anti islet 1 Search Results


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  • 94
    Abcam anti islet 1
    Effect of fluorofenidone on <t>Islet-1</t> expression in murine retinas
    Anti Islet 1, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    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: 94/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    isl1  (Abcam)
    93
    Abcam isl1
    MED17.11 express the early neuronal markers FOX3 (NeuN), <t>Isl1</t> and Tuj1, and can be transfected with GFP. Immunolabelling of MED17.11 cells cultured in permissive conditions for large T antigen expression. GFP transgene expression in MED17.11. Scale bar is 100 μm.
    Isl1, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Abcam islet1
    TDP-43 localization in patient iPSC-derived motor neurons (A) iPSC-MN stained for TDP-43 shows TDP-43 is ubiquitously expressed and nuclear in all cells including in <t>ISLET1-positive</t> neurons from control (left panel) and ALS (right panel) patients. ISLET1-positive cells from sporadic ALS iPSC-MN have nuclear staining as well as nuclear aggregates that stain with higher intensity for TDP-43 (arrowheads, right panel) but aggregates are not present in healthy controls (left panel). Fibroblasts from healthy control and sALS patients do not show nuclear aggregates. Scale bar: 30 µm. (B) Sporadic ALS patient-derived iPSC-MN cells stained with nuclear envelop marker LAMIN-A (green) and TDP-43 (red) shows TDP-43 aggregates are inside the nuclear envelope; scale bar: 20µm. (C) Quantification of healthy control IPRN.0013 and sALS IPRN.0048 clone 1 iPSC-MN cultures shows TDP-43 aggregation is present in higher fraction of ISLET1 or HB9-positive cells compared to negative cells. (30.7% of ISLET1/HB9-positive motor neurons as compared to 16.2% of ISLET1/HB9-negative cells in sALS iPSC-MN cultures. Bars are standard deviation 9% and 8% respectively; P
    Islet1, supplied by Abcam, used in various techniques. Bioz Stars score: 85/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam anti isl1 islet 1
    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
    Anti Isl1 Islet 1, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Developmental Studies Hybridoma Bank anti isl1
    Pallial boundaries during development . Micrographs of transverse sections through the telencephalon of Xenopus laevis at embryonic (A–D) , premetamorphic (E–G) and prometamorphic (H–N) stages. In each panel the developmental stage and the color code for the used markers are indicated. In the developing telencephalon, the combined immunohistochemical detection of Tbr1, expressed in the pallium, and <t>Isl1,</t> a subpallial marker, clearly allowed the identification of the boundary between both regions throughout the rostrocaudal extent (A–G,J,L–N) . The localization of Tbr1 (H) at rostral level, in comparison with GABA (I) , highlights the olfactory bulb, where GABA was very abundant, in contrast to the pallium, where the Tbr1 expression was observed (H,I) . Simultaneous labeling for GABA and Isl1 discerns the SPa from the pallium (K) . Scale bars = 50 μm (A–G) , 100 μm (H–N) . See list for abbreviations.
    Anti Isl1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 89/100, based on 205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Santa Cruz Biotechnology anti islet 1
    Green fluorescent protein (GFP) expression detected under a fluorescence microscope. The lentiviral vectors carried the GFP gene; thus, a fluorescence microscope was used to detected GFP expression in the C3H10T1/2 cells transfected with Lenti-N and the C3H10T1/2 cells transfected with <t>Lenti-Islet-1</t> at 3 days after transfection. (A) Cell morphology of the C3H10T1/2 cells transfected with Lenti-N observed under a microscope (magnification, ×20). (B) GFP expression of the C3H10T1/2 cells transfected with Lenti-N observed udner a fluorescence microscope (magnification, ×20). (C) Cell morphology of the C3H10T1/2 cells transfected with Lenti-Islet-1 observed under a microscope (magnification, ×20). (D) Cell morphology of the C3H10T1/2 cells transfected with Lenti-Islet-1 observed under a microscope (magnification, ×20). Scale bar, 20 μm. (E and F) Transfection efficiency of teh C3H10T1/2 cells transfected with Lenti-N and the C3H10T1/2 cells transfected with Lenti-Islet-1 detected by flow cytometry (FCM). The transfection efficiency of the C3H10T1/2 cells transduced with lentiviral vectors with pWPI-GFP plasmid or lentiviral vectors with pWPI-GFP-Islet-1 plasmid was detected by FCM. (E) The transfection efficiency of the C3H10T1/2 cells transfected with Lenti-N was 90.12%. (F) The transfection efficiency of the C3H10T1/2 cells transfected with Lenti-Islet-1 was 88.82%.
    Anti Islet 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Epitomics anti islet 1
