Journal: The EMBO Journal

Article Title: Mouse models to investigate in situ cell fate decisions induced by p53

doi: 10.1038/s44318-024-00189-z

Figure Lengend Snippet: ( A ) Schematic of the structure of the genetically modified Trp53 allele. The coding sequence for the triple-FLAG tag was inserted after the start codon of the Trp53 gene. ( B ) PCR analysis showing correct insertion of the coding sequences for the triple-FLAG tag into the genetically modified Trp53 allele. A band of 225 bp indicates the presence of a wt (unmodified) allele. A band of 291 bp indicates a FLAG-Trp53 allele. Each lane represents DNA taken from an individual mouse (#32, 38, 40, 44, 45) followed by DNA from a control wt mouse, control DNA from a FLAG-Trp53 KI/+ mouse, and a water-only control. ( C ) Table with observed and expected genotype distribution from inter-crosses of heterozygous FLAG-Trp53 KI/+ mice. ( D ) Table with observed and expected sex distribution from all matings that could give rise to FLAG-Trp53 KI/+ or FLAG-Trp53 KI/KI mice. Frequencies compared by Chi-square test p = 0.0262. ( E ) Kaplan–Meier survival curve showing overall survival of wt, FLAG-Trp53 KI/+ , and FLAG-Trp53 KI/KI mice. Differences in animal survival were compared using the Log-rank (Mantle–Cox) test, * p values ≤0.05. Mice were censored if harvested when healthy for use in experiments. ( F ) Thymocytes from wt, FLAG-Trp53 KI/+ and FLAG-Trp53 KI/KI mice were treated for 48 h in vitro with DMSO (vehicle control), 10 µM nutlin-3a, 1 µg/mL etoposide, 1 µg/mL ionomycin or exposed to one dose of 1.25 Gy γ-radiation and then kept for 48 h in culture. Cell viability was measured at various time points by flow cytometry after staining with fluorochrome-conjugated annexin-V and DAPI. The percentages of live, i.e. annexin-V/DAPI double-negative cells, are plotted. p values were calculated using a two-way ANOVA using Dunnett’s correction for multiple tests. Data were presented as mean ± SD. n = 4 mice of each genotype. ( G ) Mouse dermal fibroblasts (MDFs) were derived from wt, FLAG-Trp53 KI/+ and FLAG-Trp53 KI/KI mice and treated for 72 h with DMSO (vehicle control), 10 µM nutlin-3a, 1 µg/mL etoposide or 250 nM taxol. Cellular senescence was measured as a percentage of fluorescein-di-β- d -galactopyranoside (FDG) positive cells by flow cytometry. p values were calculated using a two-way ANOVA using Dunnett’s correction for multiple tests. Data were presented as mean ± SD. n = 4 mice of each genotype. ( H ) RNA was extracted from thymocytes and MDFs from wt, FLAG-Trp53 KI/+ and FLAG-Trp53 KI/KI mice after they had been treated for 6 h with DMSO (vehicle control) or 10 µM nutlin-3a in vitro. qRT-PCR was performed to determine the mRNA levels of the TRP53 target genes Bax, Mdm2, Mlh1, Pmaip1/Noxa, Cdkn1a/p21, Bbc3/Puma , and the Trp53 mRNA levels. Data were normalised using the ΔΔCT method, using Hmbs as a housekeeping gene. The data were plotted as fold-change of drug-treated compared to DMSO-treated cells. Data were presented as mean ± SD. n = 4 mice of each genotype. p values were calculated using a two-way ANOVA using Dunnett’s correction for multiple tests. All statistical tests were non-significant (i.e. had a p value >0.05). .

Article Snippet: Mlh1 , Mm00503449_m1 , Thermo Fisher Scientific.

Techniques: Genetically Modified, Sequencing, FLAG-tag, Control, In Vitro, Flow Cytometry, Staining, Derivative Assay, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: Mouse models to investigate in situ cell fate decisions induced by p53

doi: 10.1038/s44318-024-00189-z

Figure Lengend Snippet: ( A ) Next-generation sequencing results for the inserted sequences encoding the triple-FLAG tag in the F1 generation of FLAG-Trp53 KI/+ mice. Each line represents the reads from 1 independent F1 mouse. A black dot indicates a matching base in the sequencing reads compared to the reference sequence. ( B ) Representative histology from aged wt, FLAG-Trp53 KI/+ and FLAG-Trp53 KI/KI mice at the time of harvest. No tumour samples showed TRP53 staining by immunohistochemistry, indicating that they did not have mutant TRP53 driving their malignancy. Scale bar = 100 um. ( C ) RNA was extracted from thymocytes and MDFs from wt, FLAG-Trp53 KI/+ and FLAG-Trp53 KI/KI mice that had been treated for 6 h with DMSO (vehicle control) or 1.25 Gy γ-radiation in vitro. qRT-PCR analysis was performed to determine the mRNA levels of the TRP53 target genes Bax, Mdm2, Mlh1, Pmaip1/Noxa, Cdkn1a/p21, Bbc3/Puma and the Trp53 mRNA levels. Data were normalised using the ΔΔCT method, using Hmbs as a housekeeping gene. The data were plotted as fold-change compared to DMSO-treated samples. Data were presented as mean ± SD. n = 4 mice of each genotype and treatment. p values were calculated using a two-way ANOVA using Dunnett’s correction for multiple tests. All statistical tests showed that differences were not significant (had a p value >0.05).

Article Snippet: Mlh1 , Mm00503449_m1 , Thermo Fisher Scientific.

Techniques: Next-Generation Sequencing, FLAG-tag, Sequencing, Staining, Immunohistochemistry, Mutagenesis, Control, In Vitro, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: Mouse models to investigate in situ cell fate decisions induced by p53

doi: 10.1038/s44318-024-00189-z

Figure Lengend Snippet: TaqMan TM probes for qRT-PCR.

Article Snippet: Mlh1 , Mm00503449_m1 , Thermo Fisher Scientific.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Variations in the Gene Expression Profile in Atherosclerotic Patients with Non-Fatal ACS: A Preliminary Study

doi: 10.3390/ijms23095017

Figure Lengend Snippet: Screening analysis of the expression of the remaining genes associated with the function of the immune system. All results are expressed as a mean of 2 − Δ Ct (according to the reference gene encoding 18S rRNA); N/A–not available. The values of each mean gene’s expression in the study group are marked in red, while in the control group in blue.

Article Snippet: 60 , Hs00365052_m1 , FN1 , N/A , N/A.

Techniques: Expressing, Control, Gene Expression

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