cd31 pecam 1 monoclonal antibody 390  (Thermo Fisher)


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    Structured Review

    Thermo Fisher cd31 pecam 1 monoclonal antibody 390
    Effects of L6-F4-2 on retinal endothelial WNT/β-catenin signaling and vascular subtypes a-c WT (control) and Ndp KO mice were treated with PBS or L6-F4-2 by intravitreal injection (0.19 μg) at P0 and 5-7 pooled retinas for each group were harvested at P8. Retinal vessel ECs were purified by <t>anti-CD31</t> FACS and ultra-low input bulk RNA-seq analysis was performed. a Left: WT (PBS-treated) vs Ndp KO (PBS-treated). Right: Ndp KO (PBS-treated) vs Ndp KO (treated with L6-F4-2). Volcano plot depicting genes with a p-value
    Cd31 Pecam 1 Monoclonal Antibody 390, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 95 stars, based on 18 article reviews
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    cd31 pecam 1 monoclonal antibody 390 - by Bioz Stars, 2022-11
    95/100 stars

    Images

    1) Product Images from "Therapeutic modulation of the blood-brain barrier and ischemic stroke by a bioengineered FZD4-selective WNT surrogate"

    Article Title: Therapeutic modulation of the blood-brain barrier and ischemic stroke by a bioengineered FZD4-selective WNT surrogate

    Journal: bioRxiv

    doi: 10.1101/2022.10.13.510564

    Effects of L6-F4-2 on retinal endothelial WNT/β-catenin signaling and vascular subtypes a-c WT (control) and Ndp KO mice were treated with PBS or L6-F4-2 by intravitreal injection (0.19 μg) at P0 and 5-7 pooled retinas for each group were harvested at P8. Retinal vessel ECs were purified by anti-CD31 FACS and ultra-low input bulk RNA-seq analysis was performed. a Left: WT (PBS-treated) vs Ndp KO (PBS-treated). Right: Ndp KO (PBS-treated) vs Ndp KO (treated with L6-F4-2). Volcano plot depicting genes with a p-value
    Figure Legend Snippet: Effects of L6-F4-2 on retinal endothelial WNT/β-catenin signaling and vascular subtypes a-c WT (control) and Ndp KO mice were treated with PBS or L6-F4-2 by intravitreal injection (0.19 μg) at P0 and 5-7 pooled retinas for each group were harvested at P8. Retinal vessel ECs were purified by anti-CD31 FACS and ultra-low input bulk RNA-seq analysis was performed. a Left: WT (PBS-treated) vs Ndp KO (PBS-treated). Right: Ndp KO (PBS-treated) vs Ndp KO (treated with L6-F4-2). Volcano plot depicting genes with a p-value

    Techniques Used: Mouse Assay, Injection, Purification, FACS, RNA Sequencing Assay

    L6-F4-2 treatment promotes endothelial blood-brain and blood-retina barrier function a WT (control) and Ndp KO mice were treated with PBS or L6-F4-2 by i.p. injection (2.5 mg/kg) at P0, P7 and P14 and tissues were harvested at P21. Sulfo-NHS-biotin staining reveals BBB/BRB defects in cerebellum and retina, but not cerebral cortex in Ndp KO mice, while these defects can be rescued by L6-F4-2 treatment. Biotin labeling of liver and kidney parenchyma serve as a positive control. Scale bar, 500 μm. b Quantification of ( a ) for BBB/BRB leakage in Ndp KO cerebellum and retina with rescue by L6-F4-2 treatment. c Mice were treated at P21, P24 and P27 (2.5 mg/kg, i.p.) and tissues were harvested at P30. Sulfo-NHS-biotin staining reveals BBB/BRB defects in cerebellum and retina, but not cerebral cortex in Ndp KO mice with rescue by L6-F4-2. Scale bar, 200 μm. d Quantification of ( c ) with BBB/BRB leakage in Ndp KO cerebellum and retina and reversal by L6-F4-2. e L6-F4-2 rescues barrier function defects in P30 Ndp KO mice with increased expression of the tight junction component CLDN5, P30 cerebellum IF, overlay of CD31 IF and DAPI. f Quantification of ( e ). g L6-F4-2 decreases expression of the EC fenestration component PLVAP in P30 Ndp KO mice, with overlay of CD31 IF and DAPI. h Quantitation of ( g ). For e-h, scale bars represent 100 μm. To quantify CLDN5 and PLVAP in ( f , h ), the density was measured with ImageJ and normalized to vessel area (CD31). Error bars represent mean ± s.e.m., n=5, *p
    Figure Legend Snippet: L6-F4-2 treatment promotes endothelial blood-brain and blood-retina barrier function a WT (control) and Ndp KO mice were treated with PBS or L6-F4-2 by i.p. injection (2.5 mg/kg) at P0, P7 and P14 and tissues were harvested at P21. Sulfo-NHS-biotin staining reveals BBB/BRB defects in cerebellum and retina, but not cerebral cortex in Ndp KO mice, while these defects can be rescued by L6-F4-2 treatment. Biotin labeling of liver and kidney parenchyma serve as a positive control. Scale bar, 500 μm. b Quantification of ( a ) for BBB/BRB leakage in Ndp KO cerebellum and retina with rescue by L6-F4-2 treatment. c Mice were treated at P21, P24 and P27 (2.5 mg/kg, i.p.) and tissues were harvested at P30. Sulfo-NHS-biotin staining reveals BBB/BRB defects in cerebellum and retina, but not cerebral cortex in Ndp KO mice with rescue by L6-F4-2. Scale bar, 200 μm. d Quantification of ( c ) with BBB/BRB leakage in Ndp KO cerebellum and retina and reversal by L6-F4-2. e L6-F4-2 rescues barrier function defects in P30 Ndp KO mice with increased expression of the tight junction component CLDN5, P30 cerebellum IF, overlay of CD31 IF and DAPI. f Quantification of ( e ). g L6-F4-2 decreases expression of the EC fenestration component PLVAP in P30 Ndp KO mice, with overlay of CD31 IF and DAPI. h Quantitation of ( g ). For e-h, scale bars represent 100 μm. To quantify CLDN5 and PLVAP in ( f , h ), the density was measured with ImageJ and normalized to vessel area (CD31). Error bars represent mean ± s.e.m., n=5, *p

    Techniques Used: Mouse Assay, Injection, Staining, Labeling, Positive Control, Expressing, Quantitation Assay

    L6-F4-2 treatment rescues stroke phenotypes in wild-type mice a Schematic of tMCAO surgery with 45 min occlusion time, L6-F4-2 treatment time course and brain harvest at day 2 post-stroke. b TTC staining (top), and mouse IgG extravasation (mIgG) (bottom) of coronal sections at 48 hours post-stoke. c Quantification of infarct size for NIST control mAb (n=13) and L6-F4-2 (n=18). d Quantification of mouse IgG staining, NIST control n=4, L6-F4-2 n=5. e Representative images of BBB integrity in brains of the indicated mice in stroke and non-stroke regions after 45 min tMCAO, 2 days of reperfusion and two L6-F4-2 treatments (3 mg/kg, i.v.), as assessed by the Sulfo-NHS-biotin tracer extravasation assay. Scale bar, 50 μm. f Quantification of extravasated exogenous tracer Sulfo-NHS-biotin using ImageJ. n = 5. g Fractional change in brain edema for NIST control mAb (n=13) and L6-F4-2 (n=18). h Neurological scores at 48 h after tMCAO surgery for NIST control mAb (n=13) and L6-F4-2 (n=18). i Co-immunofluorescence staining for PDGFRB and CD31 in infarcted brain (stroke and non-stroke) regions and j quantification of pericyte coverage. The PDGFRB signal was normalized to CD31; n = 5. Scale bar, 100 μm. Error bars represent mean ± s.e.m., *p
    Figure Legend Snippet: L6-F4-2 treatment rescues stroke phenotypes in wild-type mice a Schematic of tMCAO surgery with 45 min occlusion time, L6-F4-2 treatment time course and brain harvest at day 2 post-stroke. b TTC staining (top), and mouse IgG extravasation (mIgG) (bottom) of coronal sections at 48 hours post-stoke. c Quantification of infarct size for NIST control mAb (n=13) and L6-F4-2 (n=18). d Quantification of mouse IgG staining, NIST control n=4, L6-F4-2 n=5. e Representative images of BBB integrity in brains of the indicated mice in stroke and non-stroke regions after 45 min tMCAO, 2 days of reperfusion and two L6-F4-2 treatments (3 mg/kg, i.v.), as assessed by the Sulfo-NHS-biotin tracer extravasation assay. Scale bar, 50 μm. f Quantification of extravasated exogenous tracer Sulfo-NHS-biotin using ImageJ. n = 5. g Fractional change in brain edema for NIST control mAb (n=13) and L6-F4-2 (n=18). h Neurological scores at 48 h after tMCAO surgery for NIST control mAb (n=13) and L6-F4-2 (n=18). i Co-immunofluorescence staining for PDGFRB and CD31 in infarcted brain (stroke and non-stroke) regions and j quantification of pericyte coverage. The PDGFRB signal was normalized to CD31; n = 5. Scale bar, 100 μm. Error bars represent mean ± s.e.m., *p

    Techniques Used: Mouse Assay, Staining, Immunofluorescence

    2) Product Images from "Rnf20 shapes the endothelial control of heart morphogenesis and function"

    Article Title: Rnf20 shapes the endothelial control of heart morphogenesis and function

    Journal: bioRxiv

    doi: 10.1101/2022.09.16.508288

    Effect of angiocrine factors, receptors on endothelial cells and extracellular matrix proteins on CM behavior, Related to Figure 7. (A) Screening: Percentage of binucleated CMs as determined by the FACS analysis with the Vybrant DyeCycle DNA dye and CD31 (to subtract HUVECs). P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors. (B) Screening: Percentage of CMs in G2/M as determined by the FACS analysis with the Vybrant DyeCycle DNA dye and CD31 (to subtract HUVECs). P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors. (C) Representative FACS analysis of co-cultures stained with the Vybrant DyeCycle DNA dye and CD31 (to subtract HUVECs). P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors for 2 days. (D) Plot of contraction amplitude and speed in spontaneously beating CMs from video sequences using MYOCYTER. P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors for 4 days.
    Figure Legend Snippet: Effect of angiocrine factors, receptors on endothelial cells and extracellular matrix proteins on CM behavior, Related to Figure 7. (A) Screening: Percentage of binucleated CMs as determined by the FACS analysis with the Vybrant DyeCycle DNA dye and CD31 (to subtract HUVECs). P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors. (B) Screening: Percentage of CMs in G2/M as determined by the FACS analysis with the Vybrant DyeCycle DNA dye and CD31 (to subtract HUVECs). P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors. (C) Representative FACS analysis of co-cultures stained with the Vybrant DyeCycle DNA dye and CD31 (to subtract HUVECs). P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors for 2 days. (D) Plot of contraction amplitude and speed in spontaneously beating CMs from video sequences using MYOCYTER. P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors for 4 days.

    Techniques Used: FACS, Cell Culture, Staining

    Rnf20-dependent EC-derived factors regulate CM proliferation, binucleation and contractility (A) Bar plot of total numbers of ligand-receptor interactions between ECs and CMs including autocrine signaling in Rnf20 iEC-KO versus control ( Skelly et al., 2018 ). (B) Circos plot of the significantly changed ligand-receptor interactions between ECs and CMs upon Rnf20 LOF in ECs. Red labeled arrow represent upregulated ligand, blue labeled downregulated ligand and arrow thickness indicates the interaction score ( Skelly et al., 2018 ). (C) Schematic representation of the experimental design: Lentivirus particles produced by overexpression of lentiviral plasmids from genome-wide libraries in HEK293 cells were used to transduce HUVECs for 2 days. HUVECs overexpressing selected factors were then co-cultured with wild-type P1 rat CM. (D) Percentage of mononucleated and binucleated CMs determined by staining with Vybrant DyeCycle DNA dye and CD31 (to exclude HUVEC cells) and subjected to FACS analysis. P1 rat CMs were co-cultured with HUVECs overexpressing selected factors for 2 or 4 days (n=3). (E) Percentage of proliferating EdU-positive CMs determined by EdU-incorporation and FACS analysis. P1 rat CMs were co-cultured with HUVECs overexpressing selected factors for 2 days. (F) Plot of contraction amplitude and speed in spontaneously beating CMs extracted from video sequences using MYOCYTER. P1 rat CMs were co-cultured with HUVECs overexpressing selected factors for 4 days. (G) Beating rate of rat CMs co-cultured with HUVECs overexpressing selected factors. Beating rate was extracted from video sequences using MYOCYTER. (H) Model of the function of Rnf20 in ECs for proper cardiac morphogenesis and function.
    Figure Legend Snippet: Rnf20-dependent EC-derived factors regulate CM proliferation, binucleation and contractility (A) Bar plot of total numbers of ligand-receptor interactions between ECs and CMs including autocrine signaling in Rnf20 iEC-KO versus control ( Skelly et al., 2018 ). (B) Circos plot of the significantly changed ligand-receptor interactions between ECs and CMs upon Rnf20 LOF in ECs. Red labeled arrow represent upregulated ligand, blue labeled downregulated ligand and arrow thickness indicates the interaction score ( Skelly et al., 2018 ). (C) Schematic representation of the experimental design: Lentivirus particles produced by overexpression of lentiviral plasmids from genome-wide libraries in HEK293 cells were used to transduce HUVECs for 2 days. HUVECs overexpressing selected factors were then co-cultured with wild-type P1 rat CM. (D) Percentage of mononucleated and binucleated CMs determined by staining with Vybrant DyeCycle DNA dye and CD31 (to exclude HUVEC cells) and subjected to FACS analysis. P1 rat CMs were co-cultured with HUVECs overexpressing selected factors for 2 or 4 days (n=3). (E) Percentage of proliferating EdU-positive CMs determined by EdU-incorporation and FACS analysis. P1 rat CMs were co-cultured with HUVECs overexpressing selected factors for 2 days. (F) Plot of contraction amplitude and speed in spontaneously beating CMs extracted from video sequences using MYOCYTER. P1 rat CMs were co-cultured with HUVECs overexpressing selected factors for 4 days. (G) Beating rate of rat CMs co-cultured with HUVECs overexpressing selected factors. Beating rate was extracted from video sequences using MYOCYTER. (H) Model of the function of Rnf20 in ECs for proper cardiac morphogenesis and function.