    DNA methylation levels and acetylation levels of the histone H3K9 site in the GATA4 and Nkx2.5 promoter regions during the differentiation process promoted by <t>Islet-1.</t> (A) The detection of methylation levels on the GATA4 promoter (1329–1489 bp) by MSP assay. (B) The detection of the methylation levels at the Nkx2.5 promoter (51–219 bp) by MSP assay. (C) ChIP results demonstrated the levels of histone acetylation on the promoter regions of GATA4 and Nkx2.5. *P
    Anti Islet 1, supplied by Epitomics, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GeneTex anti isl1
    tbx1+ cells contribute to LPM-derived cardiac lineages. a – p Representative maximum intensity projections of whole-mount tbx1 :EGFP-expressing embryos counterstained for anti-EGFP and anti-MHC ( n = 3) ( a – h ) or <t>anti-Isl1</t> ( n = 11) ( i – p ) at 26 hpf; lateral views, anterior to the left. a – d tbx1 reporter expression can be detected in the MHC-positive linear heart tube and in the MHC-negative poles at the cardiac inflow and outflow tracts (arrowheads); e – h depicts a 2.25x magnification of the framed area in a – d . tbx1 :EGFP also marks the pharyngeal arches (pa) and endothelial cells of the cranial vasculature (cv) ( d ). i – p tbx1 reporter-expressing cells at the IFT co-express the SHF marker Isl1 (asterisks n , o ); m – p depicts a 3x magnification of the framed area in i – l . q Quantification of tbx1 :EGFP/Isl1 double- compared to Isl1 single-positive cells at the IFT of the linear heart tube, n = 11 individual embryos analyzed, means ± s.d. r Lineage tracing of tbx1 and drl reporter-expressing cells, shown in representative embryos. tbx1:creERT2 ( n = 11) or drl:creERT2 ( n = 3) transgenics, respectively, were crossed to the ubiquitous hsp70l:Switch loxP tracer line, embryos were 4-OHT-induced at 90% epiboly, and heat-shocked at 3 dpf. s-a’ Live SPIM imaging of still hearts of representative lineage-traced and control embryos; maximum intensity projections of ventral views, anterior to the top, dashed outlines mark the heart with the bulbus arteriosus (BA), atrium (A), and ventricle (V). s – u tbx1 : creERT2 lineage tracing ( tbx1 > EGFP) at late gastrulation labels myl7 :DsRed2-expressing cardiomyocytes (arrowheads) in the ventricle and inflow tract of the atrium, and DAR-4M-stained cells (arrowhead) in the BA. v – x drl :creERT2 -mediated lineage tracing ( drl > EGFP) at 90% epiboly marks all cardiomyocytes (arrowheads) in the ventricle and atrium, BA cells (arrowhead), and the endocardium (arrows). y - a ’ tbx1:creERT2;hsp70l:Switch transgenics without 4-OHT treatment and heat-shocked at 3 dpf show no specific EGFP expression (asterisks mark the autofluorescent pigment cell). Scale bars 20 μm ( e – h , m – p ), 100 μm ( a – d , i – l , s – a ’)
    Anti Isl1, supplied by GeneTex, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Developmental Studies Hybridoma Bank anti islet 1 2
    Evf-2  and Dlx-2 form a complex in vivo. ( a ) Nuclear extracts made from C17 neural cells transfected with Flag-tagged Emx-1, Flag-tagged Dlx-2, or pcDNA control were analyzed for the presence of  Evf-2 –Dlx-2 complexes by immunoprecipitation with anti-Flag antibody, followed by RT–PCR against  Evf-2 -specific primers and S17 control primers. Western analysis shows that both Flag-Emx-1 and Flag-Dlx-2 are present in nuclear extracts transfected with constructs expressing these proteins. ( b ) Nuclear extracts made from rat E11.5 BAs were analyzed for the presence of  Evf-2 /Dlx-2 complexes by immunoprecipitation with anti- dll,  anti-Islet 1/2, or anti-IgG antibody, followed by RT–PCR against  Evf-2 -specific primers or GAPDH. ( c ) Single-cell suspensions made from mouse E12.5 dorsal and ventral telencephalon were dissected as shown in the schematic, centrifuged onto slides, and processed for fluorescent in situ/immunolocalization using  Evf-2  antisense RNA probe and anti- dll  antibody. DAPI staining (blue) reveals nuclei.  Evf-2  RNA (Alexa fluor 568) is in red, Dlx (Alexa fluor 488) is in green, and regions of overlap are in yellow. ( d ) A model proposing that a complex of  Evf-2  and Dlx-2 affects ei activity, ultimately affecting transcription of the Dlx-5 and Dlx-6 genes.
    Anti Islet 1 2, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 88/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    R&D Systems anti islet 1
