Journal: Tissue Engineering and Regenerative Medicine

Article Title: Optimal Stem Cell Transporting Conditions to Maintain Cell Viability and Characteristics

doi: 10.1007/s13770-018-0133-y

Figure Lengend Snippet: Real-time PCR primer sequences

Article Snippet: The antibody information was listed in Table . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Primary antibody Company (Cat. no) Dilution Collagen 1 Abcam (ab34710) 1:200 Collagen 4 Thermo ( PA5-37854) 1:500 Laminin Novus 1:500 Fibrinogen Novus 1:200 Fibronectin Abcam (ab2413) 1:200 Nestin Abcam (ab6320) 1:200 a-SMA Abcam (ab124964) 1:200 pan Cytokeratin Abcam (ab7753) 1:200 Open in a separate window Secondary antibody Company Dilution Alexa Fluor 594 Goat anti-rabbit IgG Thermo ( {"type":"entrez-nucleotide","attrs":{"text":"R37117","term_id":"794573","term_text":"R37117"}} R37117 ) 1:1000 Alexa Fluor 594 Goat anti-mouse IgG Thermo (A-11032) 1:1000 FITC Goat anti-mouse IgG Thermo (A-16079) 1:1000 Open in a separate window Antibody information in immunocytochemistry The H&E and ICC results were confirmed by real-time PCR.

Techniques: Real-time Polymerase Chain Reaction

Journal: Tissue Engineering and Regenerative Medicine

Article Title: Optimal Stem Cell Transporting Conditions to Maintain Cell Viability and Characteristics

doi: 10.1007/s13770-018-0133-y

Figure Lengend Snippet: Antibody information in immunocytochemistry

Article Snippet: The antibody information was listed in Table . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Primary antibody Company (Cat. no) Dilution Collagen 1 Abcam (ab34710) 1:200 Collagen 4 Thermo ( PA5-37854) 1:500 Laminin Novus 1:500 Fibrinogen Novus 1:200 Fibronectin Abcam (ab2413) 1:200 Nestin Abcam (ab6320) 1:200 a-SMA Abcam (ab124964) 1:200 pan Cytokeratin Abcam (ab7753) 1:200 Open in a separate window Secondary antibody Company Dilution Alexa Fluor 594 Goat anti-rabbit IgG Thermo ( {"type":"entrez-nucleotide","attrs":{"text":"R37117","term_id":"794573","term_text":"R37117"}} R37117 ) 1:1000 Alexa Fluor 594 Goat anti-mouse IgG Thermo (A-11032) 1:1000 FITC Goat anti-mouse IgG Thermo (A-16079) 1:1000 Open in a separate window Antibody information in immunocytochemistry The H&E and ICC results were confirmed by real-time PCR.

Techniques:

Journal: Experimental eye research

Article Title: Species variation and spatial differences in mucin expression from corneal epithelial cells

doi: 10.1016/j.exer.2016.09.001

Figure Lengend Snippet: PCR primer sequences and NCBI accession numbers for mucin genes/transcripts. The letter P denotes sequences that are predicted.

Article Snippet: Human MUC16 , Hs01065189_m1 , , {"type":"entrez-nucleotide","attrs":{"text":"NM_024690","term_id":"83367076","term_text":"NM_024690"}} NM_024690.

Techniques:

Journal: Experimental eye research

Article Title: Species variation and spatial differences in mucin expression from corneal epithelial cells

doi: 10.1016/j.exer.2016.09.001

Figure Lengend Snippet: The peripheral cornea expresses MUC1, MUC4 and MUC16 at higher levels than the central cornea. Epithelium was debrided from both the peripheral (~75% corneal surface area) and central (~25% corneal surface area) cornea and quantitative PCR was performed. In all species examined (A-rhesus macaque, B-canine, C-rabbit), mucin expression tended to be higher in the peripheral cornea when compared with the central cornea. Data represents relative mucin expression normalized to the respective housekeeping gene per species (Table 1, n = 6 eyes per species from different individuals). Error bars represent standard error of the mean. Asterisk indicates p ≤ 0.05.

Article Snippet: Human MUC16 , Hs01065189_m1 , , {"type":"entrez-nucleotide","attrs":{"text":"NM_024690","term_id":"83367076","term_text":"NM_024690"}} NM_024690.

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: Experimental eye research

Article Title: Species variation and spatial differences in mucin expression from corneal epithelial cells

doi: 10.1016/j.exer.2016.09.001

Figure Lengend Snippet: Corneal mucin mRNA transcript expression pattern differs between species. Quantitative PCR of mucin mRNA transcript expression in humans (A), rhesus macaques (B), dogs (C) and rabbits (D). Similar patterns of mucin mRNA expression existed in humans, rhesus macaques and rabbits with MUC16 being expressed to the highest levels, followed by MUC1 and MUC4 expression being the lowest detected. The rabbit had a unique pattern of mucin mRNA expression with MUC4 being expressed at the highest level, MUC1 and MUC16 being expressed at lower levels. Data represents relative mucin expression normalized to the respective housekeeping gene per species (Table 1, n = 5 eyes per species from different individuals). Error bars represent standard error of the mean.

Article Snippet: Human MUC16 , Hs01065189_m1 , , {"type":"entrez-nucleotide","attrs":{"text":"NM_024690","term_id":"83367076","term_text":"NM_024690"}} NM_024690.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Experimental eye research

Article Title: Species variation and spatial differences in mucin expression from corneal epithelial cells

doi: 10.1016/j.exer.2016.09.001

Figure Lengend Snippet: MUC16 glycoprotein was detectable in human, rhesus macaque and canine corneal epithelial extracts. Using a human-specific MUC16 antibody (OC125), large high molecular weight protein was detected a human, rhesus macaque and canine corneal epithelial extracts, whereas no protein was detected in rabbit epithelial extracts.

Article Snippet: Human MUC16 , Hs01065189_m1 , , {"type":"entrez-nucleotide","attrs":{"text":"NM_024690","term_id":"83367076","term_text":"NM_024690"}} NM_024690.