    Techniques Used: Derivative Assay, Labeling, Produced, Over Expression, Genome Wide, Transduction, Cell Culture, Staining, FACS

    Endothelial Rnf20 instructs CM behavior in an in vitro co-differentiation system. (A) Schematic representation of the experimental design for the establishment of a co-differentiation system from murine ESC to endocardial cells and cardiomyocytes. (B, C) Representative FACS analysis of direct Nkx2.5-GFP fluorescence and staining for the EC marker CD31 (B) and percentage of Nkx2.5+/CD31+ endocardial cells (n=4) (C). (D) Characterization of simultaneous differentiation of CMs and endocardial cells from ESCs (co-differentiation, top). FACS analysis showing similar percentage of Nkx2.5+/CD31+ endocardial cells and Nkx2.5+/CD31-CMs co-differentiated from ESCs (bottom left) and images of co-differentiated cells (bottom right). (E) Beating rate of CMs differentiated by the direct differentiation protocol of ESCs into CMs or co-differentiated with endocardial cells at day 9. Beating rate was extracted from video sequences using MYOCYTER. (F) Co-culture of Nkx2.5+/CD31+ endocardial cells and Nkx2.5-/CD31+ hemogenic ECs with human iPSC-derived CMs. Nkx2.5+/CD31+ endocardial cells were sorted from day 7 and differentiated using the protocol represented in panel A, while Nkx2.5-/CD31+ hemogenic ECs were differentiated by the addition of EC differentiation medium at mesoderm stage (protocol adapted for mouse ESCs from ( Palpant et al., 2017 ). (G) Beating rate of hiPSC-CMs co-cultured either with Nkx2.5+/CD31+ endocardial cells or Nkx2.5-/CD31+ hemogenic ECs for 2 days. Beating rate was extracted from video sequences using MYOCYTER. (H) Relative expression of CM, EC marker genes as well as genes involved in EC-CM crosstalk in CMs differentiated by the direct differentiation protocol of ESCs into CMs or co-differentiated with endocardial cells (top) or ECs differentiated by the direct differentiation protocol of ESCs into ECs or co-differentiated with CMs (bottom). (I) Schematic representation of the methodology used for the generation of control and Rnf20 iEC-KO mouse iPSC lines. (J) Representative FACS analysis of 38 days old CMs (CD31 negative cells in the co-differentiation) stained with Vybrant DyeCycle DNA dye, showing decrease in CMs in G2/M phase upon endothelial Rnf20 ablation. For this experiment as well as the data shown in (K-M), control and Rnf20 iEC-KO iPSCs were co-differentiated in CM and endothelial specific Rnf20 ablation was induced at day 5 by 4-hydroxytamoxifen (4-OHT). (K) Quantification of the percentage of CMs (left) or endocardial cells (right) in G0/G1 and G2/M phase upon EC-specific tamoxifen-mediated Rnf20 ablation (n=4). Cells were differentiated using the co-differentiation protocol and stained with Vybrant DyeCycle DNA dye, and CD31, followed by FACS analysis. (L) Quantification of the percentage of mononucleated and binucleated CMs (day 38) of cells stained with Vybrant DyeCycle DNA dye and subjected to FACS analysis (n=4). (M) Plot of contraction amplitude and speed in spontaneously beating CMs (day 18) from video sequences using MYOCYTER.
    Figure Legend Snippet: Endothelial Rnf20 instructs CM behavior in an in vitro co-differentiation system. (A) Schematic representation of the experimental design for the establishment of a co-differentiation system from murine ESC to endocardial cells and cardiomyocytes. (B, C) Representative FACS analysis of direct Nkx2.5-GFP fluorescence and staining for the EC marker CD31 (B) and percentage of Nkx2.5+/CD31+ endocardial cells (n=4) (C). (D) Characterization of simultaneous differentiation of CMs and endocardial cells from ESCs (co-differentiation, top). FACS analysis showing similar percentage of Nkx2.5+/CD31+ endocardial cells and Nkx2.5+/CD31-CMs co-differentiated from ESCs (bottom left) and images of co-differentiated cells (bottom right). (E) Beating rate of CMs differentiated by the direct differentiation protocol of ESCs into CMs or co-differentiated with endocardial cells at day 9. Beating rate was extracted from video sequences using MYOCYTER. (F) Co-culture of Nkx2.5+/CD31+ endocardial cells and Nkx2.5-/CD31+ hemogenic ECs with human iPSC-derived CMs. Nkx2.5+/CD31+ endocardial cells were sorted from day 7 and differentiated using the protocol represented in panel A, while Nkx2.5-/CD31+ hemogenic ECs were differentiated by the addition of EC differentiation medium at mesoderm stage (protocol adapted for mouse ESCs from ( Palpant et al., 2017 ). (G) Beating rate of hiPSC-CMs co-cultured either with Nkx2.5+/CD31+ endocardial cells or Nkx2.5-/CD31+ hemogenic ECs for 2 days. Beating rate was extracted from video sequences using MYOCYTER. (H) Relative expression of CM, EC marker genes as well as genes involved in EC-CM crosstalk in CMs differentiated by the direct differentiation protocol of ESCs into CMs or co-differentiated with endocardial cells (top) or ECs differentiated by the direct differentiation protocol of ESCs into ECs or co-differentiated with CMs (bottom). (I) Schematic representation of the methodology used for the generation of control and Rnf20 iEC-KO mouse iPSC lines. (J) Representative FACS analysis of 38 days old CMs (CD31 negative cells in the co-differentiation) stained with Vybrant DyeCycle DNA dye, showing decrease in CMs in G2/M phase upon endothelial Rnf20 ablation. For this experiment as well as the data shown in (K-M), control and Rnf20 iEC-KO iPSCs were co-differentiated in CM and endothelial specific Rnf20 ablation was induced at day 5 by 4-hydroxytamoxifen (4-OHT). (K) Quantification of the percentage of CMs (left) or endocardial cells (right) in G0/G1 and G2/M phase upon EC-specific tamoxifen-mediated Rnf20 ablation (n=4). Cells were differentiated using the co-differentiation protocol and stained with Vybrant DyeCycle DNA dye, and CD31, followed by FACS analysis. (L) Quantification of the percentage of mononucleated and binucleated CMs (day 38) of cells stained with Vybrant DyeCycle DNA dye and subjected to FACS analysis (n=4). (M) Plot of contraction amplitude and speed in spontaneously beating CMs (day 18) from video sequences using MYOCYTER.

    Techniques Used: In Vitro, FACS, Fluorescence, Staining, Marker, Co-Culture Assay, Derivative Assay, Cell Culture, Expressing

    Endothelial Rnf20 inhibits EndMT, CM maturation and cell cycle withdrawal. (A) Schematic representation of the experimental setup for the analysis presented in Figure 5 and Figure 6 . E14.5 control and Rnf20 iEC-KO hearts were used for scRNA-Seq and purified ECs and CMs for RNA-Seq and ATAC-Seq. (B) 10x genomics single-cell RNA-Seq of E14.5 control and Rnf20 iEC-KO . AVC CM, atrioventricular cardiomyocytes (CM); AVcu, atrioventricular cushion cells; Pro. CM, proliferating CM; EndoC, endocardial cells; EndoV, endocardial valve cells; VaMes, valve mesenchyme; SMC, smooth muscle cells; VEC, vascular ECs; EpiC, epicardial cells; MΦ, macrophages. (C) UMAP analysis of CM populations from panel B. Feature plots visualizing Mki67 -expressing proliferative CM. aCM, atrial CM; vCM, ventricular CM. (D) UMAP analysis of CD31+ endothelial and endocardial populations from panel B identifying 7 subclusters. (E) Feature plots of the expression of Mki67 and Col5a2 , showing a decrease in proliferative endocardial cells (circled population) and an increase in Col5a2 expression in subset of endocardial ECs. (F) Violin plots visualizing expression levels of collagens and extracellular matrix proteins in different endothelial and endocardial populations. (G) Gene ontology pathway enrichment analysis and representative genes in upregulated and downregulated genes in Rnf20 iEC-KO ECs compared to ECs from control hearts. n=3; Log2(FC) ≤ -0.58, ≥0.58; p-value
    Figure Legend Snippet: Endothelial Rnf20 inhibits EndMT, CM maturation and cell cycle withdrawal. (A) Schematic representation of the experimental setup for the analysis presented in Figure 5 and Figure 6 . E14.5 control and Rnf20 iEC-KO hearts were used for scRNA-Seq and purified ECs and CMs for RNA-Seq and ATAC-Seq. (B) 10x genomics single-cell RNA-Seq of E14.5 control and Rnf20 iEC-KO . AVC CM, atrioventricular cardiomyocytes (CM); AVcu, atrioventricular cushion cells; Pro. CM, proliferating CM; EndoC, endocardial cells; EndoV, endocardial valve cells; VaMes, valve mesenchyme; SMC, smooth muscle cells; VEC, vascular ECs; EpiC, epicardial cells; MΦ, macrophages. (C) UMAP analysis of CM populations from panel B. Feature plots visualizing Mki67 -expressing proliferative CM. aCM, atrial CM; vCM, ventricular CM. (D) UMAP analysis of CD31+ endothelial and endocardial populations from panel B identifying 7 subclusters. (E) Feature plots of the expression of Mki67 and Col5a2 , showing a decrease in proliferative endocardial cells (circled population) and an increase in Col5a2 expression in subset of endocardial ECs. (F) Violin plots visualizing expression levels of collagens and extracellular matrix proteins in different endothelial and endocardial populations. (G) Gene ontology pathway enrichment analysis and representative genes in upregulated and downregulated genes in Rnf20 iEC-KO ECs compared to ECs from control hearts. n=3; Log2(FC) ≤ -0.58, ≥0.58; p-value

    Techniques Used: Purification, RNA Sequencing Assay, Expressing

    3) Product Images from "Rnf20 shapes the endothelial control of heart morphogenesis and function"

    Article Title: Rnf20 shapes the endothelial control of heart morphogenesis and function

    Journal: bioRxiv

    doi: 10.1101/2022.09.16.508288

    Effect of angiocrine factors, receptors on endothelial cells and extracellular matrix proteins on CM behavior, Related to Figure 7. (A) Screening: Percentage of binucleated CMs as determined by the FACS analysis with the Vybrant DyeCycle DNA dye and CD31 (to subtract HUVECs). P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors. (B) Screening: Percentage of CMs in G2/M as determined by the FACS analysis with the Vybrant DyeCycle DNA dye and CD31 (to subtract HUVECs). P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors. (C) Representative FACS analysis of co-cultures stained with the Vybrant DyeCycle DNA dye and CD31 (to subtract HUVECs). P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors for 2 days. (D) Plot of contraction amplitude and speed in spontaneously beating CMs from video sequences using MYOCYTER. P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors for 4 days.
    Figure Legend Snippet: Effect of angiocrine factors, receptors on endothelial cells and extracellular matrix proteins on CM behavior, Related to Figure 7. (A) Screening: Percentage of binucleated CMs as determined by the FACS analysis with the Vybrant DyeCycle DNA dye and CD31 (to subtract HUVECs). P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors. (B) Screening: Percentage of CMs in G2/M as determined by the FACS analysis with the Vybrant DyeCycle DNA dye and CD31 (to subtract HUVECs). P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors. (C) Representative FACS analysis of co-cultures stained with the Vybrant DyeCycle DNA dye and CD31 (to subtract HUVECs). P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors for 2 days. (D) Plot of contraction amplitude and speed in spontaneously beating CMs from video sequences using MYOCYTER. P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors for 4 days.

    Techniques Used: FACS, Cell Culture, Staining

    Rnf20-dependent EC-derived factors regulate CM proliferation, binucleation and contractility (A) Bar plot of total numbers of ligand-receptor interactions between ECs and CMs including autocrine signaling in Rnf20 iEC-KO versus control ( Skelly et al., 2018 ). (B) Circos plot of the significantly changed ligand-receptor interactions between ECs and CMs upon Rnf20 LOF in ECs. Red labeled arrow represent upregulated ligand, blue labeled downregulated ligand and arrow thickness indicates the interaction score ( Skelly et al., 2018 ). (C) Schematic representation of the experimental design: Lentivirus particles produced by overexpression of lentiviral plasmids from genome-wide libraries in HEK293 cells were used to transduce HUVECs for 2 days. HUVECs overexpressing selected factors were then co-cultured with wild-type P1 rat CM. (D) Percentage of mononucleated and binucleated CMs determined by staining with Vybrant DyeCycle DNA dye and CD31 (to exclude HUVEC cells) and subjected to FACS analysis. P1 rat CMs were co-cultured with HUVECs overexpressing selected factors for 2 or 4 days (n=3). (E) Percentage of proliferating EdU-positive CMs determined by EdU-incorporation and FACS analysis. P1 rat CMs were co-cultured with HUVECs overexpressing selected factors for 2 days. (F) Plot of contraction amplitude and speed in spontaneously beating CMs extracted from video sequences using MYOCYTER. P1 rat CMs were co-cultured with HUVECs overexpressing selected factors for 4 days. (G) Beating rate of rat CMs co-cultured with HUVECs overexpressing selected factors. Beating rate was extracted from video sequences using MYOCYTER. (H) Model of the function of Rnf20 in ECs for proper cardiac morphogenesis and function.
    Figure Legend Snippet: Rnf20-dependent EC-derived factors regulate CM proliferation, binucleation and contractility (A) Bar plot of total numbers of ligand-receptor interactions between ECs and CMs including autocrine signaling in Rnf20 iEC-KO versus control ( Skelly et al., 2018 ). (B) Circos plot of the significantly changed ligand-receptor interactions between ECs and CMs upon Rnf20 LOF in ECs. Red labeled arrow represent upregulated ligand, blue labeled downregulated ligand and arrow thickness indicates the interaction score ( Skelly et al., 2018 ). (C) Schematic representation of the experimental design: Lentivirus particles produced by overexpression of lentiviral plasmids from genome-wide libraries in HEK293 cells were used to transduce HUVECs for 2 days. HUVECs overexpressing selected factors were then co-cultured with wild-type P1 rat CM. (D) Percentage of mononucleated and binucleated CMs determined by staining with Vybrant DyeCycle DNA dye and CD31 (to exclude HUVEC cells) and subjected to FACS analysis. P1 rat CMs were co-cultured with HUVECs overexpressing selected factors for 2 or 4 days (n=3). (E) Percentage of proliferating EdU-positive CMs determined by EdU-incorporation and FACS analysis. P1 rat CMs were co-cultured with HUVECs overexpressing selected factors for 2 days. (F) Plot of contraction amplitude and speed in spontaneously beating CMs extracted from video sequences using MYOCYTER. P1 rat CMs were co-cultured with HUVECs overexpressing selected factors for 4 days. (G) Beating rate of rat CMs co-cultured with HUVECs overexpressing selected factors. Beating rate was extracted from video sequences using MYOCYTER. (H) Model of the function of Rnf20 in ECs for proper cardiac morphogenesis and function.

    Techniques Used: Derivative Assay, Labeling, Produced, Over Expression, Genome Wide, Transduction, Cell Culture, Staining, FACS

    Endothelial Rnf20 instructs CM behavior in an in vitro co-differentiation system. (A) Schematic representation of the experimental design for the establishment of a co-differentiation system from murine ESC to endocardial cells and cardiomyocytes. (B, C) Representative FACS analysis of direct Nkx2.5-GFP fluorescence and staining for the EC marker CD31 (B) and percentage of Nkx2.5+/CD31+ endocardial cells (n=4) (C). (D) Characterization of simultaneous differentiation of CMs and endocardial cells from ESCs (co-differentiation, top). FACS analysis showing similar percentage of Nkx2.5+/CD31+ endocardial cells and Nkx2.5+/CD31-CMs co-differentiated from ESCs (bottom left) and images of co-differentiated cells (bottom right). (E) Beating rate of CMs differentiated by the direct differentiation protocol of ESCs into CMs or co-differentiated with endocardial cells at day 9. Beating rate was extracted from video sequences using MYOCYTER. (F) Co-culture of Nkx2.5+/CD31+ endocardial cells and Nkx2.5-/CD31+ hemogenic ECs with human iPSC-derived CMs. Nkx2.5+/CD31+ endocardial cells were sorted from day 7 and differentiated using the protocol represented in panel A, while Nkx2.5-/CD31+ hemogenic ECs were differentiated by the addition of EC differentiation medium at mesoderm stage (protocol adapted for mouse ESCs from ( Palpant et al., 2017 ). (G) Beating rate of hiPSC-CMs co-cultured either with Nkx2.5+/CD31+ endocardial cells or Nkx2.5-/CD31+ hemogenic ECs for 2 days. Beating rate was extracted from video sequences using MYOCYTER. (H) Relative expression of CM, EC marker genes as well as genes involved in EC-CM crosstalk in CMs differentiated by the direct differentiation protocol of ESCs into CMs or co-differentiated with endocardial cells (top) or ECs differentiated by the direct differentiation protocol of ESCs into ECs or co-differentiated with CMs (bottom). (I) Schematic representation of the methodology used for the generation of control and Rnf20 iEC-KO mouse iPSC lines. (J) Representative FACS analysis of 38 days old CMs (CD31 negative cells in the co-differentiation) stained with Vybrant DyeCycle DNA dye, showing decrease in CMs in G2/M phase upon endothelial Rnf20 ablation. For this experiment as well as the data shown in (K-M), control and Rnf20 iEC-KO iPSCs were co-differentiated in CM and endothelial specific Rnf20 ablation was induced at day 5 by 4-hydroxytamoxifen (4-OHT). (K) Quantification of the percentage of CMs (left) or endocardial cells (right) in G0/G1 and G2/M phase upon EC-specific tamoxifen-mediated Rnf20 ablation (n=4). Cells were differentiated using the co-differentiation protocol and stained with Vybrant DyeCycle DNA dye, and CD31, followed by FACS analysis. (L) Quantification of the percentage of mononucleated and binucleated CMs (day 38) of cells stained with Vybrant DyeCycle DNA dye and subjected to FACS analysis (n=4). (M) Plot of contraction amplitude and speed in spontaneously beating CMs (day 18) from video sequences using MYOCYTER.
    Figure Legend Snippet: Endothelial Rnf20 instructs CM behavior in an in vitro co-differentiation system. (A) Schematic representation of the experimental design for the establishment of a co-differentiation system from murine ESC to endocardial cells and cardiomyocytes. (B, C) Representative FACS analysis of direct Nkx2.5-GFP fluorescence and staining for the EC marker CD31 (B) and percentage of Nkx2.5+/CD31+ endocardial cells (n=4) (C). (D) Characterization of simultaneous differentiation of CMs and endocardial cells from ESCs (co-differentiation, top). FACS analysis showing similar percentage of Nkx2.5+/CD31+ endocardial cells and Nkx2.5+/CD31-CMs co-differentiated from ESCs (bottom left) and images of co-differentiated cells (bottom right). (E) Beating rate of CMs differentiated by the direct differentiation protocol of ESCs into CMs or co-differentiated with endocardial cells at day 9. Beating rate was extracted from video sequences using MYOCYTER. (F) Co-culture of Nkx2.5+/CD31+ endocardial cells and Nkx2.5-/CD31+ hemogenic ECs with human iPSC-derived CMs. Nkx2.5+/CD31+ endocardial cells were sorted from day 7 and differentiated using the protocol represented in panel A, while Nkx2.5-/CD31+ hemogenic ECs were differentiated by the addition of EC differentiation medium at mesoderm stage (protocol adapted for mouse ESCs from ( Palpant et al., 2017 ). (G) Beating rate of hiPSC-CMs co-cultured either with Nkx2.5+/CD31+ endocardial cells or Nkx2.5-/CD31+ hemogenic ECs for 2 days. Beating rate was extracted from video sequences using MYOCYTER. (H) Relative expression of CM, EC marker genes as well as genes involved in EC-CM crosstalk in CMs differentiated by the direct differentiation protocol of ESCs into CMs or co-differentiated with endocardial cells (top) or ECs differentiated by the direct differentiation protocol of ESCs into ECs or co-differentiated with CMs (bottom). (I) Schematic representation of the methodology used for the generation of control and Rnf20 iEC-KO mouse iPSC lines. (J) Representative FACS analysis of 38 days old CMs (CD31 negative cells in the co-differentiation) stained with Vybrant DyeCycle DNA dye, showing decrease in CMs in G2/M phase upon endothelial Rnf20 ablation. For this experiment as well as the data shown in (K-M), control and Rnf20 iEC-KO iPSCs were co-differentiated in CM and endothelial specific Rnf20 ablation was induced at day 5 by 4-hydroxytamoxifen (4-OHT). (K) Quantification of the percentage of CMs (left) or endocardial cells (right) in G0/G1 and G2/M phase upon EC-specific tamoxifen-mediated Rnf20 ablation (n=4). Cells were differentiated using the co-differentiation protocol and stained with Vybrant DyeCycle DNA dye, and CD31, followed by FACS analysis. (L) Quantification of the percentage of mononucleated and binucleated CMs (day 38) of cells stained with Vybrant DyeCycle DNA dye and subjected to FACS analysis (n=4). (M) Plot of contraction amplitude and speed in spontaneously beating CMs (day 18) from video sequences using MYOCYTER.