    Evf-2  and Dlx-2 form a complex in vivo. ( a ) Nuclear extracts made from C17 neural cells transfected with Flag-tagged Emx-1, Flag-tagged Dlx-2, or pcDNA control were analyzed for the presence of  Evf-2 –Dlx-2 complexes by immunoprecipitation with anti-Flag antibody, followed by RT–PCR against  Evf-2 -specific primers and S17 control primers. Western analysis shows that both Flag-Emx-1 and Flag-Dlx-2 are present in nuclear extracts transfected with constructs expressing these proteins. ( b ) Nuclear extracts made from rat E11.5 BAs were analyzed for the presence of  Evf-2 /Dlx-2 complexes by immunoprecipitation with anti- dll,  anti-Islet 1/2, or anti-IgG antibody, followed by RT–PCR against  Evf-2 -specific primers or GAPDH. ( c ) Single-cell suspensions made from mouse E12.5 dorsal and ventral telencephalon were dissected as shown in the schematic, centrifuged onto slides, and processed for fluorescent in situ/immunolocalization using  Evf-2  antisense RNA probe and anti- dll  antibody. DAPI staining (blue) reveals nuclei.  Evf-2  RNA (Alexa fluor 568) is in red, Dlx (Alexa fluor 488) is in green, and regions of overlap are in yellow. ( d ) A model proposing that a complex of  Evf-2  and Dlx-2 affects ei activity, ultimately affecting transcription of the Dlx-5 and Dlx-6 genes.
    Anti Islet 1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Developmental Studies Hybridoma Bank islet 1 isl1
    Evf-2  and Dlx-2 form a complex in vivo. ( a ) Nuclear extracts made from C17 neural cells transfected with Flag-tagged Emx-1, Flag-tagged Dlx-2, or pcDNA control were analyzed for the presence of  Evf-2 –Dlx-2 complexes by immunoprecipitation with anti-Flag antibody, followed by RT–PCR against  Evf-2 -specific primers and S17 control primers. Western analysis shows that both Flag-Emx-1 and Flag-Dlx-2 are present in nuclear extracts transfected with constructs expressing these proteins. ( b ) Nuclear extracts made from rat E11.5 BAs were analyzed for the presence of  Evf-2 /Dlx-2 complexes by immunoprecipitation with anti- dll,  anti-Islet 1/2, or anti-IgG antibody, followed by RT–PCR against  Evf-2 -specific primers or GAPDH. ( c ) Single-cell suspensions made from mouse E12.5 dorsal and ventral telencephalon were dissected as shown in the schematic, centrifuged onto slides, and processed for fluorescent in situ/immunolocalization using  Evf-2  antisense RNA probe and anti- dll  antibody. DAPI staining (blue) reveals nuclei.  Evf-2  RNA (Alexa fluor 568) is in red, Dlx (Alexa fluor 488) is in green, and regions of overlap are in yellow. ( d ) A model proposing that a complex of  Evf-2  and Dlx-2 affects ei activity, ultimately affecting transcription of the Dlx-5 and Dlx-6 genes.
    Islet 1 Isl1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Developmental Studies Hybridoma Bank mouse anti isl1
    Hb9::Cre-derived INs do not overlap with the Shox2 non-V2a population. ( A ) Co-expression of YFP (green) and <t>Isl1</t> antibody (red) in the Hb9 :: Cre;Rosa26-YFP mouse spinal cord at E11.5. Motor neurons are also labeled by Isl1 antibody (blue box). Rightmost pictures are magnifications of the white boxed area. Arrowheads indicate overlap between Isl1 (red) and Hb9::Cre-derived INs (green). Scale bars: 100 μm and 50 μm. ( B ) Co-expression of YFP (green), Shox2 antibody (red) and/or Chx10 antibody (blue) in the Hb9 :: Cre;Rosa26-YFP mouse ventral spinal cord at E11.5. Rightmost pictures are magnifications of the white boxed area. Arrowheads indicate overlap between Hb9::Cre-derived INs (green) and Shox2 + Chx10 − (red), Shox2 − Chx10 + (blue) or Shox2 + Chx10 + (pink). Scale bars: 100 μm and 50 μm. ( C ) Quantification of overlap in (A) and (B). Bar graph showing percent of overlap between Hb9::Cre-derived INs (YFP + ) and Shox2 V2a (Shox2 + Chx10 + , 4% ± 1%), Shox2 OFF V2a (Shox2 − Chx10 + , 2% ± 0.1%), Shox2 non-V2a (Shox2 + Chx10 − , 1.3% ± 0.2%), and Isl1 (Isl1 + , 6% ± 0.2%) INs. Error bars represent ± SEM. ( D ) Percent of the Shox2 non-V2a IN population (Shox2 + Chx10 − ) that overlaps with Hb9::Cre-derived INs (YFP + ) in the Hb9 :: Cre;Rosa26-YFP mouse spinal cord at E11.5. Shox2 non-V2a INs rarely co-express YFP (Shox2 + YFP + , darker grey) (12% ± 2%). Error bars represent ± SEM.
    Mouse Anti Isl1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 90/100, based on 384 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Developmental Studies Hybridoma Bank mab anti islet 1
    OTX2 is transiently expressed by differentiating RGCs and by cells differentiating into phenotypes other than RGCs. Confocal microscopic images of frontal cryostat sections of chick retinas immunostained with antiserum against OTX2 ( red ) at E3 ( A , B ), E5 ( C, D ), and E7 ( E, F ) double stained with the RA4 mAb ( green in A, C ), the <t>anti-islet-1</t> mAb ( blue in B, D ), the 3A10 mAb ( green in E ), or the 3CB2 mAb ( green in F ). The majority of OTX2-positive cells could be identified as RGCs with the RA4 mAb; only a few cells ( arrows ) were stained only for OTX2 ( A, C ). Islet-1-positive RGCs in the mantle do not express OTX2 ( C, D ). At E7 the majority of OTX2-positive cells are neurons expressing the 3A10 antigen ( E ). A few OTX2-positive cells can be identified as Müller cells ( arrows ) by their expression of the 3CB2 antigen ( F ). fl, Fiber layer; gcl, ganglion cell layer; pe, pigment epithelium. Scale bar, A–D, 30 μm; E, F, 20 μm.
    Mab Anti Islet 1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effect of fluorofenidone on Islet-1 expression in murine retinas

