anti islet 1  (Abcam)

 
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    Name:
    Anti Islet 1 antibody 1H9
    Description:

    Catalog Number:
    ab86472
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    Structured Review

    Abcam anti islet 1
    Effect of fluorofenidone on <t>Islet-1</t> expression in murine retinas

    https://www.bioz.com/result/anti islet 1/product/Abcam
    Average 98 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    anti islet 1 - by Bioz Stars, 2020-09
    98/100 stars

    Images

    1) Product Images from "Effect of pyridone agent on blood-retinal barrier in diabetic mice"

    Article Title: Effect of pyridone agent on blood-retinal barrier in diabetic mice

    Journal: International Journal of Ophthalmology

    doi: 10.18240/ijo.2017.06.09

    Effect of fluorofenidone on Islet-1 expression in murine retinas
    Figure Legend Snippet: Effect of fluorofenidone on Islet-1 expression in murine retinas

    Techniques Used: Expressing

    2) Product Images from "Islet-1 synergizes with Gcn5 to promote MSC differentiation into cardiomyocytes"

    Article Title: Islet-1 synergizes with Gcn5 to promote MSC differentiation into cardiomyocytes

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-58387-8

    The effect of Gcn5 inhibition on MSC differentiation into cardiomyocytes induced by islet-1. ( a ) FQ-PCR detected the expression levels of GATA4 and Nkx2.5 after Gcn5 was inhibited. GATA4 and Nkx2.5 expression increased after islet-1 transfection, but in the LV-islet-1 + MB-3 group, their expression did not change compared with that in the blank and control groups. *p
    Figure Legend Snippet: The effect of Gcn5 inhibition on MSC differentiation into cardiomyocytes induced by islet-1. ( a ) FQ-PCR detected the expression levels of GATA4 and Nkx2.5 after Gcn5 was inhibited. GATA4 and Nkx2.5 expression increased after islet-1 transfection, but in the LV-islet-1 + MB-3 group, their expression did not change compared with that in the blank and control groups. *p

    Techniques Used: Inhibition, Polymerase Chain Reaction, Expressing, Transfection

    The epigenetic modification model of MSC differentiation into cardiomyocytes induced by islet-1. Islet-1 guides Gcn5 binding to the GATA4 and Nkx2.5 promoters, and Gcn5 interacts with G9A, HP1, DNMT-1 and HDAC1. Then, the chromatin of the GATA4 and Nkx2.5 promoter region loosened because of the hyperacetylation of these regions, which is conducive to GATA4 and Nkx2.5 expression.
    Figure Legend Snippet: The epigenetic modification model of MSC differentiation into cardiomyocytes induced by islet-1. Islet-1 guides Gcn5 binding to the GATA4 and Nkx2.5 promoters, and Gcn5 interacts with G9A, HP1, DNMT-1 and HDAC1. Then, the chromatin of the GATA4 and Nkx2.5 promoter region loosened because of the hyperacetylation of these regions, which is conducive to GATA4 and Nkx2.5 expression.

    Techniques Used: Modification, Binding Assay, Expressing

    During MSC differentiation into cardiomyocytes induced by Islet-1, the effects of Gcn5 inhibition on other key enzymes were assessed. ( a ) Western blot was used to detect the expression of the enzymes involved in regulating GATA4/Nkx2.5 after Gcn5 was inhibited by MB-3. The enzymes assessed were Gcn5, HDAC1, G9A, DNMT-1 and HP1. Images were cropped for clarity, and full-length blots/gels are presented in Supplementary Fig. 4 . ( b ) Quantitative analysis of these enzymes. MB-3 inhibited Gcn5 expression in both the Control + MB-3 and LV-islet-1 + MB-3 groups but had no effect on the other enzymes. ( c , d ) ChIP-qPCR results of the binding levels of these enzymes in the GATA4/Nkx2.5 promoters after Gcn5 was inhibited by MB-3 during MSC differentiation. After islet-1 transfection and Gcn5 inhibition, the binding level of the other enzymes in the GATA4/Nkx2.5 promoters did not change compared with those in the blank, control and control + MB-3 promoters. *p
    Figure Legend Snippet: During MSC differentiation into cardiomyocytes induced by Islet-1, the effects of Gcn5 inhibition on other key enzymes were assessed. ( a ) Western blot was used to detect the expression of the enzymes involved in regulating GATA4/Nkx2.5 after Gcn5 was inhibited by MB-3. The enzymes assessed were Gcn5, HDAC1, G9A, DNMT-1 and HP1. Images were cropped for clarity, and full-length blots/gels are presented in Supplementary Fig. 4 . ( b ) Quantitative analysis of these enzymes. MB-3 inhibited Gcn5 expression in both the Control + MB-3 and LV-islet-1 + MB-3 groups but had no effect on the other enzymes. ( c , d ) ChIP-qPCR results of the binding levels of these enzymes in the GATA4/Nkx2.5 promoters after Gcn5 was inhibited by MB-3 during MSC differentiation. After islet-1 transfection and Gcn5 inhibition, the binding level of the other enzymes in the GATA4/Nkx2.5 promoters did not change compared with those in the blank, control and control + MB-3 promoters. *p

    Techniques Used: Inhibition, Western Blot, Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Transfection

    Co-IP experiments confirmed that islet-1 and Gcn5 bound together during MSC differentiation into cardiomyocytes induced by islet-1 overexpression. Islet-1 and IgG antibodies were chosen to pull down proteins, and the islet-1 and Gcn5 bands were then detected by Western blot. Gcn5 was detected in proteins pulled down by the islet-1 antibody, which indicated that islet-1 could form a complex with Gcn5.
    Figure Legend Snippet: Co-IP experiments confirmed that islet-1 and Gcn5 bound together during MSC differentiation into cardiomyocytes induced by islet-1 overexpression. Islet-1 and IgG antibodies were chosen to pull down proteins, and the islet-1 and Gcn5 bands were then detected by Western blot. Gcn5 was detected in proteins pulled down by the islet-1 antibody, which indicated that islet-1 could form a complex with Gcn5.

    Techniques Used: Co-Immunoprecipitation Assay, Over Expression, Western Blot

    During MSC differentiation into cardiomyocytes induced by islet-1, changes in the levels of H3K9 acetylation/methylation and DNA methylation in the GATA4 and Nkx2.5 promoters after Gcn5 inhibition were detected. ( a) ChIP-qPCR was used to detect H3K9 acetylation and methylation levels in the GATA4 and Nkx2.5 promoters. The input% showed H3K9 acetylation and methylation levels in the LV-islet-1 + MB-3 group, in contrast to those in the LV-islet-1 group. *p
    Figure Legend Snippet: During MSC differentiation into cardiomyocytes induced by islet-1, changes in the levels of H3K9 acetylation/methylation and DNA methylation in the GATA4 and Nkx2.5 promoters after Gcn5 inhibition were detected. ( a) ChIP-qPCR was used to detect H3K9 acetylation and methylation levels in the GATA4 and Nkx2.5 promoters. The input% showed H3K9 acetylation and methylation levels in the LV-islet-1 + MB-3 group, in contrast to those in the LV-islet-1 group. *p

    Techniques Used: Methylation, DNA Methylation Assay, Inhibition, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Related Articles

    Incubation:

    Article Title: Effect of pyridone agent on blood-retinal barrier in diabetic mice
    Article Snippet: .. Total protein of murine retinas were collected and resolved on sodium dodecyl sulfate (SDS)-polyacrylamide gel, then it was transferred onto a nitrocellulose membrane and incubated with anti-Islet-1 (Abcam, UK), anti-VEGF (Abcam, UK), anti-Albumin(Abcam, UK), anti-occludin (Abcam, UK) and anti-β-actin antibodies (Sigma, USA). .. Membranes were incubated with peroxidase-conjugated secondary antibodies and developed using the ECL system.