    Techniques Used: In Vitro, FACS, Fluorescence, Staining, Marker, Co-Culture Assay, Derivative Assay, Cell Culture, Expressing

    Endothelial Rnf20 inhibits EndMT, CM maturation and cell cycle withdrawal. (A) Schematic representation of the experimental setup for the analysis presented in Figure 5 and Figure 6 . E14.5 control and Rnf20 iEC-KO hearts were used for scRNA-Seq and purified ECs and CMs for RNA-Seq and ATAC-Seq. (B) 10x genomics single-cell RNA-Seq of E14.5 control and Rnf20 iEC-KO . AVC CM, atrioventricular cardiomyocytes (CM); AVcu, atrioventricular cushion cells; Pro. CM, proliferating CM; EndoC, endocardial cells; EndoV, endocardial valve cells; VaMes, valve mesenchyme; SMC, smooth muscle cells; VEC, vascular ECs; EpiC, epicardial cells; MΦ, macrophages. (C) UMAP analysis of CM populations from panel B. Feature plots visualizing Mki67 -expressing proliferative CM. aCM, atrial CM; vCM, ventricular CM. (D) UMAP analysis of CD31+ endothelial and endocardial populations from panel B identifying 7 subclusters. (E) Feature plots of the expression of Mki67 and Col5a2 , showing a decrease in proliferative endocardial cells (circled population) and an increase in Col5a2 expression in subset of endocardial ECs. (F) Violin plots visualizing expression levels of collagens and extracellular matrix proteins in different endothelial and endocardial populations. (G) Gene ontology pathway enrichment analysis and representative genes in upregulated and downregulated genes in Rnf20 iEC-KO ECs compared to ECs from control hearts. n=3; Log2(FC) ≤ -0.58, ≥0.58; p-value
    Figure Legend Snippet: Endothelial Rnf20 inhibits EndMT, CM maturation and cell cycle withdrawal. (A) Schematic representation of the experimental setup for the analysis presented in Figure 5 and Figure 6 . E14.5 control and Rnf20 iEC-KO hearts were used for scRNA-Seq and purified ECs and CMs for RNA-Seq and ATAC-Seq. (B) 10x genomics single-cell RNA-Seq of E14.5 control and Rnf20 iEC-KO . AVC CM, atrioventricular cardiomyocytes (CM); AVcu, atrioventricular cushion cells; Pro. CM, proliferating CM; EndoC, endocardial cells; EndoV, endocardial valve cells; VaMes, valve mesenchyme; SMC, smooth muscle cells; VEC, vascular ECs; EpiC, epicardial cells; MΦ, macrophages. (C) UMAP analysis of CM populations from panel B. Feature plots visualizing Mki67 -expressing proliferative CM. aCM, atrial CM; vCM, ventricular CM. (D) UMAP analysis of CD31+ endothelial and endocardial populations from panel B identifying 7 subclusters. (E) Feature plots of the expression of Mki67 and Col5a2 , showing a decrease in proliferative endocardial cells (circled population) and an increase in Col5a2 expression in subset of endocardial ECs. (F) Violin plots visualizing expression levels of collagens and extracellular matrix proteins in different endothelial and endocardial populations. (G) Gene ontology pathway enrichment analysis and representative genes in upregulated and downregulated genes in Rnf20 iEC-KO ECs compared to ECs from control hearts. n=3; Log2(FC) ≤ -0.58, ≥0.58; p-value

    Techniques Used: Purification, RNA Sequencing Assay, Expressing

    4) Product Images from "Rnf20 shapes the endothelial control of heart morphogenesis and function"

    Article Title: Rnf20 shapes the endothelial control of heart morphogenesis and function

    Journal: bioRxiv

    doi: 10.1101/2022.09.16.508288

    Effect of angiocrine factors, receptors on endothelial cells and extracellular matrix proteins on CM behavior, Related to Figure 7. (A) Screening: Percentage of binucleated CMs as determined by the FACS analysis with the Vybrant DyeCycle DNA dye and CD31 (to subtract HUVECs). P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors. (B) Screening: Percentage of CMs in G2/M as determined by the FACS analysis with the Vybrant DyeCycle DNA dye and CD31 (to subtract HUVECs). P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors. (C) Representative FACS analysis of co-cultures stained with the Vybrant DyeCycle DNA dye and CD31 (to subtract HUVECs). P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors for 2 days. (D) Plot of contraction amplitude and speed in spontaneously beating CMs from video sequences using MYOCYTER. P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors for 4 days.
    Figure Legend Snippet: Effect of angiocrine factors, receptors on endothelial cells and extracellular matrix proteins on CM behavior, Related to Figure 7. (A) Screening: Percentage of binucleated CMs as determined by the FACS analysis with the Vybrant DyeCycle DNA dye and CD31 (to subtract HUVECs). P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors. (B) Screening: Percentage of CMs in G2/M as determined by the FACS analysis with the Vybrant DyeCycle DNA dye and CD31 (to subtract HUVECs). P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors. (C) Representative FACS analysis of co-cultures stained with the Vybrant DyeCycle DNA dye and CD31 (to subtract HUVECs). P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors for 2 days. (D) Plot of contraction amplitude and speed in spontaneously beating CMs from video sequences using MYOCYTER. P1 rat cardiomyocytes were co-cultured with HUVECs overexpressing selected factors for 4 days.

    Techniques Used: FACS, Cell Culture, Staining

    Rnf20-dependent EC-derived factors regulate CM proliferation, binucleation and contractility (A) Bar plot of total numbers of ligand-receptor interactions between ECs and CMs including autocrine signaling in Rnf20 iEC-KO versus control ( Skelly et al., 2018 ). (B) Circos plot of the significantly changed ligand-receptor interactions between ECs and CMs upon Rnf20 LOF in ECs. Red labeled arrow represent upregulated ligand, blue labeled downregulated ligand and arrow thickness indicates the interaction score ( Skelly et al., 2018 ). (C) Schematic representation of the experimental design: Lentivirus particles produced by overexpression of lentiviral plasmids from genome-wide libraries in HEK293 cells were used to transduce HUVECs for 2 days. HUVECs overexpressing selected factors were then co-cultured with wild-type P1 rat CM. (D) Percentage of mononucleated and binucleated CMs determined by staining with Vybrant DyeCycle DNA dye and CD31 (to exclude HUVEC cells) and subjected to FACS analysis. P1 rat CMs were co-cultured with HUVECs overexpressing selected factors for 2 or 4 days (n=3). (E) Percentage of proliferating EdU-positive CMs determined by EdU-incorporation and FACS analysis. P1 rat CMs were co-cultured with HUVECs overexpressing selected factors for 2 days. (F) Plot of contraction amplitude and speed in spontaneously beating CMs extracted from video sequences using MYOCYTER. P1 rat CMs were co-cultured with HUVECs overexpressing selected factors for 4 days. (G) Beating rate of rat CMs co-cultured with HUVECs overexpressing selected factors. Beating rate was extracted from video sequences using MYOCYTER. (H) Model of the function of Rnf20 in ECs for proper cardiac morphogenesis and function.
    Figure Legend Snippet: Rnf20-dependent EC-derived factors regulate CM proliferation, binucleation and contractility (A) Bar plot of total numbers of ligand-receptor interactions between ECs and CMs including autocrine signaling in Rnf20 iEC-KO versus control ( Skelly et al., 2018 ). (B) Circos plot of the significantly changed ligand-receptor interactions between ECs and CMs upon Rnf20 LOF in ECs. Red labeled arrow represent upregulated ligand, blue labeled downregulated ligand and arrow thickness indicates the interaction score ( Skelly et al., 2018 ). (C) Schematic representation of the experimental design: Lentivirus particles produced by overexpression of lentiviral plasmids from genome-wide libraries in HEK293 cells were used to transduce HUVECs for 2 days. HUVECs overexpressing selected factors were then co-cultured with wild-type P1 rat CM. (D) Percentage of mononucleated and binucleated CMs determined by staining with Vybrant DyeCycle DNA dye and CD31 (to exclude HUVEC cells) and subjected to FACS analysis. P1 rat CMs were co-cultured with HUVECs overexpressing selected factors for 2 or 4 days (n=3). (E) Percentage of proliferating EdU-positive CMs determined by EdU-incorporation and FACS analysis. P1 rat CMs were co-cultured with HUVECs overexpressing selected factors for 2 days. (F) Plot of contraction amplitude and speed in spontaneously beating CMs extracted from video sequences using MYOCYTER. P1 rat CMs were co-cultured with HUVECs overexpressing selected factors for 4 days. (G) Beating rate of rat CMs co-cultured with HUVECs overexpressing selected factors. Beating rate was extracted from video sequences using MYOCYTER. (H) Model of the function of Rnf20 in ECs for proper cardiac morphogenesis and function.

    Techniques Used: Derivative Assay, Labeling, Produced, Over Expression, Genome Wide, Transduction, Cell Culture, Staining, FACS

    Endothelial Rnf20 instructs CM behavior in an in vitro co-differentiation system. (A) Schematic representation of the experimental design for the establishment of a co-differentiation system from murine ESC to endocardial cells and cardiomyocytes. (B, C) Representative FACS analysis of direct Nkx2.5-GFP fluorescence and staining for the EC marker CD31 (B) and percentage of Nkx2.5+/CD31+ endocardial cells (n=4) (C). (D) Characterization of simultaneous differentiation of CMs and endocardial cells from ESCs (co-differentiation, top). FACS analysis showing similar percentage of Nkx2.5+/CD31+ endocardial cells and Nkx2.5+/CD31-CMs co-differentiated from ESCs (bottom left) and images of co-differentiated cells (bottom right). (E) Beating rate of CMs differentiated by the direct differentiation protocol of ESCs into CMs or co-differentiated with endocardial cells at day 9. Beating rate was extracted from video sequences using MYOCYTER. (F) Co-culture of Nkx2.5+/CD31+ endocardial cells and Nkx2.5-/CD31+ hemogenic ECs with human iPSC-derived CMs. Nkx2.5+/CD31+ endocardial cells were sorted from day 7 and differentiated using the protocol represented in panel A, while Nkx2.5-/CD31+ hemogenic ECs were differentiated by the addition of EC differentiation medium at mesoderm stage (protocol adapted for mouse ESCs from ( Palpant et al., 2017 ). (G) Beating rate of hiPSC-CMs co-cultured either with Nkx2.5+/CD31+ endocardial cells or Nkx2.5-/CD31+ hemogenic ECs for 2 days. Beating rate was extracted from video sequences using MYOCYTER. (H) Relative expression of CM, EC marker genes as well as genes involved in EC-CM crosstalk in CMs differentiated by the direct differentiation protocol of ESCs into CMs or co-differentiated with endocardial cells (top) or ECs differentiated by the direct differentiation protocol of ESCs into ECs or co-differentiated with CMs (bottom). (I) Schematic representation of the methodology used for the generation of control and Rnf20 iEC-KO mouse iPSC lines. (J) Representative FACS analysis of 38 days old CMs (CD31 negative cells in the co-differentiation) stained with Vybrant DyeCycle DNA dye, showing decrease in CMs in G2/M phase upon endothelial Rnf20 ablation. For this experiment as well as the data shown in (K-M), control and Rnf20 iEC-KO iPSCs were co-differentiated in CM and endothelial specific Rnf20 ablation was induced at day 5 by 4-hydroxytamoxifen (4-OHT). (K) Quantification of the percentage of CMs (left) or endocardial cells (right) in G0/G1 and G2/M phase upon EC-specific tamoxifen-mediated Rnf20 ablation (n=4). Cells were differentiated using the co-differentiation protocol and stained with Vybrant DyeCycle DNA dye, and CD31, followed by FACS analysis. (L) Quantification of the percentage of mononucleated and binucleated CMs (day 38) of cells stained with Vybrant DyeCycle DNA dye and subjected to FACS analysis (n=4). (M) Plot of contraction amplitude and speed in spontaneously beating CMs (day 18) from video sequences using MYOCYTER.
    Figure Legend Snippet: Endothelial Rnf20 instructs CM behavior in an in vitro co-differentiation system. (A) Schematic representation of the experimental design for the establishment of a co-differentiation system from murine ESC to endocardial cells and cardiomyocytes. (B, C) Representative FACS analysis of direct Nkx2.5-GFP fluorescence and staining for the EC marker CD31 (B) and percentage of Nkx2.5+/CD31+ endocardial cells (n=4) (C). (D) Characterization of simultaneous differentiation of CMs and endocardial cells from ESCs (co-differentiation, top). FACS analysis showing similar percentage of Nkx2.5+/CD31+ endocardial cells and Nkx2.5+/CD31-CMs co-differentiated from ESCs (bottom left) and images of co-differentiated cells (bottom right). (E) Beating rate of CMs differentiated by the direct differentiation protocol of ESCs into CMs or co-differentiated with endocardial cells at day 9. Beating rate was extracted from video sequences using MYOCYTER. (F) Co-culture of Nkx2.5+/CD31+ endocardial cells and Nkx2.5-/CD31+ hemogenic ECs with human iPSC-derived CMs. Nkx2.5+/CD31+ endocardial cells were sorted from day 7 and differentiated using the protocol represented in panel A, while Nkx2.5-/CD31+ hemogenic ECs were differentiated by the addition of EC differentiation medium at mesoderm stage (protocol adapted for mouse ESCs from ( Palpant et al., 2017 ). (G) Beating rate of hiPSC-CMs co-cultured either with Nkx2.5+/CD31+ endocardial cells or Nkx2.5-/CD31+ hemogenic ECs for 2 days. Beating rate was extracted from video sequences using MYOCYTER. (H) Relative expression of CM, EC marker genes as well as genes involved in EC-CM crosstalk in CMs differentiated by the direct differentiation protocol of ESCs into CMs or co-differentiated with endocardial cells (top) or ECs differentiated by the direct differentiation protocol of ESCs into ECs or co-differentiated with CMs (bottom). (I) Schematic representation of the methodology used for the generation of control and Rnf20 iEC-KO mouse iPSC lines. (J) Representative FACS analysis of 38 days old CMs (CD31 negative cells in the co-differentiation) stained with Vybrant DyeCycle DNA dye, showing decrease in CMs in G2/M phase upon endothelial Rnf20 ablation. For this experiment as well as the data shown in (K-M), control and Rnf20 iEC-KO iPSCs were co-differentiated in CM and endothelial specific Rnf20 ablation was induced at day 5 by 4-hydroxytamoxifen (4-OHT). (K) Quantification of the percentage of CMs (left) or endocardial cells (right) in G0/G1 and G2/M phase upon EC-specific tamoxifen-mediated Rnf20 ablation (n=4). Cells were differentiated using the co-differentiation protocol and stained with Vybrant DyeCycle DNA dye, and CD31, followed by FACS analysis. (L) Quantification of the percentage of mononucleated and binucleated CMs (day 38) of cells stained with Vybrant DyeCycle DNA dye and subjected to FACS analysis (n=4). (M) Plot of contraction amplitude and speed in spontaneously beating CMs (day 18) from video sequences using MYOCYTER.