    Journal: International Journal of Ophthalmology

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

    doi: 10.18240/ijo.2017.06.09

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

    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).

    Techniques: Expressing

    Analysis of epistasis between Brn3a and Islet1 mutations

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Brn3a and Islet1 act epistatically to regulate the gene expression program of sensory differentiation

    doi: 10.1523/JNEUROSCI.0901-11.2011

    Figure Lengend Snippet: Analysis of epistasis between Brn3a and Islet1 mutations

    Article Snippet: Other antisera included: rabbit anti-Etv1/Er81 and anti-Runx3 (gifts of S. Arber, ; ); rabbit anti-Runx1 (gift of T. Jessell), anti-DRG11 (gift of D. Lima, ), anti-caspase-3 (Cell Signal Tech), anti-peripherin (Millipore) and anti-Islet1 (Abcam); guinea pig anti-Sox 10 (gift of M. Wegner), and anti-Islet2 (gift of S. Pfaff, ); goat anti-TrkB and anti-TrkC (R & D Systems), anti-cRET (Fitzgerald) and anti-βgalactosidase (Biogenesis), and rabbit monoclonal anti-tubulinβ3 (Covance).

    Techniques:

    The Brn3a/Islet1 compound sensory phenotype at mid-gestation

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Brn3a and Islet1 act epistatically to regulate the gene expression program of sensory differentiation

    doi: 10.1523/JNEUROSCI.0901-11.2011

    Figure Lengend Snippet: The Brn3a/Islet1 compound sensory phenotype at mid-gestation

    Article Snippet: Other antisera included: rabbit anti-Etv1/Er81 and anti-Runx3 (gifts of S. Arber, ; ); rabbit anti-Runx1 (gift of T. Jessell), anti-DRG11 (gift of D. Lima, ), anti-caspase-3 (Cell Signal Tech), anti-peripherin (Millipore) and anti-Islet1 (Abcam); guinea pig anti-Sox 10 (gift of M. Wegner), and anti-Islet2 (gift of S. Pfaff, ); goat anti-TrkB and anti-TrkC (R & D Systems), anti-cRET (Fitzgerald) and anti-βgalactosidase (Biogenesis), and rabbit monoclonal anti-tubulinβ3 (Covance).

    Techniques:

    Immunofluorescence and in situ hybridization of novel Brn3a/Islet1 DKO targets

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Brn3a and Islet1 act epistatically to regulate the gene expression program of sensory differentiation

    doi: 10.1523/JNEUROSCI.0901-11.2011

    Figure Lengend Snippet: Immunofluorescence and in situ hybridization of novel Brn3a/Islet1 DKO targets

    Article Snippet: Other antisera included: rabbit anti-Etv1/Er81 and anti-Runx3 (gifts of S. Arber, ; ); rabbit anti-Runx1 (gift of T. Jessell), anti-DRG11 (gift of D. Lima, ), anti-caspase-3 (Cell Signal Tech), anti-peripherin (Millipore) and anti-Islet1 (Abcam); guinea pig anti-Sox 10 (gift of M. Wegner), and anti-Islet2 (gift of S. Pfaff, ); goat anti-TrkB and anti-TrkC (R & D Systems), anti-cRET (Fitzgerald) and anti-βgalactosidase (Biogenesis), and rabbit monoclonal anti-tubulinβ3 (Covance).

    Techniques: Immunofluorescence, In Situ Hybridization

    Chromatin immunoprecipitation of Islet1 bound to the Neurod4 locus

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Brn3a and Islet1 act epistatically to regulate the gene expression program of sensory differentiation

    doi: 10.1523/JNEUROSCI.0901-11.2011

    Figure Lengend Snippet: Chromatin immunoprecipitation of Islet1 bound to the Neurod4 locus

    Article Snippet: Other antisera included: rabbit anti-Etv1/Er81 and anti-Runx3 (gifts of S. Arber, ; ); rabbit anti-Runx1 (gift of T. Jessell), anti-DRG11 (gift of D. Lima, ), anti-caspase-3 (Cell Signal Tech), anti-peripherin (Millipore) and anti-Islet1 (Abcam); guinea pig anti-Sox 10 (gift of M. Wegner), and anti-Islet2 (gift of S. Pfaff, ); goat anti-TrkB and anti-TrkC (R & D Systems), anti-cRET (Fitzgerald) and anti-βgalactosidase (Biogenesis), and rabbit monoclonal anti-tubulinβ3 (Covance).