    Article Title: Semaphorin-Plexin signaling influences early ventral telencephalic development and thalamocortical axon guidance
    Article Snippet: .. Sections were then incubated in blocking buffer (10% solution of normal serum derived from the species in which secondary antibodies were raised in PBS) for at least 1 hour at RT, or overnight at 4° C. Following blocking, the tissue was incubated with primary antibodies diluted in PBS-NaN3 0.1% for 24–48 hours at 4° C. Primary antibodies used were mouse anti-neurofilament 2H3 (1:250; Developmental Studies Hybridoma Bank), rabbit anti-Islet1 (1:500; Abcam), mouse anti-Islet1 (1:50; Developmental Studies Hybridoma Bank), goat anti-mouse Sema6A (1:100; R & D), Armenian hamster anti-PlexinA4 (Mab-A4F5, 1:500; see Suto et al. [ ]), Armenian hamster anti-PlexinA2 (Mab-A2D3, 1:50; see Suto et al. [ ]). .. The monoclonal anti-NF 165 kD (2H3) and anti-Islet1/2 homeobox (39.4D5) antibodies, obtained from the Developmental Studies Hybridoma Bank (University of Iowa), were respectively developed by T.M.

    Article Title: ISL1 Protein Transduction Promotes Cardiomyocyte Differentiation from Human Embryonic Stem Cells
    Article Snippet: .. Cells were incubated overnight at 4°C with the appropriate primary antibodies: GATA4 (1∶200, Santa Cruz, SC-1237); MHC (1∶200, Abcam, Ab15); ISL1 (1∶200, Abcam, Ab86472); NKX2.5 (1∶200, R & D Systems, AF2444); DESMIN (1∶200, Abcam, Ab8470); ACTININ (1∶200, Abcam, Ab78505); and TAT (1∶200, Cell Application Inc., CB0888). .. Cells were then washed twice with PBS/0.1% Tween-20 for 5 min and incubated with the appropriate secondary antibody in PBS.

    Immunohistochemistry:

    Article Title: ISL-1 is overexpressed in non-Hodgkin lymphoma and promotes lymphoma cell proliferation by forming a p-STAT3/p-c-Jun/ISL-1 complex
    Article Snippet: .. Collected specimens and TMA were subjected to immunohistochemistry (IHC) analysis using the Enovision Detection Kit/DAB (GK500705, DAKO A/S, Glostrup, Denmark) according to the manufacturer’s protocol with the indicated antibody: mouse monoclonal anti-ISL-1 (ab86472, Abcam, Hong Kong, China); rabbit polyclonal anti-c-Myc and anti-phospho-c-Jun (Ser63) (E1A1002 and E1A3089, EnoGene, China); rabbit monoclonal anti-phospho-Stat3 (Tyr705) (#9145, Cell Signaling Technology, Beverly, MA, USA). .. Monoclonal mouse IgG2a and ployclonal rabbit IgG (X0943 and X0936, DAKO A/S, Glostrup, Denmark) were used as isotope controls.

    Negative Control:

    Article Title: Islet-1 synergizes with Gcn5 to promote MSC differentiation into cardiomyocytes
    Article Snippet: .. An anti-islet-1 (Abcam, Cambridge, MA, UK) antibody was used to pull down islet-1, and an anti-IgG antibody was used as a negative control. .. Protein collection was analysed by Western blot using an anti-Gcn5 (Epigentek, Farmingdale, NY, USA) antibody to detect its existence in islet-1-recruiting proteins.

    Derivative Assay:

    Article Title: Semaphorin-Plexin signaling influences early ventral telencephalic development and thalamocortical axon guidance
    Article Snippet: .. Sections were then incubated in blocking buffer (10% solution of normal serum derived from the species in which secondary antibodies were raised in PBS) for at least 1 hour at RT, or overnight at 4° C. Following blocking, the tissue was incubated with primary antibodies diluted in PBS-NaN3 0.1% for 24–48 hours at 4° C. Primary antibodies used were mouse anti-neurofilament 2H3 (1:250; Developmental Studies Hybridoma Bank), rabbit anti-Islet1 (1:500; Abcam), mouse anti-Islet1 (1:50; Developmental Studies Hybridoma Bank), goat anti-mouse Sema6A (1:100; R & D), Armenian hamster anti-PlexinA4 (Mab-A4F5, 1:500; see Suto et al. [ ]), Armenian hamster anti-PlexinA2 (Mab-A2D3, 1:50; see Suto et al. [ ]). .. The monoclonal anti-NF 165 kD (2H3) and anti-Islet1/2 homeobox (39.4D5) antibodies, obtained from the Developmental Studies Hybridoma Bank (University of Iowa), were respectively developed by T.M.

    Blocking Assay:

    Article Title: Semaphorin-Plexin signaling influences early ventral telencephalic development and thalamocortical axon guidance
    Article Snippet: .. Sections were then incubated in blocking buffer (10% solution of normal serum derived from the species in which secondary antibodies were raised in PBS) for at least 1 hour at RT, or overnight at 4° C. Following blocking, the tissue was incubated with primary antibodies diluted in PBS-NaN3 0.1% for 24–48 hours at 4° C. Primary antibodies used were mouse anti-neurofilament 2H3 (1:250; Developmental Studies Hybridoma Bank), rabbit anti-Islet1 (1:500; Abcam), mouse anti-Islet1 (1:50; Developmental Studies Hybridoma Bank), goat anti-mouse Sema6A (1:100; R & D), Armenian hamster anti-PlexinA4 (Mab-A4F5, 1:500; see Suto et al. [ ]), Armenian hamster anti-PlexinA2 (Mab-A2D3, 1:50; see Suto et al. [ ]). .. The monoclonal anti-NF 165 kD (2H3) and anti-Islet1/2 homeobox (39.4D5) antibodies, obtained from the Developmental Studies Hybridoma Bank (University of Iowa), were respectively developed by T.M.

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  • 90
    Abcam mouse anti isl1
    Figure 6. Structure of cardiomyocyte colonies grown in the primary culture of rat neonatal myocardial cells. ( A–C ) Different stages of development of the colonies stemming from <t>Isl1</t> + CSCs. ( A ) Cell division, DIV 2. Isl1 + (FITC, green), GATA-4 (phycoerythrin, red). ( B ) Colony consisting of approximately 8 cells, DIV 11. Isl1 + (FITC, green), actin (rhodamine-phalloidin, red). ( C ) Large Isl1 + colony, DIV 11. Isl1 + (FITC, green), actin (rhodamine-phalloidin, red). ( D ) The optical sections of colonies formed by Isl1 + , c-kit + , and Sca1 + CSCs on the 11th DIV. Isl1 + CSCs (Alexa 405, blue), Z = 12. c-kit + CSCs (FITC, green), Z = 12. Sca1 + CSCs (Alexa 405, blue), Z = 11. Actin was stained using rhodamine-phalloidin (red). ( E ) Differentiation of c-kit + CSCs inside the colony on the 13th DIV. Overlaid optical section of transmitted light and fluorescent images in 2 emitting wavelengths: 488 nm (FITC) and 543 nm (Alexa) in the bottom (Z = 5), in the middle (Z = 10), and the top (Z = 20) parts of the colony. c-kit + expression was revealed by FITC-conjugated antibodies (green), and α-sarcomeric actinin was revealed by Alexa-conjugated antibodies (red). Confocal microscope, Leica TCS SP5 (Germany), objective ×63, oil.
    Mouse Anti Isl1, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti isl1/product/Abcam
    Average 90 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    mouse anti isl1 - by Bioz Stars, 2020-09
    90/100 stars
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    95
    Abcam rabbit isl1
    Loss of SMN reactivates the cell cycle. a Ki67 and <t>ISL1</t> immunostaining analysis of wild-type (BJ iPS and 18a), SMA Type I (1-38 G), and SMA Type II (1-51 N) motor neuron cultures at day 28. The percentages of ISL1 + Ki67 + cells amongst all ISL1 + motor neurons are shown. b Knockdown of SMN in wild-type cell line (BJ-iPS) increased the percentage of ISL1 + motor neurons co-expressing Ki67. c Co-staining of ISL1 (red) and Ki67 (green) showing increased Ki67 + cells upon SMN knockdown in BJ-iPS motor neuron cultures. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. d Ki67 and cCASP3 immunostaining analysis of wild-type motor neurons demonstrated higher cCASP3 expression in Ki67 + motor neurons than Ki67 − motor neurons. *** p
    Rabbit Isl1, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit isl1/product/Abcam
    Average 95 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    rabbit isl1 - by Bioz Stars, 2020-09
    95/100 stars
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    98
    Abcam rabbit anti isl1
    <t>ISL1</t> directly regulates a number of genes required for normal pacemaker function in mice and human.
    Rabbit Anti Isl1, supplied by Abcam, used in various techniques. Bioz Stars score: 98/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti isl1/product/Abcam
    Average 98 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    rabbit anti isl1 - by Bioz Stars, 2020-09
    98/100 stars
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    Image Search Results