    Techniques Used: In Vitro, FACS, Fluorescence, Staining, Marker, Co-Culture Assay, Derivative Assay, Cell Culture, Expressing

    Endothelial Rnf20 inhibits EndMT, CM maturation and cell cycle withdrawal. (A) Schematic representation of the experimental setup for the analysis presented in Figure 5 and Figure 6 . E14.5 control and Rnf20 iEC-KO hearts were used for scRNA-Seq and purified ECs and CMs for RNA-Seq and ATAC-Seq. (B) 10x genomics single-cell RNA-Seq of E14.5 control and Rnf20 iEC-KO . AVC CM, atrioventricular cardiomyocytes (CM); AVcu, atrioventricular cushion cells; Pro. CM, proliferating CM; EndoC, endocardial cells; EndoV, endocardial valve cells; VaMes, valve mesenchyme; SMC, smooth muscle cells; VEC, vascular ECs; EpiC, epicardial cells; MΦ, macrophages. (C) UMAP analysis of CM populations from panel B. Feature plots visualizing Mki67 -expressing proliferative CM. aCM, atrial CM; vCM, ventricular CM. (D) UMAP analysis of CD31+ endothelial and endocardial populations from panel B identifying 7 subclusters. (E) Feature plots of the expression of Mki67 and Col5a2 , showing a decrease in proliferative endocardial cells (circled population) and an increase in Col5a2 expression in subset of endocardial ECs. (F) Violin plots visualizing expression levels of collagens and extracellular matrix proteins in different endothelial and endocardial populations. (G) Gene ontology pathway enrichment analysis and representative genes in upregulated and downregulated genes in Rnf20 iEC-KO ECs compared to ECs from control hearts. n=3; Log2(FC) ≤ -0.58, ≥0.58; p-value
    Figure Legend Snippet: Endothelial Rnf20 inhibits EndMT, CM maturation and cell cycle withdrawal. (A) Schematic representation of the experimental setup for the analysis presented in Figure 5 and Figure 6 . E14.5 control and Rnf20 iEC-KO hearts were used for scRNA-Seq and purified ECs and CMs for RNA-Seq and ATAC-Seq. (B) 10x genomics single-cell RNA-Seq of E14.5 control and Rnf20 iEC-KO . AVC CM, atrioventricular cardiomyocytes (CM); AVcu, atrioventricular cushion cells; Pro. CM, proliferating CM; EndoC, endocardial cells; EndoV, endocardial valve cells; VaMes, valve mesenchyme; SMC, smooth muscle cells; VEC, vascular ECs; EpiC, epicardial cells; MΦ, macrophages. (C) UMAP analysis of CM populations from panel B. Feature plots visualizing Mki67 -expressing proliferative CM. aCM, atrial CM; vCM, ventricular CM. (D) UMAP analysis of CD31+ endothelial and endocardial populations from panel B identifying 7 subclusters. (E) Feature plots of the expression of Mki67 and Col5a2 , showing a decrease in proliferative endocardial cells (circled population) and an increase in Col5a2 expression in subset of endocardial ECs. (F) Violin plots visualizing expression levels of collagens and extracellular matrix proteins in different endothelial and endocardial populations. (G) Gene ontology pathway enrichment analysis and representative genes in upregulated and downregulated genes in Rnf20 iEC-KO ECs compared to ECs from control hearts. n=3; Log2(FC) ≤ -0.58, ≥0.58; p-value

    Techniques Used: Purification, RNA Sequencing Assay, Expressing

    5) Product Images from "Alveolar epithelial progenitor cells drive lung regeneration via dynamic changes in chromatin topology modulated by lineage-specific Nkx2-1 activity"

    Article Title: Alveolar epithelial progenitor cells drive lung regeneration via dynamic changes in chromatin topology modulated by lineage-specific Nkx2-1 activity

    Journal: bioRxiv

    doi: 10.1101/2022.08.30.505919

    In vitro gene editing of AEP-derived alveolar organoids via AAV6.2FF-Cre. AAV6.2FF-Cre experimental set-up. Live/CD31 - /CD45 - /CD326 + (EpCAM + )/TdTomato + (Axin2 + ) cells (AEPs) sorted from mice with the R26R EYFP allele (Axin2 creERT2-tDT ; R26R EYFP ) were treated with AAV6.2FF-Cre and plated with wild-type fibroblasts. (B) H E of 5 μm sections of FFPE day 29 AAV6.2FF-Cre-treated organoids, exhibiting morphology and structural complexity similar to untreated/control organoids ( Figure 1B ). (C) Whole-well brightfield and GFP images of day 29 organoids (untreated vs. AAV6.2FF-Cre-treated) at MOI=1000. (D) Comparison of cells treated with an MOI of 1000, 10000, 20000. Quantification of day 32 organoids (n=3 wells per condition) showing that an MOI of 1000 causes recombination in organoids without significant effects on colony forming efficiency (CFE). (E) Quantification (n=5 wells per condition) showing that an MOI=1000 induces significant levels of recombination without significant effects on organoid number or size. (F) Whole-mount immunofluorescence of day 32 AAV6.2FF-Cre-treated AEP-derived organoids (same experimental set-up as Figure 2 ). White box highlighting untargeted epithelial cells (YFP - /GFP - ) next to a targeted (YFP + /GFP + ) organoid in the same well, supporting clonal expansion of AAV6.2FF-Cre-treated cells. ( ns = p > 0 . 05; *P =0 . 05, **P =0 . 01, ***P =0 . 001, and ****P =0 . 0001 ). Note: EYFP was stained for using anti-GFP antibodies and imaged (whole well images) using GFP filter cubes. [ Scale bars = 50 μm ]. AAV = Adeno-Associated Virus; MOI = Multiplicity of Infection
    Figure Legend Snippet: In vitro gene editing of AEP-derived alveolar organoids via AAV6.2FF-Cre. AAV6.2FF-Cre experimental set-up. Live/CD31 - /CD45 - /CD326 + (EpCAM + )/TdTomato + (Axin2 + ) cells (AEPs) sorted from mice with the R26R EYFP allele (Axin2 creERT2-tDT ; R26R EYFP ) were treated with AAV6.2FF-Cre and plated with wild-type fibroblasts. (B) H E of 5 μm sections of FFPE day 29 AAV6.2FF-Cre-treated organoids, exhibiting morphology and structural complexity similar to untreated/control organoids ( Figure 1B ). (C) Whole-well brightfield and GFP images of day 29 organoids (untreated vs. AAV6.2FF-Cre-treated) at MOI=1000. (D) Comparison of cells treated with an MOI of 1000, 10000, 20000. Quantification of day 32 organoids (n=3 wells per condition) showing that an MOI of 1000 causes recombination in organoids without significant effects on colony forming efficiency (CFE). (E) Quantification (n=5 wells per condition) showing that an MOI=1000 induces significant levels of recombination without significant effects on organoid number or size. (F) Whole-mount immunofluorescence of day 32 AAV6.2FF-Cre-treated AEP-derived organoids (same experimental set-up as Figure 2 ). White box highlighting untargeted epithelial cells (YFP - /GFP - ) next to a targeted (YFP + /GFP + ) organoid in the same well, supporting clonal expansion of AAV6.2FF-Cre-treated cells. ( ns = p > 0 . 05; *P =0 . 05, **P =0 . 01, ***P =0 . 001, and ****P =0 . 0001 ). Note: EYFP was stained for using anti-GFP antibodies and imaged (whole well images) using GFP filter cubes. [ Scale bars = 50 μm ]. AAV = Adeno-Associated Virus; MOI = Multiplicity of Infection

    Techniques Used: In Vitro, Derivative Assay, Mouse Assay, Formalin-fixed Paraffin-Embedded, Immunofluorescence, Staining, Infection

    AEP-derived alveolar organoids clonally expand and pattern complex, polarized alveolar-like cavities. (A) Schematic of experimental design and overview. Live/CD31 - /CD45 - /CD326 + (EpCAM + )/TdTomato + (Axin2 + ) cells (AEPs) were mixed with mouse lung fibroblasts from P28 mice and cultured for up to 35 days, followed by analysis via high content imaging. (B) H E of 5 μm sections of FFPE day 35 Axin2 + organoids, showing cellular morphologies typical of both AT1 and AT2 cells. (C-G) Whole-mount immunofluorescence time course of Axin2 + organoids showing expansion of SFTPC + AT2 cells (red), increased differentiation into RAGE + AT1 cells (green) and increased structural complexity. (H) Imaris 3D reconstruction of day 35 Axin2 + organoid (z-depth = 174.13 μm) showing cellular arrangement/organization within mature organoids. (I) Click-iT EdU (green) whole-mount day 25 Axin2 + organoids, with proliferating cells primarily on outer edges or ‘buds’ growing outward from the organoid. (J-K) Electron microscopy of day 28 organoids. (J) Image of properly polarized AT2 cell with apical microvilli (black arrowhead) secreting surfactant (blue arrowhead) into a lumen. (K) Image of AT2 cell with lamellar bodies (black arrowhead) adjacent to an AT1 cell (green arrowhead, right). (L-M) Comparison of in vivo mouse lung (9-month C57BL/6J mouse) and in vitro day 25 Axin2 + organoids. [ Scale bars = 50 μm, except for electron microscopy (J, K) scale bars = 2 . 5 μm ]. ( RAGE = Receptor for Advanced Glycation End-products [AT1 cell marker]; SFTPC = Surfactant Protein C [AT2 cell marker]; EdU = 5-ethynyl-2’-deoxyuridine; FFPE = formalin-fixed, paraffin-embedded )
    Figure Legend Snippet: AEP-derived alveolar organoids clonally expand and pattern complex, polarized alveolar-like cavities. (A) Schematic of experimental design and overview. Live/CD31 - /CD45 - /CD326 + (EpCAM + )/TdTomato + (Axin2 + ) cells (AEPs) were mixed with mouse lung fibroblasts from P28 mice and cultured for up to 35 days, followed by analysis via high content imaging. (B) H E of 5 μm sections of FFPE day 35 Axin2 + organoids, showing cellular morphologies typical of both AT1 and AT2 cells. (C-G) Whole-mount immunofluorescence time course of Axin2 + organoids showing expansion of SFTPC + AT2 cells (red), increased differentiation into RAGE + AT1 cells (green) and increased structural complexity. (H) Imaris 3D reconstruction of day 35 Axin2 + organoid (z-depth = 174.13 μm) showing cellular arrangement/organization within mature organoids. (I) Click-iT EdU (green) whole-mount day 25 Axin2 + organoids, with proliferating cells primarily on outer edges or ‘buds’ growing outward from the organoid. (J-K) Electron microscopy of day 28 organoids. (J) Image of properly polarized AT2 cell with apical microvilli (black arrowhead) secreting surfactant (blue arrowhead) into a lumen. (K) Image of AT2 cell with lamellar bodies (black arrowhead) adjacent to an AT1 cell (green arrowhead, right). (L-M) Comparison of in vivo mouse lung (9-month C57BL/6J mouse) and in vitro day 25 Axin2 + organoids. [ Scale bars = 50 μm, except for electron microscopy (J, K) scale bars = 2 . 5 μm ]. ( RAGE = Receptor for Advanced Glycation End-products [AT1 cell marker]; SFTPC = Surfactant Protein C [AT2 cell marker]; EdU = 5-ethynyl-2’-deoxyuridine; FFPE = formalin-fixed, paraffin-embedded )

    Techniques Used: Derivative Assay, Mouse Assay, Cell Culture, Imaging, Formalin-fixed Paraffin-Embedded, Immunofluorescence, Electron Microscopy, In Vivo, In Vitro, Marker

    In vitro Nkx2-1 KO of AEP-derived alveolar organoids drives irreversible transition to a Krt8 + stressed transitional state. (A) AAV6.2FF-Cre experimental set-up. Live/CD31 - /CD45 - /CD326 + (EpCAM + )/TdTomato + (Axin2 + ) cells (AEPs) sorted from Axin2 creERT2-tDT ; R26R EYFP mice and Axin2 creERT2-tDT ; R26R EYFP ; Nkx2-1 fl/fl mice were treated with AAV6.2FF-Cre and plated with wild-type fibroblasts. (B) Comparison of brightfield and GFP whole-well images of organoids grown from control (AAV6.2FF-Cre-treated sorted R26R EYFP AEPs) and Nkx2-1 KO AEPs (AAV6.2FF-Cre-treated sorted R26R EYFP ; Nkx2-1 fl/fl AEPs) at day 28 of culture. Control (non-GFP) organoids with normal morphology are marked with white asterisk. (C-J) H E and immunofluorescence images of R26R EYFP ; Nkx2-1 fl/fl AEP-derived organoids that did (F-J) or did not (C-E) undergo recombination via AAV6.2FF-Cre. (C-E) Non-recombined organoids (D) express SPC (red) and Nkx2-1 (white), but do not express the YFP lineage label (green), whereas (G) recombined organoids do not express SPC or Nkx2-1 but do express the YFP lineage label. Non-recombined (E) and recombined (H) organoids maintain epithelial identify expressing CDH1. Nkx2-1 KO organoids express KRT8 and many proliferate and express Ki67 at late as day 40 of culture (J-J’’). (K-R) Integrated scRNAseq comparing epithelial cells from day 28 control organoids (Uninfected), AAV6.2FF-Cre-treated control organoids (AAV control), and AAV6.2FF-Cre-treated Nkx2-1 KO organoids (Nkx2-1 KO ). Nkx2-1 KO cells cluster separately from Uninfected and AAV control cells near Krt8 + cells (K-L), which make up a majority of cells in the Nkx2-1 KO condition (M). Marker genes for normal alveolar epithelium are lost and novel markers gained (N) in Nkx2-1 KO . (O-R) Module scoring using published gene sets for AEPs (O), Krt8/PATS/DATP/ADI cells (P), lung cancer cells (Q), and foregut endoderm (R). Compare to Figure S7 for detailed marker gene analysis.
    Figure Legend Snippet: In vitro Nkx2-1 KO of AEP-derived alveolar organoids drives irreversible transition to a Krt8 + stressed transitional state. (A) AAV6.2FF-Cre experimental set-up. Live/CD31 - /CD45 - /CD326 + (EpCAM + )/TdTomato + (Axin2 + ) cells (AEPs) sorted from Axin2 creERT2-tDT ; R26R EYFP mice and Axin2 creERT2-tDT ; R26R EYFP ; Nkx2-1 fl/fl mice were treated with AAV6.2FF-Cre and plated with wild-type fibroblasts. (B) Comparison of brightfield and GFP whole-well images of organoids grown from control (AAV6.2FF-Cre-treated sorted R26R EYFP AEPs) and Nkx2-1 KO AEPs (AAV6.2FF-Cre-treated sorted R26R EYFP ; Nkx2-1 fl/fl AEPs) at day 28 of culture. Control (non-GFP) organoids with normal morphology are marked with white asterisk. (C-J) H E and immunofluorescence images of R26R EYFP ; Nkx2-1 fl/fl AEP-derived organoids that did (F-J) or did not (C-E) undergo recombination via AAV6.2FF-Cre. (C-E) Non-recombined organoids (D) express SPC (red) and Nkx2-1 (white), but do not express the YFP lineage label (green), whereas (G) recombined organoids do not express SPC or Nkx2-1 but do express the YFP lineage label. Non-recombined (E) and recombined (H) organoids maintain epithelial identify expressing CDH1. Nkx2-1 KO organoids express KRT8 and many proliferate and express Ki67 at late as day 40 of culture (J-J’’). (K-R) Integrated scRNAseq comparing epithelial cells from day 28 control organoids (Uninfected), AAV6.2FF-Cre-treated control organoids (AAV control), and AAV6.2FF-Cre-treated Nkx2-1 KO organoids (Nkx2-1 KO ). Nkx2-1 KO cells cluster separately from Uninfected and AAV control cells near Krt8 + cells (K-L), which make up a majority of cells in the Nkx2-1 KO condition (M). Marker genes for normal alveolar epithelium are lost and novel markers gained (N) in Nkx2-1 KO . (O-R) Module scoring using published gene sets for AEPs (O), Krt8/PATS/DATP/ADI cells (P), lung cancer cells (Q), and foregut endoderm (R). Compare to Figure S7 for detailed marker gene analysis.

    Techniques Used: In Vitro, Derivative Assay, Mouse Assay, Immunofluorescence, Expressing, Marker

    6) Product Images from "PI3K signaling specifies proximal-distal fate by driving a developmental gene regulatory network in SOX9+ mouse lung progenitors"

    Article Title: PI3K signaling specifies proximal-distal fate by driving a developmental gene regulatory network in SOX9+ mouse lung progenitors

    Journal: eLife

    doi: 10.7554/eLife.67954

    FACS sorting strategy for isolation of Sox9+EPC cells. SOX9+lung epithelial progenitor cells were isolated by cell sorting CD326+/CD31-/CD45-/7-AAD-/GFP + cells isolated from Sox9-GFP reporter embryos at E11.5 and E16.5. Key single stain controls are shown.
    Figure Legend Snippet: FACS sorting strategy for isolation of Sox9+EPC cells. SOX9+lung epithelial progenitor cells were isolated by cell sorting CD326+/CD31-/CD45-/7-AAD-/GFP + cells isolated from Sox9-GFP reporter embryos at E11.5 and E16.5. Key single stain controls are shown.