    Techniques: Chromatin Immunoprecipitation

    Displacement of cranial DRG and loss of peripheral sensory projections in Brn3a/Islet1 DKO embryos

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Brn3a and Islet1 act epistatically to regulate the gene expression program of sensory differentiation

    doi: 10.1523/JNEUROSCI.0901-11.2011

    Figure Lengend Snippet: Displacement of cranial DRG and loss of peripheral sensory projections in Brn3a/Islet1 DKO embryos

    Article Snippet: Other antisera included: rabbit anti-Etv1/Er81 and anti-Runx3 (gifts of S. Arber, ; ); rabbit anti-Runx1 (gift of T. Jessell), anti-DRG11 (gift of D. Lima, ), anti-caspase-3 (Cell Signal Tech), anti-peripherin (Millipore) and anti-Islet1 (Abcam); guinea pig anti-Sox 10 (gift of M. Wegner), and anti-Islet2 (gift of S. Pfaff, ); goat anti-TrkB and anti-TrkC (R & D Systems), anti-cRET (Fitzgerald) and anti-βgalactosidase (Biogenesis), and rabbit monoclonal anti-tubulinβ3 (Covance).

    Techniques:

    Loss of subtype specification in Brn3a/Islet1 DKO embryos

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Brn3a and Islet1 act epistatically to regulate the gene expression program of sensory differentiation

    doi: 10.1523/JNEUROSCI.0901-11.2011

    Figure Lengend Snippet: Loss of subtype specification in Brn3a/Islet1 DKO embryos

    Article Snippet: Other antisera included: rabbit anti-Etv1/Er81 and anti-Runx3 (gifts of S. Arber, ; ); rabbit anti-Runx1 (gift of T. Jessell), anti-DRG11 (gift of D. Lima, ), anti-caspase-3 (Cell Signal Tech), anti-peripherin (Millipore) and anti-Islet1 (Abcam); guinea pig anti-Sox 10 (gift of M. Wegner), and anti-Islet2 (gift of S. Pfaff, ); goat anti-TrkB and anti-TrkC (R & D Systems), anti-cRET (Fitzgerald) and anti-βgalactosidase (Biogenesis), and rabbit monoclonal anti-tubulinβ3 (Covance).

    Techniques:

    ISL1 and Gata3 synergistically activate downstream genes in neuroblastoma. (A. B) Heatmap (A) and aggregation plot (B) of ChIP-Seq mapped reads of Gata3 and ISL1 at all ISL1 binding sites. (C) Overlay of ISL1 ChIP-seq and GATA3 ChIP-seq datasets in SH-SY5Y (left). Overlay of the common target genes of ISL1 and GATA3 with genes differentially expressed in ISL1 KD neuroblastoma cells. (right). (D) GO terms enriched in common direct targets of ISL1 and GATA3 upregulated or downregulated in ISL1 KD neuroblastoma (top 5 not redundant categories are shown). (E) Integrative Genomics Viewer (IGV) showing ChIP-seq tracks for ISL1 and GATA3 at the loci of representative common target genes in neuroblastoma (arrowhead showing binding peaks). (F) qPCR analysis of GATA3 KD SH-SY5Y cells, showing similar alterations in expression of selected ISL1 target genes, noting that ISL1 expression is not altered. Error bars represent ±SD, n=3, *p

    Journal: Theranostics

    Article Title: Collaborative ISL1/GATA3 interaction in controlling neuroblastoma oncogenic pathways overlapping with but distinct from MYCN

    doi: 10.7150/thno.30199

    Figure Lengend Snippet: ISL1 and Gata3 synergistically activate downstream genes in neuroblastoma. (A. B) Heatmap (A) and aggregation plot (B) of ChIP-Seq mapped reads of Gata3 and ISL1 at all ISL1 binding sites. (C) Overlay of ISL1 ChIP-seq and GATA3 ChIP-seq datasets in SH-SY5Y (left). Overlay of the common target genes of ISL1 and GATA3 with genes differentially expressed in ISL1 KD neuroblastoma cells. (right). (D) GO terms enriched in common direct targets of ISL1 and GATA3 upregulated or downregulated in ISL1 KD neuroblastoma (top 5 not redundant categories are shown). (E) Integrative Genomics Viewer (IGV) showing ChIP-seq tracks for ISL1 and GATA3 at the loci of representative common target genes in neuroblastoma (arrowhead showing binding peaks). (F) qPCR analysis of GATA3 KD SH-SY5Y cells, showing similar alterations in expression of selected ISL1 target genes, noting that ISL1 expression is not altered. Error bars represent ±SD, n=3, *p

    Article Snippet: To detect the interaction of endogenous ISL1 and GATA3, SH-SY5Y cell lysates were immunoprecipitated with anti-ISL1 antibody and Western blot using anti-GATA3 antibody was performed.