    Figure 6. Structure of cardiomyocyte colonies grown in the primary culture of rat neonatal myocardial cells. ( A–C ) Different stages of development of the colonies stemming from Isl1 + CSCs. ( A ) Cell division, DIV 2. Isl1 + (FITC, green), GATA-4 (phycoerythrin, red). ( B ) Colony consisting of approximately 8 cells, DIV 11. Isl1 + (FITC, green), actin (rhodamine-phalloidin, red). ( C ) Large Isl1 + colony, DIV 11. Isl1 + (FITC, green), actin (rhodamine-phalloidin, red). ( D ) The optical sections of colonies formed by Isl1 + , c-kit + , and Sca1 + CSCs on the 11th DIV. Isl1 + CSCs (Alexa 405, blue), Z = 12. c-kit + CSCs (FITC, green), Z = 12. Sca1 + CSCs (Alexa 405, blue), Z = 11. Actin was stained using rhodamine-phalloidin (red). ( E ) Differentiation of c-kit + CSCs inside the colony on the 13th DIV. Overlaid optical section of transmitted light and fluorescent images in 2 emitting wavelengths: 488 nm (FITC) and 543 nm (Alexa) in the bottom (Z = 5), in the middle (Z = 10), and the top (Z = 20) parts of the colony. c-kit + expression was revealed by FITC-conjugated antibodies (green), and α-sarcomeric actinin was revealed by Alexa-conjugated antibodies (red). Confocal microscope, Leica TCS SP5 (Germany), objective ×63, oil.

    Journal: Cell Cycle

    Article Title: Characterization of contracting cardiomyocyte colonies in the primary culture of neonatal rat myocardial cells

    doi: 10.4161/cc.27768

    Figure Lengend Snippet: Figure 6. Structure of cardiomyocyte colonies grown in the primary culture of rat neonatal myocardial cells. ( A–C ) Different stages of development of the colonies stemming from Isl1 + CSCs. ( A ) Cell division, DIV 2. Isl1 + (FITC, green), GATA-4 (phycoerythrin, red). ( B ) Colony consisting of approximately 8 cells, DIV 11. Isl1 + (FITC, green), actin (rhodamine-phalloidin, red). ( C ) Large Isl1 + colony, DIV 11. Isl1 + (FITC, green), actin (rhodamine-phalloidin, red). ( D ) The optical sections of colonies formed by Isl1 + , c-kit + , and Sca1 + CSCs on the 11th DIV. Isl1 + CSCs (Alexa 405, blue), Z = 12. c-kit + CSCs (FITC, green), Z = 12. Sca1 + CSCs (Alexa 405, blue), Z = 11. Actin was stained using rhodamine-phalloidin (red). ( E ) Differentiation of c-kit + CSCs inside the colony on the 13th DIV. Overlaid optical section of transmitted light and fluorescent images in 2 emitting wavelengths: 488 nm (FITC) and 543 nm (Alexa) in the bottom (Z = 5), in the middle (Z = 10), and the top (Z = 20) parts of the colony. c-kit + expression was revealed by FITC-conjugated antibodies (green), and α-sarcomeric actinin was revealed by Alexa-conjugated antibodies (red). Confocal microscope, Leica TCS SP5 (Germany), objective ×63, oil.

    Article Snippet: In the second series, the primary mouse anti-Isl1 (Abcam) and anti-Sca1 (Abcam) monoclonal antibodies were preliminary conjugated with Alexa 405 according to Zenon technology (Invitrogen) and then used for immunostaining at a 1:100 dilution.

    Techniques: Staining, Expressing, Microscopy

    Figure 7. Differentiation of Isl1 + and c-kit + CSCs inside the colonies on the 13th DIV. The optical sections of colonies on 2 levels: ( A ) Isl1 + middle (Z = 14) and ( B ) bottom (Z = 0). ( C ) c-kit + top (Z = 10) and ( D ) bottom (Z = 0). Confocal microscope, LEICA TCS SL, objective ×63, oil.

    Journal: Cell Cycle

    Article Title: Characterization of contracting cardiomyocyte colonies in the primary culture of neonatal rat myocardial cells

    doi: 10.4161/cc.27768

    Figure Lengend Snippet: Figure 7. Differentiation of Isl1 + and c-kit + CSCs inside the colonies on the 13th DIV. The optical sections of colonies on 2 levels: ( A ) Isl1 + middle (Z = 14) and ( B ) bottom (Z = 0). ( C ) c-kit + top (Z = 10) and ( D ) bottom (Z = 0). Confocal microscope, LEICA TCS SL, objective ×63, oil.

    Article Snippet: In the second series, the primary mouse anti-Isl1 (Abcam) and anti-Sca1 (Abcam) monoclonal antibodies were preliminary conjugated with Alexa 405 according to Zenon technology (Invitrogen) and then used for immunostaining at a 1:100 dilution.

    Techniques: Microscopy

    BMP signaling enhances Isl1-CPC cardiac differentiation. Freshly sorted EB day 5.5 GFP + Isl1-CPC were treated with control (A, D) or 25 ng/ml Bmp4 (B, E) for 6 days, followed by immunostaining with antibodies against cardiomyocyte-specific protein cTnT (red in A and B) or labelled with cTnT and Alexa fluor 647 for FACS analysis (D, E). Nuclei: blue (Hoechst). (C) Quantification of A and B (over 2000 cells counted from 3 independent experiments). (F) Quantification of D and E (20000 cells FACS analyzed for each of 3 independent experiments). Scale bar: 100 µm; *statistical significance p

    Journal: PLoS ONE

    Article Title: Functional Cardiomyocytes Derived from Isl1 Cardiac Progenitors via Bmp4 Stimulation

    doi: 10.1371/journal.pone.0110752

    Figure Lengend Snippet: BMP signaling enhances Isl1-CPC cardiac differentiation. Freshly sorted EB day 5.5 GFP + Isl1-CPC were treated with control (A, D) or 25 ng/ml Bmp4 (B, E) for 6 days, followed by immunostaining with antibodies against cardiomyocyte-specific protein cTnT (red in A and B) or labelled with cTnT and Alexa fluor 647 for FACS analysis (D, E). Nuclei: blue (Hoechst). (C) Quantification of A and B (over 2000 cells counted from 3 independent experiments). (F) Quantification of D and E (20000 cells FACS analyzed for each of 3 independent experiments). Scale bar: 100 µm; *statistical significance p

    Article Snippet: Immunofluorescence stainings were performed with mouse anti-Isl1 antibody (Hybridoma Bank) and rabbit anti-alpha smooth muscle actin (SMA from Abcam), cardiac troponin T (cTnT from Abcam) or CD31 (Abcam).

    Techniques: Immunostaining, FACS

    Isl1-CPC derived cultures have cardiomyocyte-like electrophysiological properties. Action potentials of differentiated Isl1-CPCs from control (A) or Bmp4-treated cultures (B) were recorded after 6 days in culture under whole-cell current clamp mode. (A, B) Representative action potentials demonstrate nodal-like, atrial-like and ventricular-like cardiomyocytes in the absence and presence of Bmp4. (C) Action potential amplitude (APA), action potential duration at the 90% of repolarization (APD 90 ) and maximum diastolic potential (MDP) for ventricular-like cardiomyocytes were quantified and depicted as bar graph for both control and Bmp4-treated cultures. A total of 29 cardiomyocytes derived from Isl1-CPCs was analyzed from 4 independent experiments. There were no significant differences for APA, MDP and APD 90 between control and Bmp4 treated samples. The details for AP measurement setting are under Materials and Methods .