Techniques: High Molecular Weight

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Flrt2 mRNA detection by fluorescence in situ hybridization (FISH) in P8 wild-type mouse retina in the optic nerve head (ONH) area, where the central artery and vein enter the retina, and in the superficial vascular plexus (SVP) (upper panel); and in P8 wild-type cerebral cortex (upper cortical layers and pial vasculature) (lower panel). Blood vessels were detected by immunostaining with podocalyxin (Podxl) (general vessel marker). (b) Coronal section of the cerebral cortex from P6 wild-type mouse stained for FLRT2 and NeuN as neuronal marker (neuronal layers I-VI annotated). Blood vessels were visualized with isolectin-B4 (IB4) staining. Arrows show FLRT2 positive blood vessels (right). (c) Flat-mounted P7-P8 retinas from control and Flrt2 iΔEC littermate mice injected with 4-hydroxytamoxifen (Tmx) from P1 to P3 and stained with IB4. (d - f) Quantification of radial vascular length ratio (d), total retinal vessel length (e), and total number of branch points (f) per retina. (g) Representative images of P7-P8 control and Flrt2 iΔEC flat-mounted retinas stained for IB4. Veins (V) and arteries (A) are indicated. (h) Quantification of capillary network density between veins and arteries. (i) Glut1 staining of the vasculature in control and Flrt2 iΔEC brain cortices from P7-P8 mice after Tmx administration from P1 to P3. ( j - l ) Quantification of vessel density (j), vessel length (k) and number of branch points (l). Scale bars: 20 μm (a), 50 μm (b), 500 μm (c), 200 μm (g), 100 μm (i). n = 7-12 (d), 6-7 (e, f), 8 (h), 7 (j-k), 5-6 (l) animals per genotype. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired t-test.

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Fluorescence, In Situ Hybridization, Immunostaining, Marker, Staining, Control, Injection

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Schematic representation of the Flrt2 endothelial specific knockout strategy in Cdh5(PAC)-CreERT2:Flrt2 lox/lox ( Flrt2 iΔEC ) mice. (b) Experimental design of Flrt2 gene deletion by Cre recombination. 4-hydroxytamoxifen (Tmx) was applied from postnatal day 1 (P1) to 3 and tissue collected at P5 and P7 or 8. Cartoon created with Biorender.com. (c, e) Flat-mounted retina (c) and neocortical brains slice (e) from P7 Cdh5-CreERT2:Rosa26tdTomato mice injected from P1 to P3 with Tmx. Cells undergoing Cre-mediated recombination expressed the fluorescent protein tdTomato, which was used to assess specificity and efficiency in blood vessels, co-stained with IB4 (c) or Podxl (e). (d, f) Quantification of the percentage of tdTomato-positive signal per IB4-positive (d) and Podxl-positive signal (f). (g) Immunoblot showing FLRT2 protein levels in primary lung ECs isolated from P8 control and Flrt2 iΔEC mice. FLRT2-specific antibody detects the full-size protein, the cleaved extracellular domain, and glycosylated forms. Pan-cadherin was used as loading control. (h) Flrt2 and Flrt3 mRNA levels from primary mouse brain ECs (pmBECs) isolated from P5 control and Flrt2 iΔEC mice. (i) pmBECs isolated from P7 control and Flrt2 iΔEC mice stained for FLRT2 and cell nuclei (DAPI). (j) FLRT2 fluorescence intensity quantification in control and Flrt2 iΔEC pmBECs. (k) Body weight measured from control and Flrt2 iΔEC male and female mice at P7-8. (l) Example of whole fixed brains from control and Flrt2 iΔEC P7-8 mice. (m, n) Quantifications of brain length (m) and width (n) of control and Flrt2 iΔEC P7-8 mice. Scale bar: 200 μm (c), 100 μm (e), 10 μm (i), 1 mm (l). n = 8 mice (d), 3 mice (f), 4-10 animals per genotype (h), 14-16 pictures per animal from 2 mice per genotype, one litter (j), 6-13 mice per genotype and sex (k), 9-10 mice per genotype (m, n). Horizontal bar shows the median value (d, f). Data are shown as mean ± SEM. **P < 0.01, ***P > 0.001, ns= not significant, unpaired t-test (h ( Flrt3 ), j, k, m, n), Mann-Whitney test (h, Flrt2 ).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Knock-Out, Injection, Staining, Western Blot, Isolation, Control, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Flat mounted retinas stained for Collagen IV (Col IV) and IB4 to visualize blood vessel regression. Col IV+ IB4-empty sleeves are marked with yellow arrows. (b) Quantification of vessel regression as Col IV+ IB4-empty sleeves per imaging field. (c) Representative images of retinas from control and Flrt2 iΔEC P7-P8 littermates stained with IB4. Arteries and veins are highlighted in pink and light blue, respectively. (d) Quantification of the total number of veins and arteries per retina in mutant and control mice at P7-8. (e) Negative Flrt2 (grey) expression in a cortical artery (A) expressing the Gkn3 marker (red) and positive Flrt2 signal in a vein (V) expressing Scl38a5 (blue). Podxl immunostaining is used as a general marker of the vasculature. (f) Representative images of recombinant tip cells in the retinal vascular front of Cdh5-CreERT2:Rosa26tdTomato (control) and Flrt2 iΔEC :Rosa26tdTomato P7-P8 mice. Upon Cre-mediated recombination ECs express tdTomato reporter protein (depicted as red). (g) Quantification of the number of filopodia per tip cell. (h) Retinal vascular fronts from control and Flrt2 iΔEC P7-P8 mice with proliferative cells labelled with EdU, EC nuclei stained with ERG, and blood vessels with IB4. (i) Quantification of % EdU + ERG + cells in total ERG + cells per image. Scale bars: 100 μm (a), 500 μm (c), 25 μm (e, f, h). n = 6-7 animals per genotype (b, d), 19-21 cells per genotype (g), 7-9 animals per genotype (i). Data are shown as mean ± SEM. ***P > 0.001, ns = not significant, two-way ANOVA (d), unpaired t-test (b, g i).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Staining, Imaging, Control, Mutagenesis, Expressing, Marker, Immunostaining, Recombinant

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Representative images of main arteries and veins (identified by their morphology) from control and Flrt2 iΔEC P7-P8 retinas stained with IB4. Red dots indicate branch points from the corresponding mother vessel. (b) Quantification of the number of branch points in arteries (left) and veins (right) per vessel length. (c) Expanded P7 retina stained for FLRT2 and collagen IV (Col IV) showing FLRT2 expression in retinal vessels. Note the expression of FLRT2 at the EC membrane in the vein but its absence in the artery. (d) Representative images of retinal vascular front from control and Flrt2 iΔEC mice stained with IB4. Red dots indicate cellular protrusions identified as angiogenic sprouts. (e) Quantification of number of sprouts per 100 μm of retinal vascular front. (f) Vascular fronts from control and Flrt2 iΔEC P7-P8 retinas showing blood vessels labelled with IB4 and EC nuclei stained for ERG. (g) Quantification of the number of tip cells per stalk cells at the vascular front. (h) Glut1 staining visualizing vessel sprouts in P7-P8 control and Flrt2 iΔEC cerebral cortices. ( i ) Quantification of the number of sprouts per vessel density in the cerebral cortex. Scale bars: 50 μm (a, f), 90 μm (c), 40 μm (d), 100 μm (h). n = 17-20 (b), 18-22 (e), 8-10 (g), 5-6 (i) animals per genotype. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001, ns = not significant, unpaired t-test.