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Expressing

    Essential role of ISL1 in regulating genes required for neuroblastoma pathogenesis. (A) Scatterplot of RNA-seq showing relative gene expression of mRNA in ISL1 KD and control SH-SY5Y cells. Red and blue represent genes upregulated or downregulated, respectively. (B, C) Gene Ontology (GO) functional clustering of genes down- and upregulated in ISL1 KD SH-SY5Y cells, highlighting pathways most significantly affected in ISL1 KD neuroblastoma cells (top 10 categories are shown). (D-F) Gene Set Enrichment Analysis (GSEA) showing downregulation of genesets involved in cell proliferation, DNA replication and mitotic nuclear division in ISL1 KD SH-SY5Y cells. (G. H) qPCR validation of selected downregulated and upregulated genes in ISL1 KD neuroblastoma, respectively Error bars represent ±SD, n=3 per group, *p

    Journal: Theranostics

    Article Title: Collaborative ISL1/GATA3 interaction in controlling neuroblastoma oncogenic pathways overlapping with but distinct from MYCN

    doi: 10.7150/thno.30199

    Figure Lengend Snippet: Essential role of ISL1 in regulating genes required for neuroblastoma pathogenesis. (A) Scatterplot of RNA-seq showing relative gene expression of mRNA in ISL1 KD and control SH-SY5Y cells. Red and blue represent genes upregulated or downregulated, respectively. (B, C) Gene Ontology (GO) functional clustering of genes down- and upregulated in ISL1 KD SH-SY5Y cells, highlighting pathways most significantly affected in ISL1 KD neuroblastoma cells (top 10 categories are shown). (D-F) Gene Set Enrichment Analysis (GSEA) showing downregulation of genesets involved in cell proliferation, DNA replication and mitotic nuclear division in ISL1 KD SH-SY5Y cells. (G. H) qPCR validation of selected downregulated and upregulated genes in ISL1 KD neuroblastoma, respectively Error bars represent ±SD, n=3 per group, *p

    Article Snippet: To detect the interaction of endogenous ISL1 and GATA3, SH-SY5Y cell lysates were immunoprecipitated with anti-ISL1 antibody and Western blot using anti-GATA3 antibody was performed.

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

    ISL1 directly regulates genes involved in neuroblastoma proliferation and differentiation. (A) Genome-wide distribution of ISL1 ChIP-Seq peaks mapped relative to their nearest TSS (transcription start site). Annotation indicates the positions of peaks are in TTS (transcription termination site, defined as from -100bp to +1Kb), exon, 5'UTR, 3'UTR, intronic or intergenic. (B) Top motifs enriched in ISL1-bound regions. (C) GO analysis of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). (D) Intersection of ISL1 ChIP-seq and ISL1 KD RNA-seq datasets revealed 389 direct downstream targets of ISL1 (186 upregulated and 203 downregulated) in neuroblastoma (DEG: differentially expressed genes). (E, F) Enriched GO terms of direct targets of ISL1 downregulated (E) and upregulated (F) in neuroblastoma (top 10 categories are shown). (G) Integrative Genomics Viewer (IGV) showing ChIP-seq tracks for H3K4me1, H3K27ac, and ISL1 at the loci of representative ISL1 target genes in neuroblastoma (arrowhead showing binding peaks).

    Journal: Theranostics

    Article Title: Collaborative ISL1/GATA3 interaction in controlling neuroblastoma oncogenic pathways overlapping with but distinct from MYCN

    doi: 10.7150/thno.30199

    Figure Lengend Snippet: ISL1 directly regulates genes involved in neuroblastoma proliferation and differentiation. (A) Genome-wide distribution of ISL1 ChIP-Seq peaks mapped relative to their nearest TSS (transcription start site). Annotation indicates the positions of peaks are in TTS (transcription termination site, defined as from -100bp to +1Kb), exon, 5'UTR, 3'UTR, intronic or intergenic. (B) Top motifs enriched in ISL1-bound regions. (C) GO analysis of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). (D) Intersection of ISL1 ChIP-seq and ISL1 KD RNA-seq datasets revealed 389 direct downstream targets of ISL1 (186 upregulated and 203 downregulated) in neuroblastoma (DEG: differentially expressed genes). (E, F) Enriched GO terms of direct targets of ISL1 downregulated (E) and upregulated (F) in neuroblastoma (top 10 categories are shown). (G) Integrative Genomics Viewer (IGV) showing ChIP-seq tracks for H3K4me1, H3K27ac, and ISL1 at the loci of representative ISL1 target genes in neuroblastoma (arrowhead showing binding peaks).

    Article Snippet: To detect the interaction of endogenous ISL1 and GATA3, SH-SY5Y cell lysates were immunoprecipitated with anti-ISL1 antibody and Western blot using anti-GATA3 antibody was performed.

