclonal antibody IHC: 1:2000 1:1000 Abcam ab246504 GAPDH monoclonal antibody e 1:5000 ProteinTech (Chicago, IL) 60004-1-Ig HA tag polyclonal antibody e 1:1000 ProteinTech 51064-2-AP MYC tag polyclonal antibody e 1:1000 ProteinTech 16286-1-AP DYKDDDDK tag recombinant antibody (binds to FLAG tag epitope) e 1:500 ProteinTech 80010-1-RR Smooth muscle actin polyclonal antibody IHC: 1:200 IF: 1:100 1:1000 ProteinTech 14395-1-AP Fibronectin monoclonal antibody IHC: 1:200 IF: 1:100 1:2000 ProteinTech 66042-1-Ig SNAIL1 polyclonal antibody e 1:500 ProteinTech 13099-1-AP PAI-1 polyclonal antibody e 1:1000 ProteinTech 13801-1-AP PCSK6 polyclonal antibody IHC: 1:100 1:500 Novus Biologicals (Centennial, CO) NBP1-05030 Collagen type I polyclonal antibody e 1:2000 ProteinTech 14695-1-AP CD31 IHC: 1:100 IF: 1:100 e Santa Cruz Biotechnology (Dallas, TX) sc-376764 PDGFRb IHC: 1:100 IF: 1:100 e Affinity Biosciences (China) AF6133 e, not applicable; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin; IF, immunofluorescence; IHC, immunohistochemical; PAI-1, plasminogen activator inhibitor 1; PCSK6, proprotein convertase subtilisin/kexin-6; PDGFRb, platelet-derived growth factor receptor b; WB, Western blot. at 2 ng/mL for different periods of time, except as otherwise indicated in some experiments.

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining

Journal: Journal of Functional Biomaterials

Article Title: Multi-Composite Bioactive Osteogenic Sponges Featuring Mesenchymal Stem Cells, Platelet-Rich Plasma, Nanoporous Silicon Enclosures, and Peptide Amphiphiles for Rapid Bone Regeneration

doi: 10.3390/jfb2020039

Figure Lengend Snippet: Number of CB MSC attached to bacterial grade (non-adherent) plastic coated with various protein-containing solutions after 1 hour. 10% PRP resulted in the greatest cell retention, while both PRP and PPP were superior to gelatin or fibronectin coatings ( A ). Percentage of cells retained by coated or gelled PCL (gray) and collagen (white) scaffolds after 24 h compared to MSC seeded onto tissue culture plastic. PRP proved to be the most potent coating agent, while PRP and PRP-PA gels retained greater than 90% of loaded cells in PRP-coated collagen scaffolds ( B ). PA gels were significantly greater for cell retention than any of the coatings (δ, p = 3.0 × 10 −7 ). PA gels retained significantly less cells in either scaffold material than the PRP only gel (Ω, p = 0.012) or the PRP + PA gel carrier (Π, p < 0.002). Collagen retained significantly more cells than PCL scaffolds when delivered via PRP only gel (†, p = 0.023) or PRP + PA gel (‡, p = 0.010).

Article Snippet: Recombinant human bone morphogenetic protein-2 (BMP2) and recombinant human fibronectin were purchased from R&D Systems (Minneapolis, MN, U.S.).

Techniques:

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: About 10 or 20 μg total protein were separated on 7.5% or 10% of TGX stain-free gels (Bio-Rad, Hercules, California, USA), transferred to PVDF membranes (Roche, Basel, Switzerland), immunostained with monoclonal mouse anti-human fibronectin (1:5000, MAB19182, R&D systems) or rabbit anti-CCM3 (1:150; IG-626, ImmunoGlobe, Himmelstadt, Germany), HRP conjugated mouse IgGκ light chain binding protein (1:30 000, sc-516102, Santa Cruz Biotechnology, Dallas, Texas, USA) or anti-rabbit HRP secondary antibody (1:30 000, sc2357, Santa Cruz Biotechnology), Precision Protein StrepTactin-HRP Conjugate (1:15 000, Bio-Rad) and detected with Clarity Western ECL (Bio-Rad).

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: About 10 or 20 μg total protein were separated on 7.5% or 10% of TGX stain-free gels (Bio-Rad, Hercules, California, USA), transferred to PVDF membranes (Roche, Basel, Switzerland), immunostained with monoclonal mouse anti-human fibronectin (1:5000, MAB19182, R&D systems) or rabbit anti-CCM3 (1:150; IG-626, ImmunoGlobe, Himmelstadt, Germany), HRP conjugated mouse IgGκ light chain binding protein (1:30 000, sc-516102, Santa Cruz Biotechnology, Dallas, Texas, USA) or anti-rabbit HRP secondary antibody (1:30 000, sc2357, Santa Cruz Biotechnology), Precision Protein StrepTactin-HRP Conjugate (1:15 000, Bio-Rad) and detected with Clarity Western ECL (Bio-Rad).