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Control, Staining, Expressing, Membrane

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) P5 flat-mounted retinas from control and Flrt2 iΔEC littermates injected with 4-hydroxytamoxifen (Tmx) from P1 to P3, stained with IB4 to visualize blood vessels. (b , c) Quantification of the vascular radial growth ratio (b), and the capillary density (c) as the area covered by IB4 staining in P5 control and mutant retinas. (d) Glut1 staining of control and Flrt2 iΔEC cortices at P4-P5 after Tmx administration from P1 to P3. (e - g) Quantification of vessel density (e), vessel length (f) and number of branch points (g) in mouse cerebral cortices. (h) Representative images of arteries and veins from control and Flrt2 iΔEC retinas stained with IB4 at P5. Red dots indicate branch points from the mother vessel. (i) Quantification of the number of branch points in main arteries (left) and veins (right) per vessel length. (j) P5 retinal vascular fronts stained with IB4. Red dots indicate cellular protrusions identified as angiogenic sprouts. (k) Quantification of the number of sprouts per 100 μm of vascular front. ( l) Control and Flrt2 iΔEC cerebral cortices at P5 stained for Glut1 to visualize vessel sprouts. ( m ) Quantification of the number of sprouts per vessel density at P5 in Flrt2 iΔEC and control littermates. Scale bars: 500 μm (a), 100 μm (d), 50 μm (h, j, l). n = 10-13 (b, i), 7-9 (c), 10-11 (e), 6 -10 (f, g), 10-12 (k), 6-10 (m) animals per genotype. Data are shown as mean ± SEM. **P < 0.01, ns = not significant, unpaired t-test.

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Control, Injection, Staining, Mutagenesis