    Techniques: Genome Wide, Chromatin Immunoprecipitation, RNA Sequencing Assay, Binding Assay

    LMO1 is a direct downstream target of ISL1 in neuroblastoma. (A) ChIP-qPCR showing binding of ISL1 at P1, P2 and P3 enhancer regions of LMO1 . Error bars represent ±SD; n=3, **p

    Journal: Theranostics

    Article Title: Collaborative ISL1/GATA3 interaction in controlling neuroblastoma oncogenic pathways overlapping with but distinct from MYCN

    doi: 10.7150/thno.30199

    Figure Lengend Snippet: LMO1 is a direct downstream target of ISL1 in neuroblastoma. (A) ChIP-qPCR showing binding of ISL1 at P1, P2 and P3 enhancer regions of LMO1 . Error bars represent ±SD; n=3, **p

    Article Snippet: To detect the interaction of endogenous ISL1 and GATA3, SH-SY5Y cell lysates were immunoprecipitated with anti-ISL1 antibody and Western blot using anti-GATA3 antibody was performed.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay

    ISL1 and MYCN act in parallel to regulate common yet distinct oncogenic pathways in neuroblastoma. (A, B) Relative mRNA expression of ISL1, MYCN, GATA3 and selected ISL1 or MYCN downstream genes in ISL1 KD and MYCN KD SK-N-BE(2) neuroblastoma cells. Error bars represent ±SD; n=3; *p

    Journal: Theranostics

    Article Title: Collaborative ISL1/GATA3 interaction in controlling neuroblastoma oncogenic pathways overlapping with but distinct from MYCN

    doi: 10.7150/thno.30199

    Figure Lengend Snippet: ISL1 and MYCN act in parallel to regulate common yet distinct oncogenic pathways in neuroblastoma. (A, B) Relative mRNA expression of ISL1, MYCN, GATA3 and selected ISL1 or MYCN downstream genes in ISL1 KD and MYCN KD SK-N-BE(2) neuroblastoma cells. Error bars represent ±SD; n=3; *p

    Article Snippet: To detect the interaction of endogenous ISL1 and GATA3, SH-SY5Y cell lysates were immunoprecipitated with anti-ISL1 antibody and Western blot using anti-GATA3 antibody was performed.

    Techniques: Activated Clotting Time Assay, Expressing

    Knockdown of ISL1 results in inhibition of neuroblastoma growth. (A, B) Validation of shRNA-mediated knockdown ISL1 by qPCR and Western blot (Arrow head) in SH-SY5Y cells. Error bars represent standard deviation (±SD), n=3, *p

    Journal: Theranostics

    Article Title: Collaborative ISL1/GATA3 interaction in controlling neuroblastoma oncogenic pathways overlapping with but distinct from MYCN

    doi: 10.7150/thno.30199

    Figure Lengend Snippet: Knockdown of ISL1 results in inhibition of neuroblastoma growth. (A, B) Validation of shRNA-mediated knockdown ISL1 by qPCR and Western blot (Arrow head) in SH-SY5Y cells. Error bars represent standard deviation (±SD), n=3, *p

    Article Snippet: To detect the interaction of endogenous ISL1 and GATA3, SH-SY5Y cell lysates were immunoprecipitated with anti-ISL1 antibody and Western blot using anti-GATA3 antibody was performed.

    Techniques: Inhibition, shRNA, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation

    Colabeling RGC with RBPMS, ISL, and Brn3 in human fetal retina, retinal organoid, and rhesus retina. (a) Colabeling RGC with RBPMS and Islet1 in fetal retina. (b) Costaining fetal retina with RBPMS and Brn3. (c) Double staining of HuD and RBPMS in fetal retina. (d) RPBMS expression shared the same pattern in retinal organoids. (e1, e2) In the human adult retina, Islet1 was expressed in retinal ganglion cells (white triangle) but these cells were Brn3 negative. (f1, f2) Human adult ganglion cells were HuD positive (white triangle) but Brn3 negative. (g1) No Brn3-positive cells were detected in rhesus retina. (g2) Retinal ganglion cells were RPBMS positive but could not been stained by Brn3 antibody. (g3) HuD antibody stained retinal ganglion cells along with RBPMS. (g4) Islet1 was positive in both GCL and INL, but RPBMS only labeled retinal ganglion cells. (h) Brn3 immunofluorescence intensity on human fetal retina, along with RBPMS. Scale bar = 100 μ m.