    Journal: PLoS ONE

    Article Title: Functional Cardiomyocytes Derived from Isl1 Cardiac Progenitors via Bmp4 Stimulation

    doi: 10.1371/journal.pone.0110752

    Figure Lengend Snippet: Isl1-CPC derived cultures have cardiomyocyte-like electrophysiological properties. Action potentials of differentiated Isl1-CPCs from control (A) or Bmp4-treated cultures (B) were recorded after 6 days in culture under whole-cell current clamp mode. (A, B) Representative action potentials demonstrate nodal-like, atrial-like and ventricular-like cardiomyocytes in the absence and presence of Bmp4. (C) Action potential amplitude (APA), action potential duration at the 90% of repolarization (APD 90 ) and maximum diastolic potential (MDP) for ventricular-like cardiomyocytes were quantified and depicted as bar graph for both control and Bmp4-treated cultures. A total of 29 cardiomyocytes derived from Isl1-CPCs was analyzed from 4 independent experiments. There were no significant differences for APA, MDP and APD 90 between control and Bmp4 treated samples. The details for AP measurement setting are under Materials and Methods .

    Article Snippet: Immunofluorescence stainings were performed with mouse anti-Isl1 antibody (Hybridoma Bank) and rabbit anti-alpha smooth muscle actin (SMA from Abcam), cardiac troponin T (cTnT from Abcam) or CD31 (Abcam).

    Techniques: Derivative Assay

    Bmp4 signaling enhances transcription factor Tbx5 and Tbx20 mRNA expression. FACS-sorted GFP+ Isl1-CPCs were cultured in the presence of Bmp4 or vehicle control. mRNA was collected following 2, 4 and 6 days of Bmp4 treatment. qRT-PCR analysis of duplicates from three independent experiments for tbx5 (A) and tbx20 (B) expression is normalized by housekeeping gene, Gapdh. *p

    Journal: PLoS ONE

    Article Title: Functional Cardiomyocytes Derived from Isl1 Cardiac Progenitors via Bmp4 Stimulation

    doi: 10.1371/journal.pone.0110752

    Figure Lengend Snippet: Bmp4 signaling enhances transcription factor Tbx5 and Tbx20 mRNA expression. FACS-sorted GFP+ Isl1-CPCs were cultured in the presence of Bmp4 or vehicle control. mRNA was collected following 2, 4 and 6 days of Bmp4 treatment. qRT-PCR analysis of duplicates from three independent experiments for tbx5 (A) and tbx20 (B) expression is normalized by housekeeping gene, Gapdh. *p

    Article Snippet: Immunofluorescence stainings were performed with mouse anti-Isl1 antibody (Hybridoma Bank) and rabbit anti-alpha smooth muscle actin (SMA from Abcam), cardiac troponin T (cTnT from Abcam) or CD31 (Abcam).

    Techniques: Expressing, FACS, Cell Culture, Quantitative RT-PCR

    Calcium transients of cardiomyocytes derived from Isl1-CPC after cell loading with Ca 2+ indicator fura-2. (A) Representative recordings for increase in [Ca 2+ ]i transient amplitude and frequency was measured following loading of control and Bmp4-treated cardiomyocyte cultures with Ca 2+ indicator fura-2. Cytosolic Ca 2+ of spontaneous beating cardiomyocyte was measured by ratio of fluorescence intensity at 340 nm and 380 nm (F340/F380). The measurements were recorded 6 days of incubation after Isl1-CPC isolation. (B) The quantitative representation of beating frequency (BF) of calcium transients per minute is shown by bar graphs (n = 3). Experiments were repeated 3 times and 10–15 cells were measured in each experiment. (C) Change of [Ca 2+ ] was calculated from Ca 2+ transient recordings for cardiomyocyte cultures differentiated in the absence or presence of Bmp4. *p

    Journal: PLoS ONE

    Article Title: Functional Cardiomyocytes Derived from Isl1 Cardiac Progenitors via Bmp4 Stimulation

    doi: 10.1371/journal.pone.0110752

    Figure Lengend Snippet: Calcium transients of cardiomyocytes derived from Isl1-CPC after cell loading with Ca 2+ indicator fura-2. (A) Representative recordings for increase in [Ca 2+ ]i transient amplitude and frequency was measured following loading of control and Bmp4-treated cardiomyocyte cultures with Ca 2+ indicator fura-2. Cytosolic Ca 2+ of spontaneous beating cardiomyocyte was measured by ratio of fluorescence intensity at 340 nm and 380 nm (F340/F380). The measurements were recorded 6 days of incubation after Isl1-CPC isolation. (B) The quantitative representation of beating frequency (BF) of calcium transients per minute is shown by bar graphs (n = 3). Experiments were repeated 3 times and 10–15 cells were measured in each experiment. (C) Change of [Ca 2+ ] was calculated from Ca 2+ transient recordings for cardiomyocyte cultures differentiated in the absence or presence of Bmp4. *p

    Article Snippet: Immunofluorescence stainings were performed with mouse anti-Isl1 antibody (Hybridoma Bank) and rabbit anti-alpha smooth muscle actin (SMA from Abcam), cardiac troponin T (cTnT from Abcam) or CD31 (Abcam).

    Techniques: Derivative Assay, Fluorescence, Incubation, Isolation

    Loss of SMN reactivates the cell cycle. a Ki67 and ISL1 immunostaining analysis of wild-type (BJ iPS and 18a), SMA Type I (1-38 G), and SMA Type II (1-51 N) motor neuron cultures at day 28. The percentages of ISL1 + Ki67 + cells amongst all ISL1 + motor neurons are shown. b Knockdown of SMN in wild-type cell line (BJ-iPS) increased the percentage of ISL1 + motor neurons co-expressing Ki67. c Co-staining of ISL1 (red) and Ki67 (green) showing increased Ki67 + cells upon SMN knockdown in BJ-iPS motor neuron cultures. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. d Ki67 and cCASP3 immunostaining analysis of wild-type motor neurons demonstrated higher cCASP3 expression in Ki67 + motor neurons than Ki67 − motor neurons. *** p

    Journal: Cell Death & Disease

    Article Title: Cell cycle inhibitors protect motor neurons in an organoid model of Spinal Muscular Atrophy

    doi: 10.1038/s41419-018-1081-0

    Figure Lengend Snippet: Loss of SMN reactivates the cell cycle. a Ki67 and ISL1 immunostaining analysis of wild-type (BJ iPS and 18a), SMA Type I (1-38 G), and SMA Type II (1-51 N) motor neuron cultures at day 28. The percentages of ISL1 + Ki67 + cells amongst all ISL1 + motor neurons are shown. b Knockdown of SMN in wild-type cell line (BJ-iPS) increased the percentage of ISL1 + motor neurons co-expressing Ki67. c Co-staining of ISL1 (red) and Ki67 (green) showing increased Ki67 + cells upon SMN knockdown in BJ-iPS motor neuron cultures. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. d Ki67 and cCASP3 immunostaining analysis of wild-type motor neurons demonstrated higher cCASP3 expression in Ki67 + motor neurons than Ki67 − motor neurons. *** p

    Article Snippet: The following primary antibodies (and their respective dilutions) were used: rabbit SOX1 (1:1000) (Abcam, ab87775), mouse Nestin (1:1000) (Abcam, ab22035), rabbit ISL1 (1:1500) (Abcam, ab109517), rabbit cleaved Caspase-3 (1:1000) (Cell Signaling Technology, #9661), mouse Ki67 (1:1500) (Cell Signaling Technology, #9449), mouse SMI-32 (1:1000) (Calbiochem, NE-1023), mouse SMN (1:400) (BD Pharmingen, 610647), rabbit Ki67 (1:250) (Abcam, ab16667), mouse TUJ1 (1:2000) (Biolegend, #801202), goat SOX10 (1:100) (Santa Cruz Biotechnologies, sc-17342), rabbit HOXB4 (1:200) (Abcam, ab133521), rabbit HOXC8 (1:200) (Abcam, ab86236), rabbit Calbindin (1:1000) (Abcam, ab11426), mouse FoxP1 (1:100) (R & D Systems, MAB45341), and sheep Chx10 (1:200) (Abcam, ab16141).