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a, b) Flat mounted retinas (a) and cortical brain slices (c) stained for apoptosis marker cleaved caspase-3 (cleaved casp-3) and blood vessels (IB4 or Podxl). (b, d) Quantification of cleaved caspase-3+ ECs in control and Flrt2 iΔEC P5 retinas (b) and P4-P5 brain neocortices (d). (e, g) Flat mounted retinas (e) and cortical brain slices (g) stained for cell cycle arrest marker p21 and blood vessels (IB4 or Podxl). (f, h) Quantification of p21+ ECs in control and Flrt2 iΔEC P5 retinas (f) and P4-P5 brain neocortices (h). Scale bars: 50 μm. n = 4-5 animals per genotype (b, f), 6-7 animals per genotype (d), 5-6 animals per genotype (h). Data are shown as mean ± SEM. *P < 0.05, ns = not significant, unpaired t-test (b, d, h), Mann-Whitney test (f).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Staining, Marker, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Proximity ligation assay (PLA) in HUVEC cultures. White puncta indicate FLRT2 and VE-cadherin being in close proximity (< 40 nm). Cells were stained with phalloidin and DAPI to visualize actin filaments and nuclei, respectively. (b) PLA signal quantification as puncta per cell number in the field (identified by DAPI nuclei). (c) Immunoprecipitation (IP) of VE-cadherin and immunodetection of FLRT2 and VE-cadherin from total mouse brain lysates. TL, total lysate. (d) Representative immunoblot showing FLRT2 protein reduced expression in HUVECs treated with Flrt2 siRNA compared to control treated cells. β-actin was used as loading control. (e) Quantification of FLRT2 protein levels in control and Flrt2 siRNA treated HUVECs. (f) Antibody feeding assay in HUVEC transfected with control and Flrt2 -specific siRNA treated with chloroquine. Cells were immunostained for internalized VE-cadherin, total VE-cadherin and cell nuclei (DAPI). Intensity of internalized VE-cadherin is shown in arbitrary units (AU, upper panels). (g) Quantification of the fluorescence intensity of internalized VE-cadherin per cell. (h) Quantification of VE-cadherin intensity per cell-junction length. (i) Representative immunoblot showing VE-cadherin, its cleaved form, and α-tubulin as loading control in HUVEC transfected with control and Flrt2 -specific siRNAs. (j) Quantification of VE-cadherin/loading control and cleaved VE-cadherin/loading control ratios. ( k) Primary mouse brain ECs (pmBEC) from control and Flrt2 iΔEC littermates stained for Calpain-2 and DAPI. (l) Quantification of the fluorescence intensity of Calpain-2 staining per cell. (m) Neocortical blood vessels stained for Calpain-2 and IB4 from control and Flrt2 iΔEC littermates. (n) Quantification of Calpain-2 fluorescence intensity in the blood vessels. Scale bars: 40 μm (a, upper panels), 15 μm (a, lower panels), 10 μm (f, k), 5 μm (m). n = 20-23 pictures per condition from three different experiments (b), 6 independent experiments (e), 84-115 cells from 3 independent experiments (g), 18-22 pictures per condition from 3 independent experiments (h), 8-11 independent experiments (j), 71-72 cells per condition, from 1 control and 1 Flrt2 iΔE littermate (l), 4-5 animals per genotype (n). Data are shown as mean ±. *P < 0.05, **P < 0.01, SEM. ***P < 0.001, ns = not significant, unpaired t-test (b, e, h, j, n), Mann-Whitney test (g, l).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Proximity Ligation Assay, Staining, Immunoprecipitation, Immunodetection, Western Blot, Expressing, Control, Feeding Assay, Transfection, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Immunoprecipitation of VE-cadherin and immunodetection of FLRT2 and VE-cadherin from HUVEC cultures. (b) PLA in primary mouse brain EC (pmBECs) cultures. White puncta indicate FLRT2 and VE-cadherin being in close proximity (< 40 nm). Cells were stained with Pecam1 and DAPI to visualize EC junctions and nuclei, respectively. Note the major presence of PLA puncta along the Pecam1+ junctions in the cells isolated form control mice, indicating that FLRT2 and VE-cadherin interact at the cell surface. (c) PLA signal quantification as puncta per cell. (d) HUVEC treated with control and Flrt2 -specific siRNAs immunostained for VE-cadherin and DAPI. (e) Quantification of VE-cadherin intensity per cellular junction length. (f) mRNA expression of Flrt2 and Cdh5 in HUVEC transfected with control and Flrt2 -specific siRNAs measured by qPCR. (g) HUVEC cultures transfected with control and Flrt2 -specific siRNAs stained for Calpain-1 and DAPI. (h) Quantification of the fluorescence intensity of Calpain-1 staining per cell. Scale bars: 10 μm (b), 15 μm (d), 20 μm (g). n = 71-72 cells per condition from 2 animals per genotype (c), 27-30 images per condition from 3 independent experiments (e), 7 independent experiments (f), 33-46 cells, 1 representative experiment from 3 independents experiments (h). Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001, Mann-Whitney test (c, h), unpaired t-test (e, f).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Immunoprecipitation, Immunodetection, Staining, Isolation, Control, Expressing, Transfection, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Proximity ligation assay (PLA) in EC cultures. White puncta indicate Numb and FLRT2 and Numb and VE-cadherin being in close proximity (< 40 nm). Cells were stained with phalloidin and DAPI to visualize actin filaments and nuclei, respectively. (b) Expansion microscopy on HUVEC cultures stained for FLRT2 and Numb showing expression of both proteins in cell-cell contacts and along filopodia extensions. (c) mRNA expression of Flrt2 and Numb in ECs transfected with control and Flrt2 -specific siRNAs measured by RT-qPCR. (d) Representative immunoblot showing FLRT2 and Numb protein levels in ECs transfected with control and Flrt2 -specific siRNAs. (e) FLRT2 and Numb protein levels quantification in ECs transfected with control and Flrt2 -specific siRNAs. (f) mRNA expression quantification of Numb from primary mouse brain ECs (pmBECs) isolated from control and Flrt2 iΔEC littermates. (g) Scheme showing the quantification of Golgi orientation in migrating cells after scratch assay and in retinal vascular fronts. Cell was classified as polarized if the angle formed between the scratch or vascular front and Golgi located within a 120°. (h) Scratch assay on HUVECs stained for VE-cadherin, Golgi apparatus (GM130) and cell nuclei (DAPI). Yellow stars in lower panels indicate cells polarized towards the wound area. The 3 first cell rows were considered for quantification. (i) Quantification of the percentage of cells per image polarized towards the wound. (j) Representative images of retinal vascular front from control and Flrt2 iΔEC littermates stained for blood vessels, EC nuclei and Golgi apparatus with IB4, ERG and GM130, respectively. White arrows indicate cellular orientation identified with GM130 position relative to ERG staining. (k) Quantification of the percentage of cells polarized towards the vascular front. The 3 first cell rows were considered for quantification. Scale bars: 20 μm (a, b, h, j). n = 4 independent experiments (c), 7 independent experiments (e), 5-10 animals per genotype (f), 27-28 images from 3 independent experiments (i), 4 animals per genotype (k). Data are shown as mean ± SEM. *P > 0.05, ***P < 0.001, unpaired t-test (c, e, f), Mann-Whitney test (i, k).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Proximity Ligation Assay, Staining, Microscopy, Expressing, Transfection, Control, Quantitative RT-PCR, Western Blot, Isolation, Wound Healing Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a and c) Vascular front of P5 wild-type retinas (a) and remodeling capillaries in cerebral cortex (c) from control and Flrt2 iΔEC littermates stained for VE-cadherin antibody. Blood vessels visualized with IB4. (b and d) Quantification of VE-cadherin activity in retina (b) and cerebral cortex (d) blood vessels. 15 x 15 μm regions of interest (ROIs) were blindly classified to a VE-cadherin activity category: low (smooth pattern), medium (irregular pattern), high (rough pattern). Example images of the three VE-cadherin activity categories in retina (b) and cortex (d) vessels are shown in correlation with the graph. Scale bars: 20 μm (a), 15 μm (c). n = 53-66 images per genotype from three litters b), 78-80 images per genotype from two litters (d). Data are shown as mean ± SEM. *P > 0.05, **P < 0.01, ns = not significant, two-way ANOVA (b and d).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Control, Staining, Activity Assay

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Expansion microscopy 3D-visualization of a large vessel stained for VE-cadherin, FLRT2 and Numb in the cerebral cortex. Higher magnification (lower panels) showing the colocalization of the three proteins at EC junction. (b and c) Expansion microscopy 3D-visualization of a large vessel stained for VE-cadherin and FLRT2 (b) and VE-cadherin and Numb (c) in the retina. (d) Expansion microscopy 3D-visualization of cerebral cortex capillaries stained for VE-cadherin and Glut1 in control (left) and Flrt2 iΔEC (right) mice. Higher magnifications (1-5) showing x-y planes (first row) and y-z planes (second row) exposing VE-cadherin pattern in a control capillary (1-2), a control tip cells (3), and a Flrt2 iΔEC capillary (4-5). Scale bars: 4 μm (a upper panel), 1 μm (a lower pannels), 15 μm (b, c), 30 μm (d).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Microscopy, Staining, Control