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: About 10 or 20 μg total protein were separated on 7.5% or 10% of TGX stain-free gels (Bio-Rad, Hercules, California, USA), transferred to PVDF membranes (Roche, Basel, Switzerland), immunostained with monoclonal mouse anti-human fibronectin (1:5000, MAB19182, R&D systems) or rabbit anti-CCM3 (1:150; IG-626, ImmunoGlobe, Himmelstadt, Germany), HRP conjugated mouse IgGκ light chain binding protein (1:30 000, sc-516102, Santa Cruz Biotechnology, Dallas, Texas, USA) or anti-rabbit HRP secondary antibody (1:30 000, sc2357, Santa Cruz Biotechnology), Precision Protein StrepTactin-HRP Conjugate (1:15 000, Bio-Rad) and detected with Clarity Western ECL (Bio-Rad).

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: About 10 or 20 μg total protein were separated on 7.5% or 10% of TGX stain-free gels (Bio-Rad, Hercules, California, USA), transferred to PVDF membranes (Roche, Basel, Switzerland), immunostained with monoclonal mouse anti-human fibronectin (1:5000, MAB19182, R&D systems) or rabbit anti-CCM3 (1:150; IG-626, ImmunoGlobe, Himmelstadt, Germany), HRP conjugated mouse IgGκ light chain binding protein (1:30 000, sc-516102, Santa Cruz Biotechnology, Dallas, Texas, USA) or anti-rabbit HRP secondary antibody (1:30 000, sc2357, Santa Cruz Biotechnology), Precision Protein StrepTactin-HRP Conjugate (1:15 000, Bio-Rad) and detected with Clarity Western ECL (Bio-Rad).

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: About 10 or 20 μg total protein were separated on 7.5% or 10% of TGX stain-free gels (Bio-Rad, Hercules, California, USA), transferred to PVDF membranes (Roche, Basel, Switzerland), immunostained with monoclonal mouse anti-human fibronectin (1:5000, MAB19182, R&D systems) or rabbit anti-CCM3 (1:150; IG-626, ImmunoGlobe, Himmelstadt, Germany), HRP conjugated mouse IgGκ light chain binding protein (1:30 000, sc-516102, Santa Cruz Biotechnology, Dallas, Texas, USA) or anti-rabbit HRP secondary antibody (1:30 000, sc2357, Santa Cruz Biotechnology), Precision Protein StrepTactin-HRP Conjugate (1:15 000, Bio-Rad) and detected with Clarity Western ECL (Bio-Rad).

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: About 10 or 20 μg total protein were separated on 7.5% or 10% of TGX stain-free gels (Bio-Rad, Hercules, California, USA), transferred to PVDF membranes (Roche, Basel, Switzerland), immunostained with monoclonal mouse anti-human fibronectin (1:5000, MAB19182, R&D systems) or rabbit anti-CCM3 (1:150; IG-626, ImmunoGlobe, Himmelstadt, Germany), HRP conjugated mouse IgGκ light chain binding protein (1:30 000, sc-516102, Santa Cruz Biotechnology, Dallas, Texas, USA) or anti-rabbit HRP secondary antibody (1:30 000, sc2357, Santa Cruz Biotechnology), Precision Protein StrepTactin-HRP Conjugate (1:15 000, Bio-Rad) and detected with Clarity Western ECL (Bio-Rad).

Techniques: Control

Journal: Cell reports

Article Title: Variation of Human Neural Stem Cells Generating Organizer States In Vitro before Committing to Cortical Excitatory or Inhibitory Neuronal Fates

doi: 10.1016/j.celrep.2020.107599

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: 1 × 10 6 cells were plated over 10 cm culture plates (Falcon, 35–3003), previously coated with poly-L-ornithine (PLO) (Sigma, P3655) and fibronectin (FN) (R&D Systems, 1030FN), and incubated at 37°C, 5% O 2 and 5% CO 2 for 5 days in DMEM/F12 medium (Mediatech 16–405-CV) plus N2 supplement, containing 25 μg/ml bovine insulin (Sigma, I6634), 100 μg/mL apotransferrin (Sigma, T2036), 20 nM progesterone (Sigma, P8783), 100 mM putrescine (Sigma, P5780), 30 nM sodium selenite (Sigma, S5261), penicillin/streptomycin (Life Technology, 15140–122).

Techniques: Virus, Plasmid Preparation, Recombinant, Transfection, Antibody Labeling, In Vitro, Microarray, Gene Expression, Derivative Assay, Software