    Techniques Used: FACS, Isolation, Staining

    7) Product Images from "PDGFRβ+ cells play a dual role as hematopoietic precursors and niche cells during mouse ontogeny"

    Article Title: PDGFRβ+ cells play a dual role as hematopoietic precursors and niche cells during mouse ontogeny

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2022.111114

    A subset of ECs, HEC/EHT, and IAHCs derived from PDGFRβ + precursors (A–D) Immunohistochemistry of E11 PDGFRβ-Cre:mTmG AGM stained with NG2 (A and B) and CD31 (C) and at E10 with Runx1 (D). (A and B) Arrowheads: DP cells; stars: GFP + mesenchymal cells. (C) Dashed lines: presumptive separation between DP, PDGFRβ-S, and DN layers; stars: hematopoietic CD31 + GFP + cells. (E–I) Flow cytometry analyses of E10 (n = 7) and E11 (n = 3) PDGFRβ-Cre; tdTomato AGMs, percentage of Tomato +/− cells within (E) PDGFRβ + cells, (F) EC (CD45 − cKit − CD31 + CD41 − ), (G) HEC/EHT-enriched population (CD45 − cKit − CD31 + CD41 + ), (H) IAHC/HSPC (CD31 + cKit + ), and (I) MP-enriched population (CD45 + CD31 − ) live cells; error bars: SD; ∗p
    Figure Legend Snippet: A subset of ECs, HEC/EHT, and IAHCs derived from PDGFRβ + precursors (A–D) Immunohistochemistry of E11 PDGFRβ-Cre:mTmG AGM stained with NG2 (A and B) and CD31 (C) and at E10 with Runx1 (D). (A and B) Arrowheads: DP cells; stars: GFP + mesenchymal cells. (C) Dashed lines: presumptive separation between DP, PDGFRβ-S, and DN layers; stars: hematopoietic CD31 + GFP + cells. (E–I) Flow cytometry analyses of E10 (n = 7) and E11 (n = 3) PDGFRβ-Cre; tdTomato AGMs, percentage of Tomato +/− cells within (E) PDGFRβ + cells, (F) EC (CD45 − cKit − CD31 + CD41 − ), (G) HEC/EHT-enriched population (CD45 − cKit − CD31 + CD41 + ), (H) IAHC/HSPC (CD31 + cKit + ), and (I) MP-enriched population (CD45 + CD31 − ) live cells; error bars: SD; ∗p

    Techniques Used: Derivative Assay, Immunohistochemistry, Staining, Flow Cytometry

    Distinct phenotypic and transcriptomic perivascular cell subsets surround the midgestation DA (A) Immunohistochemistry of E11 WT DA, showing NG2 (i, ii), PDGFRβ (i, ii), and CD31 (ii) expression. Nuclei were counterstained with DAPI. CV, cardinal veins; NC, notochord. (B) t-distributed stochastic neighbor embedding (t-SNE) plots showing 12 E11 WT cell populations and their numbers (42sp). (C) Violin plots showing the expression of genes used to identify the cell clusters DP, PDGFRβ-S, DN, and NG2-S. MP, macrophages; OBC, other blood cells; Ery/EryP, erythroid/progenitors; IAHC, intra-aortic hematopoietic clusters; HEC/EHT, hemogenic endothelial cells/endothelial-to-hematopoietic transition; EC, endothelial cells; SNS, sympathetic nervous system; SkMP, skeletal muscle progenitors. (D and E) Heatmaps showing gene expression of differentially expressed genes encoding surface (D) and intra-/extracellular (E) proteins enriched in each of the NG2 +/− PDGFRβ +/− populations. (F) Isolation of perivascular cells. (G) Fragments per kilobase of transcript per million mapped reads (FPKM) values of selected genes by bulk RNA-seq.
    Figure Legend Snippet: Distinct phenotypic and transcriptomic perivascular cell subsets surround the midgestation DA (A) Immunohistochemistry of E11 WT DA, showing NG2 (i, ii), PDGFRβ (i, ii), and CD31 (ii) expression. Nuclei were counterstained with DAPI. CV, cardinal veins; NC, notochord. (B) t-distributed stochastic neighbor embedding (t-SNE) plots showing 12 E11 WT cell populations and their numbers (42sp). (C) Violin plots showing the expression of genes used to identify the cell clusters DP, PDGFRβ-S, DN, and NG2-S. MP, macrophages; OBC, other blood cells; Ery/EryP, erythroid/progenitors; IAHC, intra-aortic hematopoietic clusters; HEC/EHT, hemogenic endothelial cells/endothelial-to-hematopoietic transition; EC, endothelial cells; SNS, sympathetic nervous system; SkMP, skeletal muscle progenitors. (D and E) Heatmaps showing gene expression of differentially expressed genes encoding surface (D) and intra-/extracellular (E) proteins enriched in each of the NG2 +/− PDGFRβ +/− populations. (F) Isolation of perivascular cells. (G) Fragments per kilobase of transcript per million mapped reads (FPKM) values of selected genes by bulk RNA-seq.

    Techniques Used: Immunohistochemistry, Expressing, Isolation, RNA Sequencing Assay

    PDGFRβ + cells from E7.5–E9.5 mouse embryo contribute to peri/vascular/hematopoietic lineages present in the E11.5 AGM (A) PDGFRβ-Cre-derived cell tracing. (B and C) Immunohistochemistry on PDGFRβ-P2A-CreERT2; mTmG E11 AGMs showing CD31 and GFP (B) and Runx1 and GFP (C). (D–G) Flow cytometry plots (D and F) and quantification (E and G) of GFP or Tomato expression within Ter119 + cells from E11 AGM (D and E) (n = 5) and YS (n = 9) (F and G); error bars: SD; ∗∗∗∗ p
    Figure Legend Snippet: PDGFRβ + cells from E7.5–E9.5 mouse embryo contribute to peri/vascular/hematopoietic lineages present in the E11.5 AGM (A) PDGFRβ-Cre-derived cell tracing. (B and C) Immunohistochemistry on PDGFRβ-P2A-CreERT2; mTmG E11 AGMs showing CD31 and GFP (B) and Runx1 and GFP (C). (D–G) Flow cytometry plots (D and F) and quantification (E and G) of GFP or Tomato expression within Ter119 + cells from E11 AGM (D and E) (n = 5) and YS (n = 9) (F and G); error bars: SD; ∗∗∗∗ p

    Techniques Used: Derivative Assay, Immunohistochemistry, Flow Cytometry, Expressing

    8) Product Images from "PDGFRβ+ cells play a dual role as hematopoietic precursors and niche cells during mouse ontogeny"

    Article Title: PDGFRβ+ cells play a dual role as hematopoietic precursors and niche cells during mouse ontogeny

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2022.111114

    A subset of ECs, HEC/EHT, and IAHCs derived from PDGFRβ + precursors (A–D) Immunohistochemistry of E11 PDGFRβ-Cre:mTmG AGM stained with NG2 (A and B) and CD31 (C) and at E10 with Runx1 (D). (A and B) Arrowheads: DP cells; stars: GFP + mesenchymal cells. (C) Dashed lines: presumptive separation between DP, PDGFRβ-S, and DN layers; stars: hematopoietic CD31 + GFP + cells. (E–I) Flow cytometry analyses of E10 (n = 7) and E11 (n = 3) PDGFRβ-Cre; tdTomato AGMs, percentage of Tomato +/− cells within (E) PDGFRβ + cells, (F) EC (CD45 − cKit − CD31 + CD41 − ), (G) HEC/EHT-enriched population (CD45 − cKit − CD31 + CD41 + ), (H) IAHC/HSPC (CD31 + cKit + ), and (I) MP-enriched population (CD45 + CD31 − ) live cells; error bars: SD; ∗p
    Figure Legend Snippet: A subset of ECs, HEC/EHT, and IAHCs derived from PDGFRβ + precursors (A–D) Immunohistochemistry of E11 PDGFRβ-Cre:mTmG AGM stained with NG2 (A and B) and CD31 (C) and at E10 with Runx1 (D). (A and B) Arrowheads: DP cells; stars: GFP + mesenchymal cells. (C) Dashed lines: presumptive separation between DP, PDGFRβ-S, and DN layers; stars: hematopoietic CD31 + GFP + cells. (E–I) Flow cytometry analyses of E10 (n = 7) and E11 (n = 3) PDGFRβ-Cre; tdTomato AGMs, percentage of Tomato +/− cells within (E) PDGFRβ + cells, (F) EC (CD45 − cKit − CD31 + CD41 − ), (G) HEC/EHT-enriched population (CD45 − cKit − CD31 + CD41 + ), (H) IAHC/HSPC (CD31 + cKit + ), and (I) MP-enriched population (CD45 + CD31 − ) live cells; error bars: SD; ∗p

    Techniques Used: Derivative Assay, Immunohistochemistry, Staining, Flow Cytometry

    Distinct phenotypic and transcriptomic perivascular cell subsets surround the midgestation DA (A) Immunohistochemistry of E11 WT DA, showing NG2 (i, ii), PDGFRβ (i, ii), and CD31 (ii) expression. Nuclei were counterstained with DAPI. CV, cardinal veins; NC, notochord. (B) t-distributed stochastic neighbor embedding (t-SNE) plots showing 12 E11 WT cell populations and their numbers (42sp). (C) Violin plots showing the expression of genes used to identify the cell clusters DP, PDGFRβ-S, DN, and NG2-S. MP, macrophages; OBC, other blood cells; Ery/EryP, erythroid/progenitors; IAHC, intra-aortic hematopoietic clusters; HEC/EHT, hemogenic endothelial cells/endothelial-to-hematopoietic transition; EC, endothelial cells; SNS, sympathetic nervous system; SkMP, skeletal muscle progenitors. (D and E) Heatmaps showing gene expression of differentially expressed genes encoding surface (D) and intra-/extracellular (E) proteins enriched in each of the NG2 +/− PDGFRβ +/− populations. (F) Isolation of perivascular cells. (G) Fragments per kilobase of transcript per million mapped reads (FPKM) values of selected genes by bulk RNA-seq.
    Figure Legend Snippet: Distinct phenotypic and transcriptomic perivascular cell subsets surround the midgestation DA (A) Immunohistochemistry of E11 WT DA, showing NG2 (i, ii), PDGFRβ (i, ii), and CD31 (ii) expression. Nuclei were counterstained with DAPI. CV, cardinal veins; NC, notochord. (B) t-distributed stochastic neighbor embedding (t-SNE) plots showing 12 E11 WT cell populations and their numbers (42sp). (C) Violin plots showing the expression of genes used to identify the cell clusters DP, PDGFRβ-S, DN, and NG2-S. MP, macrophages; OBC, other blood cells; Ery/EryP, erythroid/progenitors; IAHC, intra-aortic hematopoietic clusters; HEC/EHT, hemogenic endothelial cells/endothelial-to-hematopoietic transition; EC, endothelial cells; SNS, sympathetic nervous system; SkMP, skeletal muscle progenitors. (D and E) Heatmaps showing gene expression of differentially expressed genes encoding surface (D) and intra-/extracellular (E) proteins enriched in each of the NG2 +/− PDGFRβ +/− populations. (F) Isolation of perivascular cells. (G) Fragments per kilobase of transcript per million mapped reads (FPKM) values of selected genes by bulk RNA-seq.

    Techniques Used: Immunohistochemistry, Expressing, Isolation, RNA Sequencing Assay

    PDGFRβ + cells from E7.5–E9.5 mouse embryo contribute to peri/vascular/hematopoietic lineages present in the E11.5 AGM (A) PDGFRβ-Cre-derived cell tracing. (B and C) Immunohistochemistry on PDGFRβ-P2A-CreERT2; mTmG E11 AGMs showing CD31 and GFP (B) and Runx1 and GFP (C). (D–G) Flow cytometry plots (D and F) and quantification (E and G) of GFP or Tomato expression within Ter119 + cells from E11 AGM (D and E) (n = 5) and YS (n = 9) (F and G); error bars: SD; ∗∗∗∗ p
    Figure Legend Snippet: PDGFRβ + cells from E7.5–E9.5 mouse embryo contribute to peri/vascular/hematopoietic lineages present in the E11.5 AGM (A) PDGFRβ-Cre-derived cell tracing. (B and C) Immunohistochemistry on PDGFRβ-P2A-CreERT2; mTmG E11 AGMs showing CD31 and GFP (B) and Runx1 and GFP (C). (D–G) Flow cytometry plots (D and F) and quantification (E and G) of GFP or Tomato expression within Ter119 + cells from E11 AGM (D and E) (n = 5) and YS (n = 9) (F and G); error bars: SD; ∗∗∗∗ p

    Techniques Used: Derivative Assay, Immunohistochemistry, Flow Cytometry, Expressing

    9) Product Images from "Senescence-induced endothelial phenotypes underpin immune-mediated senescence surveillance"

    Article Title: Senescence-induced endothelial phenotypes underpin immune-mediated senescence surveillance

    Journal: Genes & Development

    doi: 10.1101/gad.349585.122

    Senescence-induced canonical NF-κB signaling in endothelial cells regulates downstream signaling and lymphocyte recruitment. ( A ) Experimental setup: direct coculture of growing or RIS ER:HRAS G12V IMR90 cells (asterisks) with HUVECs (arrowheads) expressing the IκBα superrepressor (SR) or vector control. ( B ) Representative immunofluorescence of coculture with senescence-dependent IL8 expression in both CD31 − IMR90s and CD31 + HUVECs. n = 5 biological replicates. Scale bar, 30 µm. ( C ) Separate quantification of IL8 positivity from the two cell types. Dots are individual replicates, and bars are means. Data were analyzed by one-way ANOVA with Sidak's multiple comparisons test; (****) P ≤ 0.0001. ( D ) Experimental setup: HUVECs expressing the SR or vector control were incubated in CM from growing or RIS ER:HRAS G12V IMR90 cells for 16 h before harvesting for flow cytometry for ICAM1 ( middle panel) and ICOSLG ( right panel). n ≥ 3 biological replicates. ( E ) Experimental setup: HUVECs were incubated in CM from growing or RIS ER:HRAS G12V IMR90 cells with vehicle or the indicated NF-κB inhibitors for 16 h before harvesting for flow cytometry for ICAM1. n ≥ 3 biological replicates. ( F ) Experimental setup: LSECs or HUVECs with the indicated genetic or pharmacological NF-κB inhibitors were incubated in CM from growing or RIS ER:HRAS G12V IMR90 cells for 16 h before performing a flow adhesion assay and analyses of lymphocyte adherence and trans -endothelial migration. ( G ) Representative photomicrographs of HUVECs with the indicated constructs and CM, showing adherent lymphocytes (black arrows). ( H ) Trans -endothelial migration of lymphocytes in the indicated cell lines and conditions. Dots are individual replicates, and bars are means. Data were analyzed by one-way ANOVA with Sidak's multiple comparisons test; (**) P ≤ 0.01, (****) P ≤ 0.0001.
    Figure Legend Snippet: Senescence-induced canonical NF-κB signaling in endothelial cells regulates downstream signaling and lymphocyte recruitment. ( A ) Experimental setup: direct coculture of growing or RIS ER:HRAS G12V IMR90 cells (asterisks) with HUVECs (arrowheads) expressing the IκBα superrepressor (SR) or vector control. ( B ) Representative immunofluorescence of coculture with senescence-dependent IL8 expression in both CD31 − IMR90s and CD31 + HUVECs. n = 5 biological replicates. Scale bar, 30 µm. ( C ) Separate quantification of IL8 positivity from the two cell types. Dots are individual replicates, and bars are means. Data were analyzed by one-way ANOVA with Sidak's multiple comparisons test; (****) P ≤ 0.0001. ( D ) Experimental setup: HUVECs expressing the SR or vector control were incubated in CM from growing or RIS ER:HRAS G12V IMR90 cells for 16 h before harvesting for flow cytometry for ICAM1 ( middle panel) and ICOSLG ( right panel). n ≥ 3 biological replicates. ( E ) Experimental setup: HUVECs were incubated in CM from growing or RIS ER:HRAS G12V IMR90 cells with vehicle or the indicated NF-κB inhibitors for 16 h before harvesting for flow cytometry for ICAM1. n ≥ 3 biological replicates. ( F ) Experimental setup: LSECs or HUVECs with the indicated genetic or pharmacological NF-κB inhibitors were incubated in CM from growing or RIS ER:HRAS G12V IMR90 cells for 16 h before performing a flow adhesion assay and analyses of lymphocyte adherence and trans -endothelial migration. ( G ) Representative photomicrographs of HUVECs with the indicated constructs and CM, showing adherent lymphocytes (black arrows). ( H ) Trans -endothelial migration of lymphocytes in the indicated cell lines and conditions. Dots are individual replicates, and bars are means. Data were analyzed by one-way ANOVA with Sidak's multiple comparisons test; (**) P ≤ 0.01, (****) P ≤ 0.0001.