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Representative immunoblot of the cytosolic and nuclear fractions obtained from bEnd.3 cells treated with control and Flrt2 siRNA showing protein levels of β-catenin and FoxO1, and α-tubulin and Lamin A/C as cytosolic and nuclear controls, respectively. (b and c) β-catenin (b) and FoxO1 (c) protein levels relative to the loading controls. (d) Representative immunoblot showing FLRT2 and Claudin-5, and β-actin as loading control in bEnd.3 cells treated with control and Flrt2 siRNA. (e) Quantification of FLRT2 and Claudin-5 protein levels relative to the loading control. ( f) Representative immunoblot showing Claudin 5, and α-tubulin as loading control in total brain lysates from control and Flrt2 iΔEC littermates. (g) Quantification of Claudin-5 protein levels relative to the loading control. (h) Neocortical blood vessels stained for Claudin-5 and VE-cadherin. Note the colocalization of both protein in the control vessel, compared to the split Claudin-5 signal in Flrt2 iΔEC blood vessel. (i) Quantification of the ratio of split Claudin-5 junction length to the total junctional length. (j) Representative fluorescent whole-brain images of control and Flrt2 iΔEC littermates injected with AlexaFluor TM 555-conjugated cadaverine at P7-P8. (k) Quantification of cadaverine whole-brain intensity in P7-P 8control and Flrt2 iΔEC littermates. (l) Representative images of Collagen IV (Col IV) immunostaining and cadaverine signal in control and Flrt2 iΔEC cortices showing cadaverine leakage in Flrt2 iΔEC mice. (m) TEM representative images of brain capillaries showing an EC junction in control and Flrt2 iΔEC mice. Note the abnormal junctions often associated with the presence of vacuoles (red arrows) in the Flrt2 iΔEC vessels. (n) Incidence (in percentage) of abnormal junctions (left) and junctions with vacuoles (right) in TEM images of control and Flrt2 iΔEC brain capillaries. Scale bars: 10 μm (h), 1 mm (j), 100 μm (l), 200 nm (m). n = 3 independent experiments (b, c), 5-6 independent experiments (e), 8 animals per genotype (g), 5-7 animals per genotype (i), 16-18 animals per genotype (k), 3 animals per genotype (n). Data are shown as mean ± SEM. *p < 0.05, **P < 0.01, ***P > 0.001, unpaired t-test (b, c, e, i, k), Mann-Whitney test (g), 2-way ANOVA (n).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Western Blot, Control, Staining, Injection, Immunostaining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) mRNA fold change levels of Flrt2 and tight junction protein claudin5 ( Cldn5 ) in bEnd.3 cells treated with control and Flrt2 siRNA. (b) mRNA expression quantification of Cldn5 in control and Flrt2 iΔEC total brains at P7-P8. (c, e) Representative immunoblots from total brain lysates of control and Flrt2 iΔEC mice showing tight junctions proteins ZO-1 (c) and JAM-A (e) levels. (d, f) Quantification of protein ZO-1 (d) and JAM-A (f) levels in total brain lysates from control and Flrt2 iΔEC mice. (g, h) Neocortical blood vessels stained for ZO-1 (g) and JAM-A (h) showing no differences in tight junction proteins distribution between control and Flrt2 mutant mice. (i) Representative fluorescent whole-brain images of control and Flrt2 iΔEC littermates injected with AlexaFluor555nm-conjugated ovalbumin (45 kDa) at P7-8. (j) Quantification of ovalbumin whole-brain intensity in control and Flrt2 iΔEC mice at P7-8. (k) Representative fluorescent whole-brain images of control and Flrt2 iΔEC littermates injected with AlexaFluor555nm-conjugated cadaverine at P5. (l) Quantification of cadaverine whole-brain intensity in control and Flrt2 iΔEC mice at P5. Scale bars: 10 μm (g, h), 1 mm (i, k). n = 5 independent experiments (a), 6 animals per genotype (b), 5 animals per genotype (d), 4 animals per genotype (f, j), 7-8 animals per genotype (l). Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ns = not significant, unpaired t-test.

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Control, Expressing, Western Blot, Staining, Mutagenesis, Injection

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a, c, e,) Cerebral cortices of control and vascular Flrt2 mutant mice stained with the pericyte marker PDGFRβ (a), astrocytic end-feet marker Aquaporin-4 (Aqp4) (c) and extracellular matrix marker Collagen IV (Col IV) (e) and blood vessel markers Glut1 (a) or Podxl (c, e). (b, d, f) Quantification of PDGFRβ (b), Aqp4 (d) or Col IV (f) coverage of cortical vasculature. Scale bars: 50 μm (a), 10 μm (c, e). n = 7 animals per genotype (b), 6 animals per genotype (d, f). Data are shown as mean ± SEM. ns = not significant, unpaired t-test.

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Control, Mutagenesis, Staining, Marker

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: Representative image of an endothelial-specific Flrt2 mutant cerebral cortex section showing a leakage area where the punch was performed and further processed for transmission electron microscopy (TEM) analysis. After assessing the quality of the tissue, imaging was processed focusing in capillary EC tight junctions. Cartoon created with Biorender.com. Scale bars: 1mm, 250 μm, 2500 nm, 250 nm

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Mutagenesis, Transmission Assay, Electron Microscopy, Imaging

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Schematic timeline representation of the vascular events derived from FLRT2 deletion in ECs. (b) Schematic representation of the molecular mechanisms regulated by FLRT2 in postnatal CNS vasculature. In control conditions, FLRT2 forms a complex with VE-cadherin and the endocytic adaptor Numb which allows the dynamic turnover of VE-cadherin necessary for angiogenic sprouting. By contrast, in FLRT2-deficient cells VE-cadherin cytoplasmic tail is cleaved by Calpains and subsequently fated to lysosomal degradation, while a compensatory biosynthesis accumulates VE-cadherin at the cell membrane impairing vascular sprouts. In addition, FLRT2 deletion facilitates the nuclear translocation of β-catenin, repressing Claudin-5 expression and triggering increased size-selective BBB permeability.

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Derivative Assay, Control, Membrane, Translocation Assay, Expressing, Permeability

Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: Identification of candidates involved in the trafficking of MMP2. (A) Scheme of the MMP2 RUSH construct. SS-Flag-MMP2-HA-SBP-eGFP was used as a reporter. Fluorescence images show HeLa cells expressing MMP2-SBP-eGFP counterstained against TGN46 (red). Without biotin, MMP2 is retained in the ER (0 min). It reaches the Golgi 15 min after biotin addition and is sorted into vesicles (arrowheads) at 30 and 45 min, respectively. Scale bars, 5 µm. (B) MS strategy to identify MMP2 interacting partners in the Golgi. HeLa cells expressing MMP2-SBP-eGFP or SS-SBP-eGFP were incubated for 20 min with biotin to enrich reporter proteins at the Golgi. After GFP IP, samples were analyzed using MS ( n = 3). (C) Volcano plot highlights significantly enriched MMP2 interactors in pink. 42 sorting-related candidates were found, among them TIMP2, a known inhibitor of MMP2, and NUCB1. Two-sample t test, false discovery rate = 0.3, minimum fold change = 0.5. (D) Fluorescence images of HeLa cells labeled with endogenous NUCB1 (green) and GM130 or TGN46 (red). Scale bars, 5 µm; zoom, 2 µm. (E) HEK 293T cells expressing SS-MMP2-SBP-eGFP or SS-SBP-eGFP were processed for GFP IP and WB analysis. (F) Semiquantitative analysis of the normalized NUCB1 to GFP signal from two independent experiments. Significance: one-sample t test. (G) His-tag coIP of recombinant rNUCB1-His. Endogenous MMP2 from HeLa Golgi membranes coimmunoprecipitated with rNUCB1-His but not rGFP-His. (H) Semiquantitative analysis of the MMP2 signal from three independent experiments. Bars, mean ± SD. Paired t test: *, P < 0.05; ***, P < 0.001.