    Techniques: Immunostaining, Expressing, Staining

    Inhibition of CDKs prolongs SMA motor neuron survival. a Graphical representation of ISL1 + SMA type I motor neurons (1-38 G) treated with various CDKs inhibitors treatment. The blue dotted line indicates percentage of motor neurons relative to DMSO-treated motor neurons. b Quantification of ISL1 + SMA type II motor neurons (1-51 N) treated with various CDKs inhibitors treatment. The blue dotted line indicates percentage of motor neurons relative to DMSO-treated motor neurons. c ISL1 immunostaining analysis of various CDKs knockdown in SMA type II motor neurons. The blue dotted line indicates percentage of motor neurons survival relative to non-targeting siRNA treated motor neurons. d Representative images of SMA type II motor neurons treated with various CDKs siRNA and stained with ISL1 (red) and SMI-32 (green). Cellular nuclei were counterstained with DAPI. Scale bars, 50 μm. e Western blot of SMA type II motor neurons treated with various CDKs inhibitors, indicating that SMN levels remained the same. f Quantification of SMN levels of SMA type II motor neurons treated with various CDKs inhibitors relative to α-tubulin expression. The values were not significant. * p

    Journal: Cell Death & Disease

    Article Title: Cell cycle inhibitors protect motor neurons in an organoid model of Spinal Muscular Atrophy

    doi: 10.1038/s41419-018-1081-0

    Figure Lengend Snippet: Inhibition of CDKs prolongs SMA motor neuron survival. a Graphical representation of ISL1 + SMA type I motor neurons (1-38 G) treated with various CDKs inhibitors treatment. The blue dotted line indicates percentage of motor neurons relative to DMSO-treated motor neurons. b Quantification of ISL1 + SMA type II motor neurons (1-51 N) treated with various CDKs inhibitors treatment. The blue dotted line indicates percentage of motor neurons relative to DMSO-treated motor neurons. c ISL1 immunostaining analysis of various CDKs knockdown in SMA type II motor neurons. The blue dotted line indicates percentage of motor neurons survival relative to non-targeting siRNA treated motor neurons. d Representative images of SMA type II motor neurons treated with various CDKs siRNA and stained with ISL1 (red) and SMI-32 (green). Cellular nuclei were counterstained with DAPI. Scale bars, 50 μm. e Western blot of SMA type II motor neurons treated with various CDKs inhibitors, indicating that SMN levels remained the same. f Quantification of SMN levels of SMA type II motor neurons treated with various CDKs inhibitors relative to α-tubulin expression. The values were not significant. * p

    Article Snippet: The following primary antibodies (and their respective dilutions) were used: rabbit SOX1 (1:1000) (Abcam, ab87775), mouse Nestin (1:1000) (Abcam, ab22035), rabbit ISL1 (1:1500) (Abcam, ab109517), rabbit cleaved Caspase-3 (1:1000) (Cell Signaling Technology, #9661), mouse Ki67 (1:1500) (Cell Signaling Technology, #9449), mouse SMI-32 (1:1000) (Calbiochem, NE-1023), mouse SMN (1:400) (BD Pharmingen, 610647), rabbit Ki67 (1:250) (Abcam, ab16667), mouse TUJ1 (1:2000) (Biolegend, #801202), goat SOX10 (1:100) (Santa Cruz Biotechnologies, sc-17342), rabbit HOXB4 (1:200) (Abcam, ab133521), rabbit HOXC8 (1:200) (Abcam, ab86236), rabbit Calbindin (1:1000) (Abcam, ab11426), mouse FoxP1 (1:100) (R & D Systems, MAB45341), and sheep Chx10 (1:200) (Abcam, ab16141).

    Techniques: Inhibition, Immunostaining, Staining, Western Blot, Expressing

    CDK inhibitor reversed motor neuron death in SMA spinal organoids. a Co-staining of ISL1 (red) and SMI-32 (green) in SMA type I spinal organoids treated with DMSO and PD0332991. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. b SMA type I and c SMA type II spinal organoids shows increased MN survival. * p

    Journal: Cell Death & Disease

    Article Title: Cell cycle inhibitors protect motor neurons in an organoid model of Spinal Muscular Atrophy

    doi: 10.1038/s41419-018-1081-0

    Figure Lengend Snippet: CDK inhibitor reversed motor neuron death in SMA spinal organoids. a Co-staining of ISL1 (red) and SMI-32 (green) in SMA type I spinal organoids treated with DMSO and PD0332991. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. b SMA type I and c SMA type II spinal organoids shows increased MN survival. * p

    Article Snippet: The following primary antibodies (and their respective dilutions) were used: rabbit SOX1 (1:1000) (Abcam, ab87775), mouse Nestin (1:1000) (Abcam, ab22035), rabbit ISL1 (1:1500) (Abcam, ab109517), rabbit cleaved Caspase-3 (1:1000) (Cell Signaling Technology, #9661), mouse Ki67 (1:1500) (Cell Signaling Technology, #9449), mouse SMI-32 (1:1000) (Calbiochem, NE-1023), mouse SMN (1:400) (BD Pharmingen, 610647), rabbit Ki67 (1:250) (Abcam, ab16667), mouse TUJ1 (1:2000) (Biolegend, #801202), goat SOX10 (1:100) (Santa Cruz Biotechnologies, sc-17342), rabbit HOXB4 (1:200) (Abcam, ab133521), rabbit HOXC8 (1:200) (Abcam, ab86236), rabbit Calbindin (1:1000) (Abcam, ab11426), mouse FoxP1 (1:100) (R & D Systems, MAB45341), and sheep Chx10 (1:200) (Abcam, ab16141).

    Techniques: Staining

    Generation of three-dimensional spinal organoids from human iPSCs. a Schematic illustration of spinal organoids differentiation from iPSC. b Co-staining of SOX1 (red) and Nestin (green) illustrating successful generation of neural progenitors in BJ-iPS motor neuron cultures. Cellular nuclei were counterstained with DAPI. Scale bars, 50 μm. c Representative images BJ-iPS spinal organoids at respective time points stained with SOX1 (red) and TUJ1 (green). Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. d Quantification of SOX1 + levels percentage of BJ-iPS spinal organoids at respective time points relative to total cell number. e Representative images of BJ-iPS spinal organoids demonstrating SOX1 + (green) and ISL1 + (red) in an apical-to-basal patterning. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. f Representative images of BJ-iPS spinal organoids at respective time points stained with ISL1 (red) and SMI-32 (green). Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. g Graph shows percentage of ISL1 + at day 21, 28, and 35 in BJ-iPS spinal organoids relative to total cell number. * p

    Journal: Cell Death & Disease

    Article Title: Cell cycle inhibitors protect motor neurons in an organoid model of Spinal Muscular Atrophy

    doi: 10.1038/s41419-018-1081-0

    Figure Lengend Snippet: Generation of three-dimensional spinal organoids from human iPSCs. a Schematic illustration of spinal organoids differentiation from iPSC. b Co-staining of SOX1 (red) and Nestin (green) illustrating successful generation of neural progenitors in BJ-iPS motor neuron cultures. Cellular nuclei were counterstained with DAPI. Scale bars, 50 μm. c Representative images BJ-iPS spinal organoids at respective time points stained with SOX1 (red) and TUJ1 (green). Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. d Quantification of SOX1 + levels percentage of BJ-iPS spinal organoids at respective time points relative to total cell number. e Representative images of BJ-iPS spinal organoids demonstrating SOX1 + (green) and ISL1 + (red) in an apical-to-basal patterning. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. f Representative images of BJ-iPS spinal organoids at respective time points stained with ISL1 (red) and SMI-32 (green). Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. g Graph shows percentage of ISL1 + at day 21, 28, and 35 in BJ-iPS spinal organoids relative to total cell number. * p

    Article Snippet: The following primary antibodies (and their respective dilutions) were used: rabbit SOX1 (1:1000) (Abcam, ab87775), mouse Nestin (1:1000) (Abcam, ab22035), rabbit ISL1 (1:1500) (Abcam, ab109517), rabbit cleaved Caspase-3 (1:1000) (Cell Signaling Technology, #9661), mouse Ki67 (1:1500) (Cell Signaling Technology, #9449), mouse SMI-32 (1:1000) (Calbiochem, NE-1023), mouse SMN (1:400) (BD Pharmingen, 610647), rabbit Ki67 (1:250) (Abcam, ab16667), mouse TUJ1 (1:2000) (Biolegend, #801202), goat SOX10 (1:100) (Santa Cruz Biotechnologies, sc-17342), rabbit HOXB4 (1:200) (Abcam, ab133521), rabbit HOXC8 (1:200) (Abcam, ab86236), rabbit Calbindin (1:1000) (Abcam, ab11426), mouse FoxP1 (1:100) (R & D Systems, MAB45341), and sheep Chx10 (1:200) (Abcam, ab16141).