    Techniques Used: Expressing, Plasmid Preparation, Immunofluorescence, Incubation, Flow Cytometry, Cell Adhesion Assay, Migration, Construct

    10) Product Images from "Recruitment of α4β7 monocytes and neutrophils to the brain in experimental colitis is associated with elevated cytokines and anxiety-like behavior"

    Article Title: Recruitment of α4β7 monocytes and neutrophils to the brain in experimental colitis is associated with elevated cytokines and anxiety-like behavior

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-022-02431-z

    Colitis induces the rolling and adhering of leukocytes in cerebral endothelial cells. Intravital microscopy was performed using a spinning disc confocal microscope with a 20X/0.95 NA water objective. Videos were captured and analyzed to identify rolling and adhering leukocytes in control and colitic female mice. A Colitic mice showed a significant increase in rolling ( t = 2.8, df 11, * P = 0.02; n = 6–7 mice/group) and adhering ( t = 3.1, df 11, * P = 0.01; n = 6–7 mice/group) leukocytes on cerebral endothelial cells. B Representative images of intravital imaging. CD31 was used to label cerebral endothelial cells (blue), Rho6G was used to label leukocytes (red). Scale bar: 25 µm
    Figure Legend Snippet: Colitis induces the rolling and adhering of leukocytes in cerebral endothelial cells. Intravital microscopy was performed using a spinning disc confocal microscope with a 20X/0.95 NA water objective. Videos were captured and analyzed to identify rolling and adhering leukocytes in control and colitic female mice. A Colitic mice showed a significant increase in rolling ( t = 2.8, df 11, * P = 0.02; n = 6–7 mice/group) and adhering ( t = 3.1, df 11, * P = 0.01; n = 6–7 mice/group) leukocytes on cerebral endothelial cells. B Representative images of intravital imaging. CD31 was used to label cerebral endothelial cells (blue), Rho6G was used to label leukocytes (red). Scale bar: 25 µm

    Techniques Used: Intravital Microscopy, Microscopy, Mouse Assay, Imaging

    11) Product Images from "Isolation and characterization of peritoneal microvascular pericytes"

    Article Title: Isolation and characterization of peritoneal microvascular pericytes

    Journal: FEBS Open Bio

    doi: 10.1002/2211-5463.13386

    Anatomical location of mouse mesenteric pericytes. (A) Schematic manifestation of pericytes and vascular endothelial cells in microvasculature. (B) Mesenteric tissue extracted from the mouse bowel system (displayed on glass slides). Scale bar = 1 cm. (C) Multiplex immunofluorescence staining with anti‐CD31 and anti‐PDGFR‐β antibodies to validate mouse mesenteric pericytes. Original magnification: 50×. Scale bar = 50 μm.
    Figure Legend Snippet: Anatomical location of mouse mesenteric pericytes. (A) Schematic manifestation of pericytes and vascular endothelial cells in microvasculature. (B) Mesenteric tissue extracted from the mouse bowel system (displayed on glass slides). Scale bar = 1 cm. (C) Multiplex immunofluorescence staining with anti‐CD31 and anti‐PDGFR‐β antibodies to validate mouse mesenteric pericytes. Original magnification: 50×. Scale bar = 50 μm.

    Techniques Used: Multiplex Assay, Immunofluorescence, Staining

    Flow cytometry was used to determine the purity of primary pericytes enriched by different methods. (A) Cells collected by enzymatic digestion alone. (B) Cells screened after pericyte conditional medium. (C) Copurification by conditional culture combined with MACs. (D) The rate of PDGFR‐β + cells (left) and PDGFR‐β + /CD31 – cells (right) in different protocols ( n = 3 for each group). Values are expressed as the mean ± SEM. **P
    Figure Legend Snippet: Flow cytometry was used to determine the purity of primary pericytes enriched by different methods. (A) Cells collected by enzymatic digestion alone. (B) Cells screened after pericyte conditional medium. (C) Copurification by conditional culture combined with MACs. (D) The rate of PDGFR‐β + cells (left) and PDGFR‐β + /CD31 – cells (right) in different protocols ( n = 3 for each group). Values are expressed as the mean ± SEM. **P

    Techniques Used: Flow Cytometry, Copurification, Magnetic Cell Separation

    12) Product Images from "Tet-mediated DNA demethylation regulates specification of hematopoietic stem and progenitor cells during mammalian embryogenesis"

    Article Title: Tet-mediated DNA demethylation regulates specification of hematopoietic stem and progenitor cells during mammalian embryogenesis

    Journal: Science Advances

    doi: 10.1126/sciadv.abm3470

    Absence of CD31 + c-Kit + hematopoietic clusters and floating HSPCs in AGMs of Tet-deficient embryos. ( A ) Whole-mount three-dimensional (3D) images of E11.5 AGM of indicated genotypes stained with CD31 and c-Kit antibodies. CD31 + ECs (magenta) and c-Kit + HSPCs (green) within the dorsal aorta region are shown at ×10 magnification. Inset: CD31 and c-Kit double-positive hematopoietic (Hem.) clusters are shown at ×100 magnification. ( B ) Whole-mount 3D images of E11.5 FLs of indicated genotypes stained with CD31 and c-Kit antibodies. CD31 + ECs (magenta) and c-Kit + HSPCs (green) are shown at ×40 magnification.
    Figure Legend Snippet: Absence of CD31 + c-Kit + hematopoietic clusters and floating HSPCs in AGMs of Tet-deficient embryos. ( A ) Whole-mount three-dimensional (3D) images of E11.5 AGM of indicated genotypes stained with CD31 and c-Kit antibodies. CD31 + ECs (magenta) and c-Kit + HSPCs (green) within the dorsal aorta region are shown at ×10 magnification. Inset: CD31 and c-Kit double-positive hematopoietic (Hem.) clusters are shown at ×100 magnification. ( B ) Whole-mount 3D images of E11.5 FLs of indicated genotypes stained with CD31 and c-Kit antibodies. CD31 + ECs (magenta) and c-Kit + HSPCs (green) are shown at ×40 magnification.

    Techniques Used: Staining

    Loss of Tet1/2/3 early in embryogenesis leads to aberrant hematopoiesis. ( A ) Representative gross images of E10.5 to E14.5 embryos of indicated genotypes treated with tamoxifen at E7.5. Total number of normal and dead embryos for each developmental stage is summarized in the table. ( B ) Representative gross images of E11.5 embryos of indicated genotypes attached to yolk sac (YS) and placenta treated with tamoxifen at E7.5. Note the lack of blood in the Tet1/2/3 f/f ;R26 +/CreER YSs. ( C ) Representative gross images of E11.5 embryonic livers of indicated genotypes treated with tamoxifen at E7.5. Note the smaller size and lack of blood in the Tet1/2/3 f/f ;R26 +/CreER fetal liver (FL). ( D ) Quantitation of % HSPCs and % ECs in E9.5 YS and E11.5 AGM of Tet1/2/3 f/f ;R26 +/CreER and littermate control Tet1/2/3 f/f ;R26 +/+ embryos by flow cytometry. E9.5 YS HSPCs (Lin − CD31 + c-Kit + ), ECs (Lin − CD31 + c-Kit − ), E11.5 AGM HSPCs (CD31 + c-Kit + ), and ECs (CD31 + c-Kit − ). Lin, lineage. ( E and F ) Quantitation of the percent and total number of CD45 + hematopoietic cells and Ter-119 + erythrocytes in E11.5 FL of Tet1/2/3 f/f ;R26 +/CreER and littermate control Tet1/2/3 f/f ;R26 +/+ embryos by flow cytometry. For all panels, data are presented as means ± SEM. Statistically significant (* P
    Figure Legend Snippet: Loss of Tet1/2/3 early in embryogenesis leads to aberrant hematopoiesis. ( A ) Representative gross images of E10.5 to E14.5 embryos of indicated genotypes treated with tamoxifen at E7.5. Total number of normal and dead embryos for each developmental stage is summarized in the table. ( B ) Representative gross images of E11.5 embryos of indicated genotypes attached to yolk sac (YS) and placenta treated with tamoxifen at E7.5. Note the lack of blood in the Tet1/2/3 f/f ;R26 +/CreER YSs. ( C ) Representative gross images of E11.5 embryonic livers of indicated genotypes treated with tamoxifen at E7.5. Note the smaller size and lack of blood in the Tet1/2/3 f/f ;R26 +/CreER fetal liver (FL). ( D ) Quantitation of % HSPCs and % ECs in E9.5 YS and E11.5 AGM of Tet1/2/3 f/f ;R26 +/CreER and littermate control Tet1/2/3 f/f ;R26 +/+ embryos by flow cytometry. E9.5 YS HSPCs (Lin − CD31 + c-Kit + ), ECs (Lin − CD31 + c-Kit − ), E11.5 AGM HSPCs (CD31 + c-Kit + ), and ECs (CD31 + c-Kit − ). Lin, lineage. ( E and F ) Quantitation of the percent and total number of CD45 + hematopoietic cells and Ter-119 + erythrocytes in E11.5 FL of Tet1/2/3 f/f ;R26 +/CreER and littermate control Tet1/2/3 f/f ;R26 +/+ embryos by flow cytometry. For all panels, data are presented as means ± SEM. Statistically significant (* P

    Techniques Used: Quantitation Assay, Flow Cytometry

    13) Product Images from "Tet-mediated DNA demethylation regulates specification of hematopoietic stem and progenitor cells during mammalian embryogenesis"

    Article Title: Tet-mediated DNA demethylation regulates specification of hematopoietic stem and progenitor cells during mammalian embryogenesis

    Journal: Science Advances

    doi: 10.1126/sciadv.abm3470

    Absence of CD31 + c-Kit + hematopoietic clusters and floating HSPCs in AGMs of Tet-deficient embryos. ( A ) Whole-mount three-dimensional (3D) images of E11.5 AGM of indicated genotypes stained with CD31 and c-Kit antibodies. CD31 + ECs (magenta) and c-Kit + HSPCs (green) within the dorsal aorta region are shown at ×10 magnification. Inset: CD31 and c-Kit double-positive hematopoietic (Hem.) clusters are shown at ×100 magnification. ( B ) Whole-mount 3D images of E11.5 FLs of indicated genotypes stained with CD31 and c-Kit antibodies. CD31 + ECs (magenta) and c-Kit + HSPCs (green) are shown at ×40 magnification.
    Figure Legend Snippet: Absence of CD31 + c-Kit + hematopoietic clusters and floating HSPCs in AGMs of Tet-deficient embryos. ( A ) Whole-mount three-dimensional (3D) images of E11.5 AGM of indicated genotypes stained with CD31 and c-Kit antibodies. CD31 + ECs (magenta) and c-Kit + HSPCs (green) within the dorsal aorta region are shown at ×10 magnification. Inset: CD31 and c-Kit double-positive hematopoietic (Hem.) clusters are shown at ×100 magnification. ( B ) Whole-mount 3D images of E11.5 FLs of indicated genotypes stained with CD31 and c-Kit antibodies. CD31 + ECs (magenta) and c-Kit + HSPCs (green) are shown at ×40 magnification.

    Techniques Used: Staining

    Loss of Tet1/2/3 early in embryogenesis leads to aberrant hematopoiesis. ( A ) Representative gross images of E10.5 to E14.5 embryos of indicated genotypes treated with tamoxifen at E7.5. Total number of normal and dead embryos for each developmental stage is summarized in the table. ( B ) Representative gross images of E11.5 embryos of indicated genotypes attached to yolk sac (YS) and placenta treated with tamoxifen at E7.5. Note the lack of blood in the Tet1/2/3 f/f ;R26 +/CreER YSs. ( C ) Representative gross images of E11.5 embryonic livers of indicated genotypes treated with tamoxifen at E7.5. Note the smaller size and lack of blood in the Tet1/2/3 f/f ;R26 +/CreER fetal liver (FL). ( D ) Quantitation of % HSPCs and % ECs in E9.5 YS and E11.5 AGM of Tet1/2/3 f/f ;R26 +/CreER and littermate control Tet1/2/3 f/f ;R26 +/+ embryos by flow cytometry. E9.5 YS HSPCs (Lin − CD31 + c-Kit + ), ECs (Lin − CD31 + c-Kit − ), E11.5 AGM HSPCs (CD31 + c-Kit + ), and ECs (CD31 + c-Kit − ). Lin, lineage. ( E and F ) Quantitation of the percent and total number of CD45 + hematopoietic cells and Ter-119 + erythrocytes in E11.5 FL of Tet1/2/3 f/f ;R26 +/CreER and littermate control Tet1/2/3 f/f ;R26 +/+ embryos by flow cytometry. For all panels, data are presented as means ± SEM. Statistically significant (* P
    Figure Legend Snippet: Loss of Tet1/2/3 early in embryogenesis leads to aberrant hematopoiesis. ( A ) Representative gross images of E10.5 to E14.5 embryos of indicated genotypes treated with tamoxifen at E7.5. Total number of normal and dead embryos for each developmental stage is summarized in the table. ( B ) Representative gross images of E11.5 embryos of indicated genotypes attached to yolk sac (YS) and placenta treated with tamoxifen at E7.5. Note the lack of blood in the Tet1/2/3 f/f ;R26 +/CreER YSs. ( C ) Representative gross images of E11.5 embryonic livers of indicated genotypes treated with tamoxifen at E7.5. Note the smaller size and lack of blood in the Tet1/2/3 f/f ;R26 +/CreER fetal liver (FL). ( D ) Quantitation of % HSPCs and % ECs in E9.5 YS and E11.5 AGM of Tet1/2/3 f/f ;R26 +/CreER and littermate control Tet1/2/3 f/f ;R26 +/+ embryos by flow cytometry. E9.5 YS HSPCs (Lin − CD31 + c-Kit + ), ECs (Lin − CD31 + c-Kit − ), E11.5 AGM HSPCs (CD31 + c-Kit + ), and ECs (CD31 + c-Kit − ). Lin, lineage. ( E and F ) Quantitation of the percent and total number of CD45 + hematopoietic cells and Ter-119 + erythrocytes in E11.5 FL of Tet1/2/3 f/f ;R26 +/CreER and littermate control Tet1/2/3 f/f ;R26 +/+ embryos by flow cytometry. For all panels, data are presented as means ± SEM. Statistically significant (* P

    Techniques Used: Quantitation Assay, Flow Cytometry

    14) Product Images from "Tet-mediated DNA demethylation regulates specification of hematopoietic stem and progenitor cells during mammalian embryogenesis"

    Article Title: Tet-mediated DNA demethylation regulates specification of hematopoietic stem and progenitor cells during mammalian embryogenesis

    Journal: Science Advances

    doi: 10.1126/sciadv.abm3470

    Absence of CD31 + c-Kit + hematopoietic clusters and floating HSPCs in AGMs of Tet-deficient embryos. ( A ) Whole-mount three-dimensional (3D) images of E11.5 AGM of indicated genotypes stained with CD31 and c-Kit antibodies. CD31 + ECs (magenta) and c-Kit + HSPCs (green) within the dorsal aorta region are shown at ×10 magnification. Inset: CD31 and c-Kit double-positive hematopoietic (Hem.) clusters are shown at ×100 magnification. ( B ) Whole-mount 3D images of E11.5 FLs of indicated genotypes stained with CD31 and c-Kit antibodies. CD31 + ECs (magenta) and c-Kit + HSPCs (green) are shown at ×40 magnification.
    Figure Legend Snippet: Absence of CD31 + c-Kit + hematopoietic clusters and floating HSPCs in AGMs of Tet-deficient embryos. ( A ) Whole-mount three-dimensional (3D) images of E11.5 AGM of indicated genotypes stained with CD31 and c-Kit antibodies. CD31 + ECs (magenta) and c-Kit + HSPCs (green) within the dorsal aorta region are shown at ×10 magnification. Inset: CD31 and c-Kit double-positive hematopoietic (Hem.) clusters are shown at ×100 magnification. ( B ) Whole-mount 3D images of E11.5 FLs of indicated genotypes stained with CD31 and c-Kit antibodies. CD31 + ECs (magenta) and c-Kit + HSPCs (green) are shown at ×40 magnification.