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: Construct, Fluorescence, Expressing, Incubation, Labeling, Recombinant

Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: MMP2-eGFP secretion and evaluation of CRISPR NUCB1-KO clones . (A) HeLa cells stably expressing SS-MMP2-eGFP were seeded on glass slides and incubated at 37°C for 3 d to evaluate MMP2-eGFP secretion. After fixation, cells were incubated with GFP antibody and Alexa Fluor 594. Confocal fluorescence images show colocalization of MMP2-eGFP and GFP antibody of nonpermeabilized cells, evidencing secretion of MMP2-eGFP to the extracellular space. Scale bars, 10 µm; zoom bar, 2 µm. (B and C) NUCB1-KO cells were generated using the CRISPR-Cas9 system with three different gRNAs and selection of single colonies. After puromycin selection, three NUCB1-KO clones were identified by WB (B) and later confirmed by immunofluorescence (C). *, unspecific band; KO, HeLa NUCB1-KO cells; CN, HeLa control. Semiquantitative analysis shows normalized NUCB1-to-β-actin signal.

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: CRISPR, Clone Assay, Stable Transfection, Expressing, Incubation, Fluorescence, Generated, Selection, Immunofluorescence

Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: MMP2 is partially sorted in LyzC-positive secretory vesicles. HeLa cells expressing MMP2-eGFP were immunolabeled with a-Rab5, a-Rab7, or Rab11 antibodies (red). MMP2-eGFP–expressing cells were cotransfected with mCherry (mCh)-lysosomes or LyzC-mCherry to label lysosomes or LyzC-positive secretory vesicles, respectively. Rab6-GFP or Rab8-GFP constructs were cotransfected with MMP2-tagRFP. Images were acquired by confocal microscopy. White arrowheads point to distinct vesicles; magenta arrowheads point to colocalizing vesicles. Bars, 10 µm; zoom, 2 µm.

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: Expressing, Immunolabeling, Construct, Confocal Microscopy

Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: Protein purification and evaluation of the direct interaction between MMP2 and NUCB1. (A) Coomassie-stained SDS-PAGE for the evaluation of His-tag purified recombinant NUCB1-His (rNUCB1-His). (B) Anti-NUCB1 WB analysis of the elution fraction shown in line 4 from A. (C) WB analysis of purified His-SUMO-MMP2 using MMP2 antibody. (D) Recombinant His-SUMO-MMP2 (rHS-MMP2) was bioconjugated with Cy3 via maleimide labeling and subsequently analyzed by AUC. The lowest panel shows peak of sedimentation of rHS-MMP2 at 4.705 S. (E) AUC profile of rHis-SUMO-MMP2-Cy3 and NUCB1-His. The lowest panel shows a peak at 3.189 S, indicating a change in the sedimentation velocity associated to a direct interaction of NUCB1 and MMP2. (F) Coomassie-stained SDS-PAGE of purified His-tagged NUCB1 Ca 2+ binding mutant (rNUCB1mEFh1+2). (G) WB analysis of the elution fraction shown in line 4 of F using NUCB1 antibody. (H) CD measurement of rNUCB1-His and rNUCB1mEFh1+2-His under presence or absence of 1 mM Ca 2+ . rNUCB1-mEF1+2 molar ellipticity is lower compared with rNUCB1-His. Evaluation of the CD spectra using CONTIN showed an increase in rNUCB1-His α-helicity upon Ca 2+ addition (from 0.385 to 0.413) that was not observed in rNUCB1-mEFh1+2 (from 0.256 to 0.147). Instead, an increase in β-sheet content (from 0.151 to 0.322) was observed. These findings are in accordance with the results described by .

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: Protein Purification, Staining, SDS Page, Purification, Recombinant, Labeling, Sedimentation, Binding Assay, Mutagenesis

Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: NUCB1-KO impairs the trafficking of MMP2. (A) Fluorescent images of HeLa or NUCB1-KO cells expressing SS-MMP2-SBP-eGFP with or without NUCB1-WT, counterstained against NUCB1 (red) and captured after 0, 15, 30, and 45 min of biotin incubation. Arrowheads, cytoplasmic vesicles. Scale bars, 5 µm. (B) Cytoplasmic vesicle counts as described in A are plotted as number of vesicles per cell ( n ≥ 90 cells, median ± IQR of two independent experiments; ***, P < 0.001; n.s., not significant). (C) Confocal microscopy images of HeLa or NUCB1-KO cells expressing LyzC-SBP-eGFP and counterstained against NUCB1 (red) after 0, 20, 40, and 60 min of biotin incubation. Arrowheads, cytoplasmic vesicles. Scale bars, 5 µm. (D) Cytoplasmic vesicle counts from C of two independent experiments ( n ≥ 42 cells, median ± IQR). (E) Secretion assay of HeLa or NUCB1-KO cells expressing SS-MMP2-SBP-eGFP or LyzC-SBP-EGFP and incubated with biotin for 45 or 60 min, respectively. WCL, whole-cell lysates. [SNs], 10×-concentrated supernatants. (F) Semiquantitative analysis from three independent experiments, one-sample t test. Bars, mean ± SD. (G) GFP-coIP of HeLa or NUCB1-KO cells expressing LyzC-eGFP, with or without NUCB1-WT. GFP-HA, negative control; CN, HeLa control; KO, NUCB1-KO. (H) Semiquantitative analysis of NUCB1 to GFP signal from three independent experiments. Bars, mean ± SD; paired t test.