    Techniques: Staining

    Spinal organoids consists of various spinal cord cell types. Representative images illustrating the presence of a HOXB4 + and b HOXC8 + cells in spinal organoids. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. c Co-staining of FOXP1 (green) and ISL1 (red) demonstrates presence of limb-innervating neurons in spinal organoids. Scale bars, 100 μm. d Representative images of spinal organoids at day 42 stained with ISL1 (red) and ChAT (green). Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. Spinal organoids are stained with e CHX10 + cells (RED) and f CALB + cells (green). Scale bars, 100 μm. g Co-staining of S100β and TUJ1 shows presence of astrocytes in spinal organoids. Scale bars, 100 μm. h Quantitative-PCR analysis demonstrates a lack of dorsal cell types in the spinal organoids generated

    Journal: Cell Death & Disease

    Article Title: Cell cycle inhibitors protect motor neurons in an organoid model of Spinal Muscular Atrophy

    doi: 10.1038/s41419-018-1081-0

    Figure Lengend Snippet: Spinal organoids consists of various spinal cord cell types. Representative images illustrating the presence of a HOXB4 + and b HOXC8 + cells in spinal organoids. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. c Co-staining of FOXP1 (green) and ISL1 (red) demonstrates presence of limb-innervating neurons in spinal organoids. Scale bars, 100 μm. d Representative images of spinal organoids at day 42 stained with ISL1 (red) and ChAT (green). Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. Spinal organoids are stained with e CHX10 + cells (RED) and f CALB + cells (green). Scale bars, 100 μm. g Co-staining of S100β and TUJ1 shows presence of astrocytes in spinal organoids. Scale bars, 100 μm. h Quantitative-PCR analysis demonstrates a lack of dorsal cell types in the spinal organoids generated

    Article Snippet: The following primary antibodies (and their respective dilutions) were used: rabbit SOX1 (1:1000) (Abcam, ab87775), mouse Nestin (1:1000) (Abcam, ab22035), rabbit ISL1 (1:1500) (Abcam, ab109517), rabbit cleaved Caspase-3 (1:1000) (Cell Signaling Technology, #9661), mouse Ki67 (1:1500) (Cell Signaling Technology, #9449), mouse SMI-32 (1:1000) (Calbiochem, NE-1023), mouse SMN (1:400) (BD Pharmingen, 610647), rabbit Ki67 (1:250) (Abcam, ab16667), mouse TUJ1 (1:2000) (Biolegend, #801202), goat SOX10 (1:100) (Santa Cruz Biotechnologies, sc-17342), rabbit HOXB4 (1:200) (Abcam, ab133521), rabbit HOXC8 (1:200) (Abcam, ab86236), rabbit Calbindin (1:1000) (Abcam, ab11426), mouse FoxP1 (1:100) (R & D Systems, MAB45341), and sheep Chx10 (1:200) (Abcam, ab16141).

    Techniques: Staining, Real-time Polymerase Chain Reaction, Generated

    Cell cycle genes are upregulated in SMA motor neurons. a Motor neurons were purified based on HB9 immunoreactivity. mRNA expression levels of CDKs and cyclins measured by RNA-seq and qPCR respectively. Graph shows fold change comparing SMA HB9 + motor neurons to wild-type HB9 + motor neurons. The blue dotted line indicates relative expression of wild-type HB9 + motor neurons. b qPCR analysis of ISL1 + motor neurons derived from day 28 organoids. Graph shows mRNA fold change, relative to BJ ISL1 + motor neurons. c Knockdown of SMN in wild-type motor neuron cultures revealed upregulation of cell cycle genes. * p

    Journal: Cell Death & Disease

    Article Title: Cell cycle inhibitors protect motor neurons in an organoid model of Spinal Muscular Atrophy

    doi: 10.1038/s41419-018-1081-0

    Figure Lengend Snippet: Cell cycle genes are upregulated in SMA motor neurons. a Motor neurons were purified based on HB9 immunoreactivity. mRNA expression levels of CDKs and cyclins measured by RNA-seq and qPCR respectively. Graph shows fold change comparing SMA HB9 + motor neurons to wild-type HB9 + motor neurons. The blue dotted line indicates relative expression of wild-type HB9 + motor neurons. b qPCR analysis of ISL1 + motor neurons derived from day 28 organoids. Graph shows mRNA fold change, relative to BJ ISL1 + motor neurons. c Knockdown of SMN in wild-type motor neuron cultures revealed upregulation of cell cycle genes. * p

    Article Snippet: The following primary antibodies (and their respective dilutions) were used: rabbit SOX1 (1:1000) (Abcam, ab87775), mouse Nestin (1:1000) (Abcam, ab22035), rabbit ISL1 (1:1500) (Abcam, ab109517), rabbit cleaved Caspase-3 (1:1000) (Cell Signaling Technology, #9661), mouse Ki67 (1:1500) (Cell Signaling Technology, #9449), mouse SMI-32 (1:1000) (Calbiochem, NE-1023), mouse SMN (1:400) (BD Pharmingen, 610647), rabbit Ki67 (1:250) (Abcam, ab16667), mouse TUJ1 (1:2000) (Biolegend, #801202), goat SOX10 (1:100) (Santa Cruz Biotechnologies, sc-17342), rabbit HOXB4 (1:200) (Abcam, ab133521), rabbit HOXC8 (1:200) (Abcam, ab86236), rabbit Calbindin (1:1000) (Abcam, ab11426), mouse FoxP1 (1:100) (R & D Systems, MAB45341), and sheep Chx10 (1:200) (Abcam, ab16141).

    Techniques: Purification, Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Derivative Assay

    SMA organoids shows reduced motor neuron survival. a Co-staining of SOX1 (red) and TUJ1 (green) in SMA Type I (1-38 G) and SMA Type II (1-51 N) spinal organoids at respective time points. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. b Quantification of SOX1 + levels percentage of SMA Type I and Type II spinal organoids at respective time points relative to total cell number. The values were not significant. c Representative images of SMA Type I and Type II spinal organoids at respective time points stained with ISL1 (red) and SMI-32 (green). Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. d Graph shows percentage of ISL1 + at day 21, 28, and 35 in SMA Type I and Type II spinal organoids relative to total cell number. ** p

    Journal: Cell Death & Disease

    Article Title: Cell cycle inhibitors protect motor neurons in an organoid model of Spinal Muscular Atrophy

    doi: 10.1038/s41419-018-1081-0

    Figure Lengend Snippet: SMA organoids shows reduced motor neuron survival. a Co-staining of SOX1 (red) and TUJ1 (green) in SMA Type I (1-38 G) and SMA Type II (1-51 N) spinal organoids at respective time points. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. b Quantification of SOX1 + levels percentage of SMA Type I and Type II spinal organoids at respective time points relative to total cell number. The values were not significant. c Representative images of SMA Type I and Type II spinal organoids at respective time points stained with ISL1 (red) and SMI-32 (green). Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. d Graph shows percentage of ISL1 + at day 21, 28, and 35 in SMA Type I and Type II spinal organoids relative to total cell number. ** p

    Article Snippet: The following primary antibodies (and their respective dilutions) were used: rabbit SOX1 (1:1000) (Abcam, ab87775), mouse Nestin (1:1000) (Abcam, ab22035), rabbit ISL1 (1:1500) (Abcam, ab109517), rabbit cleaved Caspase-3 (1:1000) (Cell Signaling Technology, #9661), mouse Ki67 (1:1500) (Cell Signaling Technology, #9449), mouse SMI-32 (1:1000) (Calbiochem, NE-1023), mouse SMN (1:400) (BD Pharmingen, 610647), rabbit Ki67 (1:250) (Abcam, ab16667), mouse TUJ1 (1:2000) (Biolegend, #801202), goat SOX10 (1:100) (Santa Cruz Biotechnologies, sc-17342), rabbit HOXB4 (1:200) (Abcam, ab133521), rabbit HOXC8 (1:200) (Abcam, ab86236), rabbit Calbindin (1:1000) (Abcam, ab11426), mouse FoxP1 (1:100) (R & D Systems, MAB45341), and sheep Chx10 (1:200) (Abcam, ab16141).