    Techniques Used: Staining

    Loss of Tet1/2/3 early in embryogenesis leads to aberrant hematopoiesis. ( A ) Representative gross images of E10.5 to E14.5 embryos of indicated genotypes treated with tamoxifen at E7.5. Total number of normal and dead embryos for each developmental stage is summarized in the table. ( B ) Representative gross images of E11.5 embryos of indicated genotypes attached to yolk sac (YS) and placenta treated with tamoxifen at E7.5. Note the lack of blood in the Tet1/2/3 f/f ;R26 +/CreER YSs. ( C ) Representative gross images of E11.5 embryonic livers of indicated genotypes treated with tamoxifen at E7.5. Note the smaller size and lack of blood in the Tet1/2/3 f/f ;R26 +/CreER fetal liver (FL). ( D ) Quantitation of % HSPCs and % ECs in E9.5 YS and E11.5 AGM of Tet1/2/3 f/f ;R26 +/CreER and littermate control Tet1/2/3 f/f ;R26 +/+ embryos by flow cytometry. E9.5 YS HSPCs (Lin − CD31 + c-Kit + ), ECs (Lin − CD31 + c-Kit − ), E11.5 AGM HSPCs (CD31 + c-Kit + ), and ECs (CD31 + c-Kit − ). Lin, lineage. ( E and F ) Quantitation of the percent and total number of CD45 + hematopoietic cells and Ter-119 + erythrocytes in E11.5 FL of Tet1/2/3 f/f ;R26 +/CreER and littermate control Tet1/2/3 f/f ;R26 +/+ embryos by flow cytometry. For all panels, data are presented as means ± SEM. Statistically significant (* P
    Figure Legend Snippet: Loss of Tet1/2/3 early in embryogenesis leads to aberrant hematopoiesis. ( A ) Representative gross images of E10.5 to E14.5 embryos of indicated genotypes treated with tamoxifen at E7.5. Total number of normal and dead embryos for each developmental stage is summarized in the table. ( B ) Representative gross images of E11.5 embryos of indicated genotypes attached to yolk sac (YS) and placenta treated with tamoxifen at E7.5. Note the lack of blood in the Tet1/2/3 f/f ;R26 +/CreER YSs. ( C ) Representative gross images of E11.5 embryonic livers of indicated genotypes treated with tamoxifen at E7.5. Note the smaller size and lack of blood in the Tet1/2/3 f/f ;R26 +/CreER fetal liver (FL). ( D ) Quantitation of % HSPCs and % ECs in E9.5 YS and E11.5 AGM of Tet1/2/3 f/f ;R26 +/CreER and littermate control Tet1/2/3 f/f ;R26 +/+ embryos by flow cytometry. E9.5 YS HSPCs (Lin − CD31 + c-Kit + ), ECs (Lin − CD31 + c-Kit − ), E11.5 AGM HSPCs (CD31 + c-Kit + ), and ECs (CD31 + c-Kit − ). Lin, lineage. ( E and F ) Quantitation of the percent and total number of CD45 + hematopoietic cells and Ter-119 + erythrocytes in E11.5 FL of Tet1/2/3 f/f ;R26 +/CreER and littermate control Tet1/2/3 f/f ;R26 +/+ embryos by flow cytometry. For all panels, data are presented as means ± SEM. Statistically significant (* P

    Techniques Used: Quantitation Assay, Flow Cytometry

    15) Product Images from "Tet-mediated DNA demethylation regulates specification of hematopoietic stem and progenitor cells during mammalian embryogenesis"

    Article Title: Tet-mediated DNA demethylation regulates specification of hematopoietic stem and progenitor cells during mammalian embryogenesis

    Journal: Science Advances

    doi: 10.1126/sciadv.abm3470

    Absence of CD31 + c-Kit + hematopoietic clusters and floating HSPCs in AGMs of Tet-deficient embryos. ( A ) Whole-mount three-dimensional (3D) images of E11.5 AGM of indicated genotypes stained with CD31 and c-Kit antibodies. CD31 + ECs (magenta) and c-Kit + HSPCs (green) within the dorsal aorta region are shown at ×10 magnification. Inset: CD31 and c-Kit double-positive hematopoietic (Hem.) clusters are shown at ×100 magnification. ( B ) Whole-mount 3D images of E11.5 FLs of indicated genotypes stained with CD31 and c-Kit antibodies. CD31 + ECs (magenta) and c-Kit + HSPCs (green) are shown at ×40 magnification.
    Figure Legend Snippet: Absence of CD31 + c-Kit + hematopoietic clusters and floating HSPCs in AGMs of Tet-deficient embryos. ( A ) Whole-mount three-dimensional (3D) images of E11.5 AGM of indicated genotypes stained with CD31 and c-Kit antibodies. CD31 + ECs (magenta) and c-Kit + HSPCs (green) within the dorsal aorta region are shown at ×10 magnification. Inset: CD31 and c-Kit double-positive hematopoietic (Hem.) clusters are shown at ×100 magnification. ( B ) Whole-mount 3D images of E11.5 FLs of indicated genotypes stained with CD31 and c-Kit antibodies. CD31 + ECs (magenta) and c-Kit + HSPCs (green) are shown at ×40 magnification.

    Techniques Used: Staining

    Loss of Tet1/2/3 early in embryogenesis leads to aberrant hematopoiesis. ( A ) Representative gross images of E10.5 to E14.5 embryos of indicated genotypes treated with tamoxifen at E7.5. Total number of normal and dead embryos for each developmental stage is summarized in the table. ( B ) Representative gross images of E11.5 embryos of indicated genotypes attached to yolk sac (YS) and placenta treated with tamoxifen at E7.5. Note the lack of blood in the Tet1/2/3 f/f ;R26 +/CreER YSs. ( C ) Representative gross images of E11.5 embryonic livers of indicated genotypes treated with tamoxifen at E7.5. Note the smaller size and lack of blood in the Tet1/2/3 f/f ;R26 +/CreER fetal liver (FL). ( D ) Quantitation of % HSPCs and % ECs in E9.5 YS and E11.5 AGM of Tet1/2/3 f/f ;R26 +/CreER and littermate control Tet1/2/3 f/f ;R26 +/+ embryos by flow cytometry. E9.5 YS HSPCs (Lin − CD31 + c-Kit + ), ECs (Lin − CD31 + c-Kit − ), E11.5 AGM HSPCs (CD31 + c-Kit + ), and ECs (CD31 + c-Kit − ). Lin, lineage. ( E and F ) Quantitation of the percent and total number of CD45 + hematopoietic cells and Ter-119 + erythrocytes in E11.5 FL of Tet1/2/3 f/f ;R26 +/CreER and littermate control Tet1/2/3 f/f ;R26 +/+ embryos by flow cytometry. For all panels, data are presented as means ± SEM. Statistically significant (* P
    Figure Legend Snippet: Loss of Tet1/2/3 early in embryogenesis leads to aberrant hematopoiesis. ( A ) Representative gross images of E10.5 to E14.5 embryos of indicated genotypes treated with tamoxifen at E7.5. Total number of normal and dead embryos for each developmental stage is summarized in the table. ( B ) Representative gross images of E11.5 embryos of indicated genotypes attached to yolk sac (YS) and placenta treated with tamoxifen at E7.5. Note the lack of blood in the Tet1/2/3 f/f ;R26 +/CreER YSs. ( C ) Representative gross images of E11.5 embryonic livers of indicated genotypes treated with tamoxifen at E7.5. Note the smaller size and lack of blood in the Tet1/2/3 f/f ;R26 +/CreER fetal liver (FL). ( D ) Quantitation of % HSPCs and % ECs in E9.5 YS and E11.5 AGM of Tet1/2/3 f/f ;R26 +/CreER and littermate control Tet1/2/3 f/f ;R26 +/+ embryos by flow cytometry. E9.5 YS HSPCs (Lin − CD31 + c-Kit + ), ECs (Lin − CD31 + c-Kit − ), E11.5 AGM HSPCs (CD31 + c-Kit + ), and ECs (CD31 + c-Kit − ). Lin, lineage. ( E and F ) Quantitation of the percent and total number of CD45 + hematopoietic cells and Ter-119 + erythrocytes in E11.5 FL of Tet1/2/3 f/f ;R26 +/CreER and littermate control Tet1/2/3 f/f ;R26 +/+ embryos by flow cytometry. For all panels, data are presented as means ± SEM. Statistically significant (* P

    Techniques Used: Quantitation Assay, Flow Cytometry

    16) Product Images from "Tet-mediated DNA demethylation regulates specification of hematopoietic stem and progenitor cells during mammalian embryogenesis"

    Article Title: Tet-mediated DNA demethylation regulates specification of hematopoietic stem and progenitor cells during mammalian embryogenesis

    Journal: Science Advances

    doi: 10.1126/sciadv.abm3470

    Absence of CD31 + c-Kit + hematopoietic clusters and floating HSPCs in AGMs of Tet-deficient embryos. ( A ) Whole-mount three-dimensional (3D) images of E11.5 AGM of indicated genotypes stained with CD31 and c-Kit antibodies. CD31 + ECs (magenta) and c-Kit + HSPCs (green) within the dorsal aorta region are shown at ×10 magnification. Inset: CD31 and c-Kit double-positive hematopoietic (Hem.) clusters are shown at ×100 magnification. ( B ) Whole-mount 3D images of E11.5 FLs of indicated genotypes stained with CD31 and c-Kit antibodies. CD31 + ECs (magenta) and c-Kit + HSPCs (green) are shown at ×40 magnification.
    Figure Legend Snippet: Absence of CD31 + c-Kit + hematopoietic clusters and floating HSPCs in AGMs of Tet-deficient embryos. ( A ) Whole-mount three-dimensional (3D) images of E11.5 AGM of indicated genotypes stained with CD31 and c-Kit antibodies. CD31 + ECs (magenta) and c-Kit + HSPCs (green) within the dorsal aorta region are shown at ×10 magnification. Inset: CD31 and c-Kit double-positive hematopoietic (Hem.) clusters are shown at ×100 magnification. ( B ) Whole-mount 3D images of E11.5 FLs of indicated genotypes stained with CD31 and c-Kit antibodies. CD31 + ECs (magenta) and c-Kit + HSPCs (green) are shown at ×40 magnification.

    Techniques Used: Staining

    Loss of Tet1/2/3 early in embryogenesis leads to aberrant hematopoiesis. ( A ) Representative gross images of E10.5 to E14.5 embryos of indicated genotypes treated with tamoxifen at E7.5. Total number of normal and dead embryos for each developmental stage is summarized in the table. ( B ) Representative gross images of E11.5 embryos of indicated genotypes attached to yolk sac (YS) and placenta treated with tamoxifen at E7.5. Note the lack of blood in the Tet1/2/3 f/f ;R26 +/CreER YSs. ( C ) Representative gross images of E11.5 embryonic livers of indicated genotypes treated with tamoxifen at E7.5. Note the smaller size and lack of blood in the Tet1/2/3 f/f ;R26 +/CreER fetal liver (FL). ( D ) Quantitation of % HSPCs and % ECs in E9.5 YS and E11.5 AGM of Tet1/2/3 f/f ;R26 +/CreER and littermate control Tet1/2/3 f/f ;R26 +/+ embryos by flow cytometry. E9.5 YS HSPCs (Lin − CD31 + c-Kit + ), ECs (Lin − CD31 + c-Kit − ), E11.5 AGM HSPCs (CD31 + c-Kit + ), and ECs (CD31 + c-Kit − ). Lin, lineage. ( E and F ) Quantitation of the percent and total number of CD45 + hematopoietic cells and Ter-119 + erythrocytes in E11.5 FL of Tet1/2/3 f/f ;R26 +/CreER and littermate control Tet1/2/3 f/f ;R26 +/+ embryos by flow cytometry. For all panels, data are presented as means ± SEM. Statistically significant (* P
    Figure Legend Snippet: Loss of Tet1/2/3 early in embryogenesis leads to aberrant hematopoiesis. ( A ) Representative gross images of E10.5 to E14.5 embryos of indicated genotypes treated with tamoxifen at E7.5. Total number of normal and dead embryos for each developmental stage is summarized in the table. ( B ) Representative gross images of E11.5 embryos of indicated genotypes attached to yolk sac (YS) and placenta treated with tamoxifen at E7.5. Note the lack of blood in the Tet1/2/3 f/f ;R26 +/CreER YSs. ( C ) Representative gross images of E11.5 embryonic livers of indicated genotypes treated with tamoxifen at E7.5. Note the smaller size and lack of blood in the Tet1/2/3 f/f ;R26 +/CreER fetal liver (FL). ( D ) Quantitation of % HSPCs and % ECs in E9.5 YS and E11.5 AGM of Tet1/2/3 f/f ;R26 +/CreER and littermate control Tet1/2/3 f/f ;R26 +/+ embryos by flow cytometry. E9.5 YS HSPCs (Lin − CD31 + c-Kit + ), ECs (Lin − CD31 + c-Kit − ), E11.5 AGM HSPCs (CD31 + c-Kit + ), and ECs (CD31 + c-Kit − ). Lin, lineage. ( E and F ) Quantitation of the percent and total number of CD45 + hematopoietic cells and Ter-119 + erythrocytes in E11.5 FL of Tet1/2/3 f/f ;R26 +/CreER and littermate control Tet1/2/3 f/f ;R26 +/+ embryos by flow cytometry. For all panels, data are presented as means ± SEM. Statistically significant (* P

    Techniques Used: Quantitation Assay, Flow Cytometry

    17) Product Images from "Endothelial Cells Potentially Participate in the Metastasis of Triple-Negative Breast Cancer"

    Article Title: Endothelial Cells Potentially Participate in the Metastasis of Triple-Negative Breast Cancer

    Journal: Journal of Immunology Research

    doi: 10.1155/2022/5412007

    scRNA-seq analysis showed three subclusters in breast-associated biopsies. (a) UMAP plot showed integration of endothelial cells isolated from normal breast and TNBC tissues. Three subclusters were identified based on genetic profiles. (b) Violin plots showed the expression pattern of three feature genes ATP1B3 , HSPA1B , and KRT7. (c) Immunofluorescent staining of 3 endothelial cell subpopulations: EC (ATP1B3), EC (HSPA1B), and EC (KRT7). Endothelial cells were labelled with CD31. (d) Heatmap showed expression pattern of top 20 genes from each endothelial cell subclusters. (e) Dot plot revealed that each endothelial cell subcluster possesses distinct biological features.
    Figure Legend Snippet: scRNA-seq analysis showed three subclusters in breast-associated biopsies. (a) UMAP plot showed integration of endothelial cells isolated from normal breast and TNBC tissues. Three subclusters were identified based on genetic profiles. (b) Violin plots showed the expression pattern of three feature genes ATP1B3 , HSPA1B , and KRT7. (c) Immunofluorescent staining of 3 endothelial cell subpopulations: EC (ATP1B3), EC (HSPA1B), and EC (KRT7). Endothelial cells were labelled with CD31. (d) Heatmap showed expression pattern of top 20 genes from each endothelial cell subclusters. (e) Dot plot revealed that each endothelial cell subcluster possesses distinct biological features.

    Techniques Used: Isolation, Expressing, Staining

    18) Product Images from "Macrophage IL-1β promotes arteriogenesis by autocrine STAT3- and NF-κB-mediated transcription of pro-angiogenic VEGF-A"

    Article Title: Macrophage IL-1β promotes arteriogenesis by autocrine STAT3- and NF-κB-mediated transcription of pro-angiogenic VEGF-A

    Journal: Cell reports

    doi: 10.1016/j.celrep.2022.110309

    Clodronate liposome treatment followed by BMDM transplant in the context of acute hindlimb ischemia confirms the important contribution of IL-1β from macrophages relative to other leukocytes (A and B) Laser Doppler images of flow in the ischemic (I) and contralateral control (C) hindlimbs from myeloid IL-1β -deleted mice (mIL-1β KO) that underwent clodronate liposome macrophage depletion followed by transplant of tdTomato-labeled BMDMs from either wild-type (Control Mφ) or IL-1β -deleted (mIL-1β KO Mφ) Ai9 mice along with quantitative analysis (B) (***, p ≤ 0.0003 compared between control Mφ and mIL-1β KO Mφ for each time point by ANOVA; n = 6 mice total, three males and three females). (C–E) Immunofluorescence micrographs of ischemic muscle tissue at day 3 post femoral artery ligation mice treated as in (A) along with quantitation of DAPI + tdTomato + (D) and DAPI + CD31 + (E) cells (***, p = 0.0008 by t test; n = 6 mice, three males and three females). Bar, 100 microns. Data, mean ± SD.
    Figure Legend Snippet: Clodronate liposome treatment followed by BMDM transplant in the context of acute hindlimb ischemia confirms the important contribution of IL-1β from macrophages relative to other leukocytes (A and B) Laser Doppler images of flow in the ischemic (I) and contralateral control (C) hindlimbs from myeloid IL-1β -deleted mice (mIL-1β KO) that underwent clodronate liposome macrophage depletion followed by transplant of tdTomato-labeled BMDMs from either wild-type (Control Mφ) or IL-1β -deleted (mIL-1β KO Mφ) Ai9 mice along with quantitative analysis (B) (***, p ≤ 0.0003 compared between control Mφ and mIL-1β KO Mφ for each time point by ANOVA; n = 6 mice total, three males and three females). (C–E) Immunofluorescence micrographs of ischemic muscle tissue at day 3 post femoral artery ligation mice treated as in (A) along with quantitation of DAPI + tdTomato + (D) and DAPI + CD31 + (E) cells (***, p = 0.0008 by t test; n = 6 mice, three males and three females). Bar, 100 microns. Data, mean ± SD.