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: Expressing, Incubation, Confocal Microscopy, Negative Control

Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: MMP2 IG trafficking is exclusively dependent on Golgi-localized NUCB1, which also impairs IG trafficking of MT1-MMP. (A) HeLa or NUCB1-KO cells expressing SS-SBP-MMP2-eGFP alone or with a cytosolic variant of NUCB1 lacking its SS (NUCB1-cyto) were fixed after 0, 15, 30, and 45 min of biotin incubation. Maximal Z-projection analysis of confocal microscopy images shows no differences in MMP2 trafficking of NUCB1-cyto transfected cells compared with NUCB1-KO cells (arrowheads). Scale bars, 10 µm. (B) Quantification of cytoplasmic MMP2 vesicles from cells in A. n > 18 cells; mean ± SD; two independent experiments. Significant differences with P < 0.05 were analyzed via nonparametric Kruskal–Wallis test with Dunn’s multiple comparison, **, P < 0.01. (C) mCherry-tagged MT1-MMP RUSH construct (SS-MT1-MMP-SBP-mCh). Cyto, cytosolic domain. (D) Confocal fluorescence images of HeLa or NUCB1-KO cells transfected with or without NUCB1-WT and fixed after 30, 60, and 90 min of biotin incubation. Arrowheads, cytoplasmic vesicles. Scale bars, 5 µm. (E) Quantification of cytoplasmic vesicles observed in A. n = 24 cells; two independent experiments; median ± IQR; ***, P < 0.001; n.s., non-significant. (F) Cell surface biotinylation assay coupled with streptavidin pull-down. HeLa or NUCB1-KO cells were untreated (time 0) or incubated with sulfo-NHS-Biotin for 90 min to label cell surface proteins, and then pulled down with Neutravidin beads. WB analysis shows a reduction in the amount of endogenous active MT1-MMP at the surface of NUCB1-KO cells compared with HeLa control. β-1 integrin was used as loading control. (G) Semiquantitative analysis of surface labeled active MT1-MMP from F represented as % of normalized MT1-MMP intensity to β-1 integrin in comparison to control (100%). n = 3 independent experiments; one-sample t test, **, P < 0.01. Bars, mean ± SD.

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: Expressing, Variant Assay, Incubation, Confocal Microscopy, Transfection, Construct, Fluorescence, Cell Surface Biotinylation Assay, Labeling

Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: NUCB1 does not affect MMP2 activation nor trafficking of other cargoes such as HRP and Cathepsin D. (A) Zymography assay of HeLa cells expressing SS-MMP2-SBP-eGFP. Untsf HeLa, Hela without transfection; [SN], 10×-concentrated supernatants; CN, HeLa control; KO, NUCB1-KO. (B) Semiquantitative analysis of experiment shown in A. n = 3 independent experiments; one-sample t test; n.s., nonsignificant. (C) Whole-cell lysates of HeLa and NUCB1-KO cells stably expressing SS-HRP-FLAG were analyzed by anti-FLAG, anti-NUCB1, and anti-β-actin WB. SS-HRP-FLAG is expressed in HeLa and NUCB1-KO cells to similar levels. (D) Cell culture supernatants of cells described in C were analyzed for HRP activity by chemiluminescence after 4-h secretion. BFA served as a positive control for perturbed secretion and was added for 1 h before HRP secretion analysis. No significant differences were observed between NUCB1-KO and HeLa control cells. *, P < 0.05. (E) HeLa or NUCB1-KO cells expressing SS-SBP-eGFP-Cathepsin D were fixed 20, 40, and 60 min after biotin addition. Representative maximum Z-projection images show Cathepsin D trafficking from Golgi to cytoplasmic vesicles (arrowheads). Scale bars, 10 µm. (F) Quantification of cytoplasmic Cathepsin D vesicles from cells shown in E. n > 30 HeLa and NUCB1-KO cells per time point; two independent experiments; mean ± SD. Statistical analysis was performed using a nonparametric Kruskal–Wallis test with Dunn’s multiple comparison test. No significant differences with P < 0.05 were detected.

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: Activation Assay, Zymography, Expressing, Transfection, Stable Transfection, Cell Culture, Activity Assay, Positive Control

Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: MMP2 trafficking delay occurs at the cis-Golgi. (A) Fluorescence images of HeLa or NUCB1-KO cells transiently expressing SS-MMP2-SBP-eGFP, fixed at 2.5, 5, and 7.5 min after biotin addition, and counterstained against ERGIC53 (red). Scale bars, 5 µm. (B) Average PC per time point. (C) Colocalization of HeLa or NUCB1-KO cells expressing SS-MMP2-SBP-eGFP with GM130 (red) after 10, 15, 20, and 25 min of biotin incubation. Scale bars, 5 µm. (D) Average PC illustrates decreased colocalization at 10, 15, and 20 min after biotin addition. (E) Colocalization of SS-MMP2-SBP-eGFP with TGN46 (red) expressed in HeLa or NUCB1-KO cells at 20, 25, 30, 35, and 40 min after biotin addition. Scale bars, 5 µm. (F) Average PC shows that MMP2 is equally colocalizing with TGN46 in HeLa and NUCB1-KO cells upon arrival at the TGN. Error bars represent SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant.

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: Fluorescence, Expressing, Incubation

Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: MMP2 trafficking is exclusively delayed at the Golgi in living cells. (A) HeLa or NUCB1-KO cells expressing SS-SBP-MMP2-eGFP were analyzed by live-cell wide-field microscopy. Representative images of MMP2 trafficking after 0, 30, 35, and 40 min of biotin incubation. Images were acquired in 1-min frames for each analyzed cell. Arrowheads, cytoplasmic MMP2 vesicles. Scale bars, 10 µm. (B) Quantification of cytoplasmic MMP2 vesicles per frame from cells shown in A. n.s., nonsignificant. *, P < 0.05; **, P < 0.01. (C) Schematic representation of ER–Golgi cargo transport analysis, measured as normalized Golgi area over time in cells shown in A. (D) Normalized Golgi area for each time point (median ± IQR). A reduced Golgi compaction was observed in the time range 15–23 min in NUCB1-KO cells compared with HeLa control. *, P < 0.05. (E and F) HeLa or NUCB1-KO cells ( n = 11) expressing SS-SBP-MMP2-eGFP fixed without biotin addition and immunostained for ER exit site marker Sec16 (red). Scale bar, 10 µm; zoom, 2 µm. Retained MMP2 in the ER partially colocalized with Sec16 in both control and NUCB1-KO cells to the same extent (F). Magenta arrowheads, MMP2 structures that colocalized with ER exit sites; white arrowheads, ER exit sites. t test: P < 0.05.