    Techniques: Staining

    ISL1 directly regulates a number of genes required for normal pacemaker function in mice and human.

    Journal: The Journal of Clinical Investigation

    Article Title: Transcription factor ISL1 is essential for pacemaker development and function

    doi: 10.1172/JCI68257

    Figure Lengend Snippet: ISL1 directly regulates a number of genes required for normal pacemaker function in mice and human.

    Article Snippet: The following primary antibodies were used: mouse monoclonal anti-ISL1/2 (39.4D5, Developmental Studies Hybridoma Bank [DSHB]), rabbit anti-ISL1 (ab20670, Abcam), rat anti-HCN4 (ab32675, Abcam), rabbit anti-Cx40 (sc-28658, Santa Cruz), goat anti-TBX3 (sc-17871, Santa Cruz Biotechnology Inc.), and rat anti-BrdU (ab6326, Abcam).

    Techniques: Mouse Assay

    Bradycardia and reduced TBX3 and HCN4 expression following ablation of Isl1 during later SAN morphogenesis. ( A ) Ablation of Isl1 at E11.5 led to significantly slower heart rate at E12.5 and E14.5 ( n = 15 per group, P

    Journal: The Journal of Clinical Investigation

    Article Title: Transcription factor ISL1 is essential for pacemaker development and function

    doi: 10.1172/JCI68257

    Figure Lengend Snippet: Bradycardia and reduced TBX3 and HCN4 expression following ablation of Isl1 during later SAN morphogenesis. ( A ) Ablation of Isl1 at E11.5 led to significantly slower heart rate at E12.5 and E14.5 ( n = 15 per group, P

    Article Snippet: The following primary antibodies were used: mouse monoclonal anti-ISL1/2 (39.4D5, Developmental Studies Hybridoma Bank [DSHB]), rabbit anti-ISL1 (ab20670, Abcam), rat anti-HCN4 (ab32675, Abcam), rabbit anti-Cx40 (sc-28658, Santa Cruz), goat anti-TBX3 (sc-17871, Santa Cruz Biotechnology Inc.), and rat anti-BrdU (ab6326, Abcam).

    Techniques: Expressing

    Bradycardia and loss of SAN cells in Isl1 compound mutants. ISL1-nLacZ was expressed in SV myocardium, including the SAN region (red arrow), and mesocardium at E9.5 ( A and C ) and E11.5 ( G and I ). Expression of ISL1 and HCN4 in the SV region of Isl1 compound mutant embryos was significantly reduced ( E and F ). Expression of ISL1 and the number of ISL1-expressing cells in the SV, SAN (red arrow), and DM was markedly reduced in Isl1 compound mutant embryos at E9.5 ( B and D ) and E11.5 ( H and J ). BrdU staining revealed significantly reduced proliferation of SV myocardium in Isl1 compound mutants at E9.5 ( K – M ). TUNEL labeling showed significantly increased cell death in the SV of Isl1 compound mutant embryos at E10.5 ( N – P ) ( n = 4 per group. Scale bars as shown). Echocardiography revealed a significant reduction in the heart rate of Isl1 compound mutant embryos at E9.5 and E11.5 ( Q ). n = 15 per group; * P

    Journal: The Journal of Clinical Investigation

    Article Title: Transcription factor ISL1 is essential for pacemaker development and function

    doi: 10.1172/JCI68257

    Figure Lengend Snippet: Bradycardia and loss of SAN cells in Isl1 compound mutants. ISL1-nLacZ was expressed in SV myocardium, including the SAN region (red arrow), and mesocardium at E9.5 ( A and C ) and E11.5 ( G and I ). Expression of ISL1 and HCN4 in the SV region of Isl1 compound mutant embryos was significantly reduced ( E and F ). Expression of ISL1 and the number of ISL1-expressing cells in the SV, SAN (red arrow), and DM was markedly reduced in Isl1 compound mutant embryos at E9.5 ( B and D ) and E11.5 ( H and J ). BrdU staining revealed significantly reduced proliferation of SV myocardium in Isl1 compound mutants at E9.5 ( K – M ). TUNEL labeling showed significantly increased cell death in the SV of Isl1 compound mutant embryos at E10.5 ( N – P ) ( n = 4 per group. Scale bars as shown). Echocardiography revealed a significant reduction in the heart rate of Isl1 compound mutant embryos at E9.5 and E11.5 ( Q ). n = 15 per group; * P

    Article Snippet: The following primary antibodies were used: mouse monoclonal anti-ISL1/2 (39.4D5, Developmental Studies Hybridoma Bank [DSHB]), rabbit anti-ISL1 (ab20670, Abcam), rat anti-HCN4 (ab32675, Abcam), rabbit anti-Cx40 (sc-28658, Santa Cruz), goat anti-TBX3 (sc-17871, Santa Cruz Biotechnology Inc.), and rat anti-BrdU (ab6326, Abcam).

    Techniques: Expressing, Mutagenesis, BrdU Staining, TUNEL Assay, Labeling

    Reduced expression of Hcn4 , Tbx3 , and Shox2 in the SAN region of Isl1 compound mutant embryos. At E9.5, Hcn4 and Shox2 were expressed in the SV, and SAN region (red arrow; A , C , E , and G ). Tbx3 was expressed in the SV and surrounding mesenchyme (red arrow; I and K ). In Isl1 compound mutant embryos, expression of Hcn4, Shox2, and Tbx3 in the SV and SAN region was markedly reduced ( B , D , F , H , J , and L ). Cx40 and Nkx2-5 were expressed in working myocardium but not in the SAN region ( M , O , Q , and S In Isl1 compound mutant embryos, expression of Cx40 and Nkx2-5 was markedly reduced in atrial myocardium, but no expansion or ectopic expression of Cx40 or Nkx2-5 was observed in the SAN region ( N , P , R , and T ). n = 4 per group, Scale bars as shown.

    Journal: The Journal of Clinical Investigation

    Article Title: Transcription factor ISL1 is essential for pacemaker development and function

    doi: 10.1172/JCI68257

    Figure Lengend Snippet: Reduced expression of Hcn4 , Tbx3 , and Shox2 in the SAN region of Isl1 compound mutant embryos. At E9.5, Hcn4 and Shox2 were expressed in the SV, and SAN region (red arrow; A , C , E , and G ). Tbx3 was expressed in the SV and surrounding mesenchyme (red arrow; I and K ). In Isl1 compound mutant embryos, expression of Hcn4, Shox2, and Tbx3 in the SV and SAN region was markedly reduced ( B , D , F , H , J , and L ). Cx40 and Nkx2-5 were expressed in working myocardium but not in the SAN region ( M , O , Q , and S In Isl1 compound mutant embryos, expression of Cx40 and Nkx2-5 was markedly reduced in atrial myocardium, but no expansion or ectopic expression of Cx40 or Nkx2-5 was observed in the SAN region ( N , P , R , and T ). n = 4 per group, Scale bars as shown.

    Article Snippet: The following primary antibodies were used: mouse monoclonal anti-ISL1/2 (39.4D5, Developmental Studies Hybridoma Bank [DSHB]), rabbit anti-ISL1 (ab20670, Abcam), rat anti-HCN4 (ab32675, Abcam), rabbit anti-Cx40 (sc-28658, Santa Cruz), goat anti-TBX3 (sc-17871, Santa Cruz Biotechnology Inc.), and rat anti-BrdU (ab6326, Abcam).