    Techniques Used: Mouse Assay, Labeling, Immunofluorescence, Ligation, Quantitation Assay

    19) Product Images from "Lymphatics constitute a novel component of the intestinal stem cell niche"

    Article Title: Lymphatics constitute a novel component of the intestinal stem cell niche

    Journal: bioRxiv

    doi: 10.1101/2022.01.28.478205

    LECs and RGFs are the major source of mucosal Rspo3 ( A ) Flow cytometry of EpCAM − CD45 − stromal cells using Rspo3-GFP and CD31 in the small intestine and colon. The plots represent one of > 10 biological replicates. ( B ) The heatmap of RNA-seq on CD31- Rspo3-GFP- , CD31+ Rspo3-GFP- , CD31- Rspo3-GFP+ , and CD31+ Rspo3-GFP+ cells in the small intestine and colon. n = 3 mice per group. (C) Schematic of Rspo3-GFP; Grem1-tdTomato mouse. ( D-E) Confocal microscopy images of immunofluorescence for Rspo3-GFP, Grem1-tdTomato, and LYVE1 in the small intestine ( D ) and colon ( E ). Yellow arrowheads indicate RGFs. White arrows indicate LEC. The image represents one of 6 biological replicates. (F-I) Flow cytometry of EpCAM − CD45 − stromal cells in the small intestine and colon of Rspo3-GFP; Grem1-tdTomato mice. The plots represent one of 3 biological replicates. ( J ) Schematic of Rspo3 and/or Grem1 positive cells in the small intestine and colon. Scale bar, 10 μm ( D ) and 50 μm ( E ).
    Figure Legend Snippet: LECs and RGFs are the major source of mucosal Rspo3 ( A ) Flow cytometry of EpCAM − CD45 − stromal cells using Rspo3-GFP and CD31 in the small intestine and colon. The plots represent one of > 10 biological replicates. ( B ) The heatmap of RNA-seq on CD31- Rspo3-GFP- , CD31+ Rspo3-GFP- , CD31- Rspo3-GFP+ , and CD31+ Rspo3-GFP+ cells in the small intestine and colon. n = 3 mice per group. (C) Schematic of Rspo3-GFP; Grem1-tdTomato mouse. ( D-E) Confocal microscopy images of immunofluorescence for Rspo3-GFP, Grem1-tdTomato, and LYVE1 in the small intestine ( D ) and colon ( E ). Yellow arrowheads indicate RGFs. White arrows indicate LEC. The image represents one of 6 biological replicates. (F-I) Flow cytometry of EpCAM − CD45 − stromal cells in the small intestine and colon of Rspo3-GFP; Grem1-tdTomato mice. The plots represent one of 3 biological replicates. ( J ) Schematic of Rspo3 and/or Grem1 positive cells in the small intestine and colon. Scale bar, 10 μm ( D ) and 50 μm ( E ).

    Techniques Used: Flow Cytometry, RNA Sequencing Assay, Mouse Assay, Confocal Microscopy, Immunofluorescence

    LECs and RGFs expand to facilitate epithelial regeneration after irradiation induced damage ( A) Immunofluorescence (IF) for LYVE1 in the small intestine post-irradiation. The images represent one of 6 biological replicates per group. (B-C) Flow cytometry ( B ) and quantification ( C ) of CD31+Rspo3-GFP+ LECs and CD31-Rspo3-GFP+ RGFs from the small intestinal EpCAM − CD45 − stromal cells. n = 5 mice per group. (D) Confocal microscopy of IF for Rspo3-GFP, Grem1-tdTomato, and LYVE1 in the small intestine of Rspo3-GFP; Grem1-tdTomato mouse day 3 post-irradiation. Yellow arrowheads indicate expanded RGFs. White arrows indicate LECs. The images represent one of 3 biological replicates. (E) UMAP of scRNA-seq of sorted small intestinal Rspo3-GFP+ cells 3 days post-irradiation (Control, n = 2 mice and 6,171 cells; irradiation, n = 2 mice and 11,706 cells; multi-dataset integration methods). RGF, RGFs. (F) Violin plots for Rspo3 expression. (G) Violin plots for Il1r1 expression. (H) GSEA of IL-1-mediated signaling pathway genes. (I) Schematic of IL-1a administration to RGFs. (J) qRT-PCR of Rspo3 mRNA expression from RGFs after IL-1a administration. n = 3 mice per group. (K) Violin plots for Igf1, Fgf2, and Ereg expression. (L) Model of how LECs and RGFs support ISCs in homeostasis and injury. RGF 1 control, 2,226 cells from 2 mice; RGF 1 irradiation 3,471 cells from 2 mice; RGF 2 control, 812 cells from 2 mice; RGF 2 irradiation, 1,380 cells from 2 mice ( F , G , K ). Unpaired two-tailed t-tests ( C , J ). Wilcox test ( F , G , K ). Data are mean ± SD. *p
    Figure Legend Snippet: LECs and RGFs expand to facilitate epithelial regeneration after irradiation induced damage ( A) Immunofluorescence (IF) for LYVE1 in the small intestine post-irradiation. The images represent one of 6 biological replicates per group. (B-C) Flow cytometry ( B ) and quantification ( C ) of CD31+Rspo3-GFP+ LECs and CD31-Rspo3-GFP+ RGFs from the small intestinal EpCAM − CD45 − stromal cells. n = 5 mice per group. (D) Confocal microscopy of IF for Rspo3-GFP, Grem1-tdTomato, and LYVE1 in the small intestine of Rspo3-GFP; Grem1-tdTomato mouse day 3 post-irradiation. Yellow arrowheads indicate expanded RGFs. White arrows indicate LECs. The images represent one of 3 biological replicates. (E) UMAP of scRNA-seq of sorted small intestinal Rspo3-GFP+ cells 3 days post-irradiation (Control, n = 2 mice and 6,171 cells; irradiation, n = 2 mice and 11,706 cells; multi-dataset integration methods). RGF, RGFs. (F) Violin plots for Rspo3 expression. (G) Violin plots for Il1r1 expression. (H) GSEA of IL-1-mediated signaling pathway genes. (I) Schematic of IL-1a administration to RGFs. (J) qRT-PCR of Rspo3 mRNA expression from RGFs after IL-1a administration. n = 3 mice per group. (K) Violin plots for Igf1, Fgf2, and Ereg expression. (L) Model of how LECs and RGFs support ISCs in homeostasis and injury. RGF 1 control, 2,226 cells from 2 mice; RGF 1 irradiation 3,471 cells from 2 mice; RGF 2 control, 812 cells from 2 mice; RGF 2 irradiation, 1,380 cells from 2 mice ( F , G , K ). Unpaired two-tailed t-tests ( C , J ). Wilcox test ( F , G , K ). Data are mean ± SD. *p

    Techniques Used: Irradiation, Immunofluorescence, Flow Cytometry, Mouse Assay, Confocal Microscopy, Expressing, Quantitative RT-PCR, Two Tailed Test

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    Thermo Fisher cd31 pecam 1 monoclonal antibody 390
    Effects of L6-F4-2 on retinal endothelial WNT/β-catenin signaling and vascular subtypes a-c WT (control) and Ndp KO mice were treated with PBS or L6-F4-2 by intravitreal injection (0.19 μg) at P0 and 5-7 pooled retinas for each group were harvested at P8. Retinal vessel ECs were purified by <t>anti-CD31</t> FACS and ultra-low input bulk RNA-seq analysis was performed. a Left: WT (PBS-treated) vs Ndp KO (PBS-treated). Right: Ndp KO (PBS-treated) vs Ndp KO (treated with L6-F4-2). Volcano plot depicting genes with a p-value
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    Effects of L6-F4-2 on retinal endothelial WNT/β-catenin signaling and vascular subtypes a-c WT (control) and Ndp KO mice were treated with PBS or L6-F4-2 by intravitreal injection (0.19 μg) at P0 and 5-7 pooled retinas for each group were harvested at P8. Retinal vessel ECs were purified by anti-CD31 FACS and ultra-low input bulk RNA-seq analysis was performed. a Left: WT (PBS-treated) vs Ndp KO (PBS-treated). Right: Ndp KO (PBS-treated) vs Ndp KO (treated with L6-F4-2). Volcano plot depicting genes with a p-value

    Journal: bioRxiv

    Article Title: Therapeutic modulation of the blood-brain barrier and ischemic stroke by a bioengineered FZD4-selective WNT surrogate

    doi: 10.1101/2022.10.13.510564

    Figure Lengend Snippet: Effects of L6-F4-2 on retinal endothelial WNT/β-catenin signaling and vascular subtypes a-c WT (control) and Ndp KO mice were treated with PBS or L6-F4-2 by intravitreal injection (0.19 μg) at P0 and 5-7 pooled retinas for each group were harvested at P8. Retinal vessel ECs were purified by anti-CD31 FACS and ultra-low input bulk RNA-seq analysis was performed. a Left: WT (PBS-treated) vs Ndp KO (PBS-treated). Right: Ndp KO (PBS-treated) vs Ndp KO (treated with L6-F4-2). Volcano plot depicting genes with a p-value

    Article Snippet: Endothelial cells were labeled with PE-Cy7 rat anti-mouse CD31 (#25-0311-82, eBiosciences, CA).

    Techniques: Mouse Assay, Injection, Purification, FACS, RNA Sequencing Assay

    L6-F4-2 treatment promotes endothelial blood-brain and blood-retina barrier function a WT (control) and Ndp KO mice were treated with PBS or L6-F4-2 by i.p. injection (2.5 mg/kg) at P0, P7 and P14 and tissues were harvested at P21. Sulfo-NHS-biotin staining reveals BBB/BRB defects in cerebellum and retina, but not cerebral cortex in Ndp KO mice, while these defects can be rescued by L6-F4-2 treatment. Biotin labeling of liver and kidney parenchyma serve as a positive control. Scale bar, 500 μm. b Quantification of ( a ) for BBB/BRB leakage in Ndp KO cerebellum and retina with rescue by L6-F4-2 treatment. c Mice were treated at P21, P24 and P27 (2.5 mg/kg, i.p.) and tissues were harvested at P30. Sulfo-NHS-biotin staining reveals BBB/BRB defects in cerebellum and retina, but not cerebral cortex in Ndp KO mice with rescue by L6-F4-2. Scale bar, 200 μm. d Quantification of ( c ) with BBB/BRB leakage in Ndp KO cerebellum and retina and reversal by L6-F4-2. e L6-F4-2 rescues barrier function defects in P30 Ndp KO mice with increased expression of the tight junction component CLDN5, P30 cerebellum IF, overlay of CD31 IF and DAPI. f Quantification of ( e ). g L6-F4-2 decreases expression of the EC fenestration component PLVAP in P30 Ndp KO mice, with overlay of CD31 IF and DAPI. h Quantitation of ( g ). For e-h, scale bars represent 100 μm. To quantify CLDN5 and PLVAP in ( f , h ), the density was measured with ImageJ and normalized to vessel area (CD31). Error bars represent mean ± s.e.m., n=5, *p

    Journal: bioRxiv

    Article Title: Therapeutic modulation of the blood-brain barrier and ischemic stroke by a bioengineered FZD4-selective WNT surrogate

    doi: 10.1101/2022.10.13.510564

    Figure Lengend Snippet: L6-F4-2 treatment promotes endothelial blood-brain and blood-retina barrier function a WT (control) and Ndp KO mice were treated with PBS or L6-F4-2 by i.p. injection (2.5 mg/kg) at P0, P7 and P14 and tissues were harvested at P21. Sulfo-NHS-biotin staining reveals BBB/BRB defects in cerebellum and retina, but not cerebral cortex in Ndp KO mice, while these defects can be rescued by L6-F4-2 treatment. Biotin labeling of liver and kidney parenchyma serve as a positive control. Scale bar, 500 μm. b Quantification of ( a ) for BBB/BRB leakage in Ndp KO cerebellum and retina with rescue by L6-F4-2 treatment. c Mice were treated at P21, P24 and P27 (2.5 mg/kg, i.p.) and tissues were harvested at P30. Sulfo-NHS-biotin staining reveals BBB/BRB defects in cerebellum and retina, but not cerebral cortex in Ndp KO mice with rescue by L6-F4-2. Scale bar, 200 μm. d Quantification of ( c ) with BBB/BRB leakage in Ndp KO cerebellum and retina and reversal by L6-F4-2. e L6-F4-2 rescues barrier function defects in P30 Ndp KO mice with increased expression of the tight junction component CLDN5, P30 cerebellum IF, overlay of CD31 IF and DAPI. f Quantification of ( e ). g L6-F4-2 decreases expression of the EC fenestration component PLVAP in P30 Ndp KO mice, with overlay of CD31 IF and DAPI. h Quantitation of ( g ). For e-h, scale bars represent 100 μm. To quantify CLDN5 and PLVAP in ( f , h ), the density was measured with ImageJ and normalized to vessel area (CD31). Error bars represent mean ± s.e.m., n=5, *p

    Article Snippet: Endothelial cells were labeled with PE-Cy7 rat anti-mouse CD31 (#25-0311-82, eBiosciences, CA).

    Techniques: Mouse Assay, Injection, Staining, Labeling, Positive Control, Expressing, Quantitation Assay

    L6-F4-2 treatment rescues stroke phenotypes in wild-type mice a Schematic of tMCAO surgery with 45 min occlusion time, L6-F4-2 treatment time course and brain harvest at day 2 post-stroke. b TTC staining (top), and mouse IgG extravasation (mIgG) (bottom) of coronal sections at 48 hours post-stoke. c Quantification of infarct size for NIST control mAb (n=13) and L6-F4-2 (n=18). d Quantification of mouse IgG staining, NIST control n=4, L6-F4-2 n=5. e Representative images of BBB integrity in brains of the indicated mice in stroke and non-stroke regions after 45 min tMCAO, 2 days of reperfusion and two L6-F4-2 treatments (3 mg/kg, i.v.), as assessed by the Sulfo-NHS-biotin tracer extravasation assay. Scale bar, 50 μm. f Quantification of extravasated exogenous tracer Sulfo-NHS-biotin using ImageJ. n = 5. g Fractional change in brain edema for NIST control mAb (n=13) and L6-F4-2 (n=18). h Neurological scores at 48 h after tMCAO surgery for NIST control mAb (n=13) and L6-F4-2 (n=18). i Co-immunofluorescence staining for PDGFRB and CD31 in infarcted brain (stroke and non-stroke) regions and j quantification of pericyte coverage. The PDGFRB signal was normalized to CD31; n = 5. Scale bar, 100 μm. Error bars represent mean ± s.e.m., *p

    Journal: bioRxiv

    Article Title: Therapeutic modulation of the blood-brain barrier and ischemic stroke by a bioengineered FZD4-selective WNT surrogate

    doi: 10.1101/2022.10.13.510564

    Figure Lengend Snippet: L6-F4-2 treatment rescues stroke phenotypes in wild-type mice a Schematic of tMCAO surgery with 45 min occlusion time, L6-F4-2 treatment time course and brain harvest at day 2 post-stroke. b TTC staining (top), and mouse IgG extravasation (mIgG) (bottom) of coronal sections at 48 hours post-stoke. c Quantification of infarct size for NIST control mAb (n=13) and L6-F4-2 (n=18). d Quantification of mouse IgG staining, NIST control n=4, L6-F4-2 n=5. e Representative images of BBB integrity in brains of the indicated mice in stroke and non-stroke regions after 45 min tMCAO, 2 days of reperfusion and two L6-F4-2 treatments (3 mg/kg, i.v.), as assessed by the Sulfo-NHS-biotin tracer extravasation assay. Scale bar, 50 μm. f Quantification of extravasated exogenous tracer Sulfo-NHS-biotin using ImageJ. n = 5. g Fractional change in brain edema for NIST control mAb (n=13) and L6-F4-2 (n=18). h Neurological scores at 48 h after tMCAO surgery for NIST control mAb (n=13) and L6-F4-2 (n=18). i Co-immunofluorescence staining for PDGFRB and CD31 in infarcted brain (stroke and non-stroke) regions and j quantification of pericyte coverage. The PDGFRB signal was normalized to CD31; n = 5. Scale bar, 100 μm. Error bars represent mean ± s.e.m., *p

    Article Snippet: Endothelial cells were labeled with PE-Cy7 rat anti-mouse CD31 (#25-0311-82, eBiosciences, CA).

    Techniques: Mouse Assay, Staining, Immunofluorescence