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: Expressing, Microscopy, Incubation, Marker

Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: NUCB1 EFhs are essential for Golgi trafficking of MMP2. (A) Protein alignment of human NUCB1 (Q02818, aa 241–400), CaM (P0DP23), Calumenin (O43852), and Cab45 (Q9BRK5). Pink boxes, NUCB1 EFhs. (B) NUCB1 adapted PDB protein model (accession no. 1SNL ); NUCB1 EFhs, cyan; NUCB1-WT, EFhs with first and last amino acid of the domain in dark blue; NUCB1-mEFh1+2, amino acid substitutions E264Q and E316Q in pink. (C) CoIP of MMP2-eGFP transiently expressed in NUCB1-KO cells transfected with NUCB1-WT or NUCB1-mEFh1+2. n = 4 biological replicates. (D) Semiquantitative analysis of NUCB1 signal per sample normalized to the one of NUCB1-KO cells reexpressing NUCB1-WT. Bars, mean ± SD; one-sample t test. (E) Confocal fluorescence images of HeLa or NUCB1-KO cells expressing SS-MMP2-SBP-eGFP and cotransfected with or without NUCB1-WT or NUCB1-mEFh1+2. After 15, 30, and 45 min of biotin incubation, cells were fixed and costained with NUCB1 antibody (red). Scale bars, 5 µm. Arrowheads, cytoplasmic vesicles. (F) Quantification of cytoplasmic vesicles as in E from two independent experiments (median ± IQR), n ≥ 19 cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: Transfection, Fluorescence, Expressing, Incubation

Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: NUCB1 depletion impairs ECM invasion and degradation in MDA-MB-231 cells. (A) Expression levels of NUCB1 after siRNA-mediated silencing ( n = 3 independent experiments: R1, R2, and R3). *, unspecific band. (B) Semiquantitative analysis of normalized NUCB1 signal from A in silenced cells compared with control. Bars, mean ± SD. (C) Quantitative PCR analysis of relative MMP2 expression in siRNA-treated MDA-MB-231 cells ( n = 3 independent experiments, one-sample t test). (D) Secretion assay of endogenous MMP2 in MDA-MB-231 cells. [SN], 20×-concentrated supernatant; WCL, whole cell lysates. (E) Semiquantitative analysis of three independent experiments. Bars, mean ± SD. Significance, one-sample t test. (F and G) Representative pictures of Matrigel-coated Transwell invasion (F) or gelatin degradation (G) experiments. Scale bars, 150 µm. (H and I) Quantification of the number of migrating cells (H) and degraded gelatin area (I). Both invasion and degradation were reduced in siNUCB1 cells. Data: median ± IQR; n = 3 independent experiments. Paired t test: *, P < 0.05; **, P < 0.01; n.s., not significant.

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Cureus

Article Title: Re-irradiation of Recurrent Pineal Germ Cell Tumors with Radiosurgery: Report of Two Cases and Review of Literature

doi: 10.7759/cureus.585

Figure Lengend Snippet: Six Degrees of Freedom Couch Shifts Based on Daily Image Guidance with CBCT and OSMS *Representative real-time delta shifts across non-coplanar treatment couch angles OSMS = optical surface monitoring system; CBCT = cone beam CT; FSRT = fractionated stereotactic radiotherapy

Article Snippet: Intrafraction real-time optical surface monitoring system (OSMS) was performed with surface imaging using AlignRT (VisionRT, London, UK).

Techniques:

Journal: Molecules

Article Title: Anti-Fibrosis Effects of Magnesium Lithospermate B in Experimental Pulmonary Fibrosis: By Inhibiting TGF-βRI/Smad Signaling

doi: 10.3390/molecules26061715

Figure Lengend Snippet: Figure 3. MLB inhibited collagen production in A549 cells. Quiescent cells were treated with MLB alone (50 µM), TGF-β, or both TGF-β and MLB (30, 50 µM) or pirfenidone (PFD) (30 µM) for 24 h. (a) Cell toxicity of MLB was measured by Cell Counting Kit-8 (CCK-8). (b) Relative mRNA expression of Col 1A1, Col 3A1, E-cadherin (E-Cad) and vimentin (Vim) was determined by real-time quantitative PCR. (c,d) Relative protein levels of Col 1A1, fibronectin and E-Cad were determined by Western blot and analyzed by densitometry. (e) Immunofluorescence staining for Col 1A1 and fibronectin. Bars = 130 µm, magnification: 4×. (f) The release of endogenous TGF-β was measured by using an ELISA kit. Four replicates were assessed and analyzed in each group. Data are presented as the means ± SEM of the group and compared by one-way ANOVA. The experiments were replicated four times; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Control; n.s p > 0.05, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. TGF-β.

Article Snippet: Anti-Col 1A1, anti-actin alpha 2 (ACTA2, α-SMA), anti-E-Cadherin (ECad), anti-Smad3, and anti-fibronectin antibodies were obtained from Proteintech (Wuhan, China).

Techniques: Cell Counting, CCK-8 Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: Molecules

Article Title: Anti-Fibrosis Effects of Magnesium Lithospermate B in Experimental Pulmonary Fibrosis: By Inhibiting TGF-βRI/Smad Signaling

doi: 10.3390/molecules26061715

Figure Lengend Snippet: Figure 4. MLB inhibited TGF-β-induced myofibroblast transdifferentiation in human lung fibroblast cell line (MRC-5) cells. Quiescent cells were treated with MLB (50 µM) alone, TGF-β, or both TGF-β and MLB (30, 50 µM) or PFD (30 µM) for 48 h. (a) Cell toxicity of MLB was measured by CCK 8. (b) Relative mRNA expression of Vim, α-SMA, fibronectin, and TGF-β was determined by real-time quantitative PCR. (c,d) Relative protein levels of α-SMA and fibronectin were determined by Western blot and analyzed by densitometry. (e) Immunofluorescence staining for α-SMA and fibronectin. Bars = 130 µm, magnification: 4×. (f) The release of endogenous TGF-β was measured using an ELISA kit. Four replicates were assessed and analyzed in each group. Data are presented as the means ± SEM of the group and compared by one-way ANOVA. The experiments were replicated four times; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Control; n.s p > 0.05, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. TGF-β.

Article Snippet: Anti-Col 1A1, anti-actin alpha 2 (ACTA2, α-SMA), anti-E-Cadherin (ECad), anti-Smad3, and anti-fibronectin antibodies were obtained from Proteintech (Wuhan, China).

Techniques: CCK-8 Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Control