    Techniques: Expressing, Mutagenesis

    Bradycardia and loss of SAN cells following ablation of Isl1 in SAN during early developmental stages using Hcn4-CreERT2 . Isl1 mutant ( Hcn4-CreERT2 Isl1 fl/fl ) and control ( Hcn4-CreERT2 Isl1 fl/+ or +/+ ) embryos were given tamoxifen at E9.5. Embryos were analyzed 36 and 48 hours after induction. ( A ) Echocardiography revealed that the heart rate of Isl1 mutants was significantly reduced at E11 and was further reduced at E11.5 ( n = 20 per group). ( B – D ) Whole-mount X-gal staining and quantitative analysis revealed a significantly reduced number of X-gal + and Tomato + cells in the SAN (red arrow) of Isl1 mutants relative to control littermates at E11.5 ( n = 4. Scale bars as shown). ( D – H ) Immunostaining demonstrated significantly reduced expression of HCN4 and TBX3 in the SAN of Isl1 mutants compared with controls marked by Tomato + at E11.5. However, a slight but not significant reduction in the number of Hcn4 lineage–labeled cells in Isl1 mutant SAN region was observed when analyzed at E11 ( D , I , and J ). ( K – M ) TUNEL revealed increased cell death in Isl1 mutant SAN marked by Tomato + . ( N – P ) BrdU revealed decreased proliferation in Isl1 mutant SAN marked by Tomato. n = 4; * P

    Journal: The Journal of Clinical Investigation

    Article Title: Transcription factor ISL1 is essential for pacemaker development and function

    doi: 10.1172/JCI68257

    Figure Lengend Snippet: Bradycardia and loss of SAN cells following ablation of Isl1 in SAN during early developmental stages using Hcn4-CreERT2 . Isl1 mutant ( Hcn4-CreERT2 Isl1 fl/fl ) and control ( Hcn4-CreERT2 Isl1 fl/+ or +/+ ) embryos were given tamoxifen at E9.5. Embryos were analyzed 36 and 48 hours after induction. ( A ) Echocardiography revealed that the heart rate of Isl1 mutants was significantly reduced at E11 and was further reduced at E11.5 ( n = 20 per group). ( B – D ) Whole-mount X-gal staining and quantitative analysis revealed a significantly reduced number of X-gal + and Tomato + cells in the SAN (red arrow) of Isl1 mutants relative to control littermates at E11.5 ( n = 4. Scale bars as shown). ( D – H ) Immunostaining demonstrated significantly reduced expression of HCN4 and TBX3 in the SAN of Isl1 mutants compared with controls marked by Tomato + at E11.5. However, a slight but not significant reduction in the number of Hcn4 lineage–labeled cells in Isl1 mutant SAN region was observed when analyzed at E11 ( D , I , and J ). ( K – M ) TUNEL revealed increased cell death in Isl1 mutant SAN marked by Tomato + . ( N – P ) BrdU revealed decreased proliferation in Isl1 mutant SAN marked by Tomato. n = 4; * P

    Article Snippet: The following primary antibodies were used: mouse monoclonal anti-ISL1/2 (39.4D5, Developmental Studies Hybridoma Bank [DSHB]), rabbit anti-ISL1 (ab20670, Abcam), rat anti-HCN4 (ab32675, Abcam), rabbit anti-Cx40 (sc-28658, Santa Cruz), goat anti-TBX3 (sc-17871, Santa Cruz Biotechnology Inc.), and rat anti-BrdU (ab6326, Abcam).

    Techniques: Mutagenesis, Staining, Immunostaining, Expressing, Labeling, TUNEL Assay

    RNA-seq analyses reveal dysregulation of a number of genes important for SAN function in Hcn4-CreERT2 Isl1 fl/fl mutants. ( A ) Scatterplot illustrating relative gene expression of polyA-selected RNA transcripts from RNA-seq comparison of control and Hcn4-CreERT2 Isl1 fl/fl mutant SAN cells. Genes upregulated or downregulated 1.5-fold in Isl1 mutant SAN cells are shown in red and green, respectively. Values are presented as log2 of tag counts normalized to 10 7 uniquely mapped tags. ( B ) RNA-seq comparison of control and Hcn4-CreERT2 Isl1 fl/fl mutant SAN transcriptomes revealed a total of 12,441 genes expressed (RPKM ≥ 1) in SAN cells, of which 1,035 upregulated and 3,690 downregulated in Isl1 mutant SAN cells ( |fold-change mutant vs. ctrl| ≥ 1.5). ( C ) GO functional clustering of genes down- and upregulated in Isl1 mutant, highlighting cellular processes most significantly affected in mutant SAN (top 10 not redundant categories are shown). ( D ) qPCR validation analysis. mRNA expression of ion channels and associated genes, and genes involved in transcription regulation, cell cycle, and signaling pathways are shown. ( E ) qRT-PCR validation analysis. mRNA expression of atrial myocardial specific genes. Results are shown as fold-change Isl1 mutant vs. ctrl. n = 4 per group, P

    Journal: The Journal of Clinical Investigation

    Article Title: Transcription factor ISL1 is essential for pacemaker development and function

    doi: 10.1172/JCI68257

    Figure Lengend Snippet: RNA-seq analyses reveal dysregulation of a number of genes important for SAN function in Hcn4-CreERT2 Isl1 fl/fl mutants. ( A ) Scatterplot illustrating relative gene expression of polyA-selected RNA transcripts from RNA-seq comparison of control and Hcn4-CreERT2 Isl1 fl/fl mutant SAN cells. Genes upregulated or downregulated 1.5-fold in Isl1 mutant SAN cells are shown in red and green, respectively. Values are presented as log2 of tag counts normalized to 10 7 uniquely mapped tags. ( B ) RNA-seq comparison of control and Hcn4-CreERT2 Isl1 fl/fl mutant SAN transcriptomes revealed a total of 12,441 genes expressed (RPKM ≥ 1) in SAN cells, of which 1,035 upregulated and 3,690 downregulated in Isl1 mutant SAN cells ( |fold-change mutant vs. ctrl| ≥ 1.5). ( C ) GO functional clustering of genes down- and upregulated in Isl1 mutant, highlighting cellular processes most significantly affected in mutant SAN (top 10 not redundant categories are shown). ( D ) qPCR validation analysis. mRNA expression of ion channels and associated genes, and genes involved in transcription regulation, cell cycle, and signaling pathways are shown. ( E ) qRT-PCR validation analysis. mRNA expression of atrial myocardial specific genes. Results are shown as fold-change Isl1 mutant vs. ctrl. n = 4 per group, P

    Article Snippet: The following primary antibodies were used: mouse monoclonal anti-ISL1/2 (39.4D5, Developmental Studies Hybridoma Bank [DSHB]), rabbit anti-ISL1 (ab20670, Abcam), rat anti-HCN4 (ab32675, Abcam), rabbit anti-Cx40 (sc-28658, Santa Cruz), goat anti-TBX3 (sc-17871, Santa Cruz Biotechnology Inc.), and rat anti-BrdU (ab6326, Abcam).

    Techniques: RNA Sequencing Assay, Expressing, Mutagenesis, Functional Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Expression of ISL1 in pacemaker cells of the SAN during development and after birth. ISL1 was coexpressed with HCN4 in myocardium of the SV at E9.5 ( A ), and in the majority of SAN cells from E10.5–P7 ( B – G ). ISL1 expression did not overlap with Cx40, which is expressed in atrial myocardium ( E and G ). The boxed area in H delineates regions depicted in F and G . The fraction of HCN4 cells that expressed Isl1 remained constant at early stages from E11.5–E14.5, but decreased at E18 ( I ). After birth, the fraction of HCN4 cells that expressed Isl1 decreased significantly ( I ). n = 4, P

    Journal: The Journal of Clinical Investigation

    Article Title: Transcription factor ISL1 is essential for pacemaker development and function

    doi: 10.1172/JCI68257

    Figure Lengend Snippet: Expression of ISL1 in pacemaker cells of the SAN during development and after birth. ISL1 was coexpressed with HCN4 in myocardium of the SV at E9.5 ( A ), and in the majority of SAN cells from E10.5–P7 ( B – G ). ISL1 expression did not overlap with Cx40, which is expressed in atrial myocardium ( E and G ). The boxed area in H delineates regions depicted in F and G . The fraction of HCN4 cells that expressed Isl1 remained constant at early stages from E11.5–E14.5, but decreased at E18 ( I ). After birth, the fraction of HCN4 cells that expressed Isl1 decreased significantly ( I ). n = 4, P

    Article Snippet: The following primary antibodies were used: mouse monoclonal anti-ISL1/2 (39.4D5, Developmental Studies Hybridoma Bank [DSHB]), rabbit anti-ISL1 (ab20670, Abcam), rat anti-HCN4 (ab32675, Abcam), rabbit anti-Cx40 (sc-28658, Santa Cruz), goat anti-TBX3 (sc-17871, Santa Cruz Biotechnology Inc.), and rat anti-BrdU (ab6326, Abcam).

    Techniques: Expressing