    Journal: Stem Cells International

    Article Title: Islet1 and Brn3 Expression Pattern Study in Human Retina and hiPSC-Derived Retinal Organoid

    doi: 10.1155/2019/8786396

    Figure Lengend Snippet: Colabeling RGC with RBPMS, ISL, and Brn3 in human fetal retina, retinal organoid, and rhesus retina. (a) Colabeling RGC with RBPMS and Islet1 in fetal retina. (b) Costaining fetal retina with RBPMS and Brn3. (c) Double staining of HuD and RBPMS in fetal retina. (d) RPBMS expression shared the same pattern in retinal organoids. (e1, e2) In the human adult retina, Islet1 was expressed in retinal ganglion cells (white triangle) but these cells were Brn3 negative. (f1, f2) Human adult ganglion cells were HuD positive (white triangle) but Brn3 negative. (g1) No Brn3-positive cells were detected in rhesus retina. (g2) Retinal ganglion cells were RPBMS positive but could not been stained by Brn3 antibody. (g3) HuD antibody stained retinal ganglion cells along with RBPMS. (g4) Islet1 was positive in both GCL and INL, but RPBMS only labeled retinal ganglion cells. (h) Brn3 immunofluorescence intensity on human fetal retina, along with RBPMS. Scale bar = 100 μ m.

    Article Snippet: The following primary antibodies were diluted in 2% (wt/vol) donkey serum, 0.04% (vol/vol) Triton X-100 in PBS: anti-Islet1 antibody (mouse, 1 : 200, Abcam, Cambridge, UK), anti-MCM2 antibody (rabbit, 1 : 200, Abcam), anti-VSX2 antibody (sheep, 1 : 500, Millipore, Billerica, MA, USA), anti-Brn3 antibody (goat, 1 : 200, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Brn3a antibody (mouse, 1 : 20, Santa Cruz Biotechnology, Dallas, TX, USA), anti-RPBMS antibody (rabbit, 1 : 200, Abcam), anti-HuD antibody (mouse, 1 : 100, Santa Cruz Biotechnology), anti-AP2α antibody (goat, 1 : 200, Abcam), anti-Recoverin antibody (rabbit, 1 : 500, Abcam), anti-Rhodopsin antibody (mouse, 1 : 200, Abcam), anti-Ki67 (rabbit, 1 : 50, Boster, Wuhan, China), anti-S-opsin (rabbit, 1 : 5000), and anti-L/M-opsin (rabbit, 1 : 5000), which were donated by Jeremy Nathans (Department of Molecular Biology and Genetics, Neuroscience, and Ophthalmology, the Johns Hopkins University School of Medicine).

    Techniques: Double Staining, Expressing, Staining, Labeling, Immunofluorescence

    Islet1 coexpressed with Brn3 in retinal ganglion cells. (a–f) Islet1 and Brn3 colabeled retinal ganglion cells in the human fetal retina. From Fwk 20, Brn3 expression gradually decreased and was negative in Fwk 27. (g–i) Islet1-/Brn3-positive retinal ganglion cells were located on the basal side of the retinal organoid but were not laminated well. (j–l) Only sporadic Islet1-positive cells were on the basal side and were Brn3 negative. (m) Most Brn3-positive retinal ganglion cells exited the cell cycle in Fwk 9 retina. The high magnification showed one cell with Brn3 expression but still in the cell cycle. (n1) Triple staining with Islet1, Brn3, and progenitor marker MCM2 in Fwk 9 retina. (n2–n5) Showing high magnification of the white square in (n1) (scale bar = 100 μ m).

    Journal: Stem Cells International

    Article Title: Islet1 and Brn3 Expression Pattern Study in Human Retina and hiPSC-Derived Retinal Organoid

    doi: 10.1155/2019/8786396

    Figure Lengend Snippet: Islet1 coexpressed with Brn3 in retinal ganglion cells. (a–f) Islet1 and Brn3 colabeled retinal ganglion cells in the human fetal retina. From Fwk 20, Brn3 expression gradually decreased and was negative in Fwk 27. (g–i) Islet1-/Brn3-positive retinal ganglion cells were located on the basal side of the retinal organoid but were not laminated well. (j–l) Only sporadic Islet1-positive cells were on the basal side and were Brn3 negative. (m) Most Brn3-positive retinal ganglion cells exited the cell cycle in Fwk 9 retina. The high magnification showed one cell with Brn3 expression but still in the cell cycle. (n1) Triple staining with Islet1, Brn3, and progenitor marker MCM2 in Fwk 9 retina. (n2–n5) Showing high magnification of the white square in (n1) (scale bar = 100 μ m).

    Article Snippet: The following primary antibodies were diluted in 2% (wt/vol) donkey serum, 0.04% (vol/vol) Triton X-100 in PBS: anti-Islet1 antibody (mouse, 1 : 200, Abcam, Cambridge, UK), anti-MCM2 antibody (rabbit, 1 : 200, Abcam), anti-VSX2 antibody (sheep, 1 : 500, Millipore, Billerica, MA, USA), anti-Brn3 antibody (goat, 1 : 200, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Brn3a antibody (mouse, 1 : 20, Santa Cruz Biotechnology, Dallas, TX, USA), anti-RPBMS antibody (rabbit, 1 : 200, Abcam), anti-HuD antibody (mouse, 1 : 100, Santa Cruz Biotechnology), anti-AP2α antibody (goat, 1 : 200, Abcam), anti-Recoverin antibody (rabbit, 1 : 500, Abcam), anti-Rhodopsin antibody (mouse, 1 : 200, Abcam), anti-Ki67 (rabbit, 1 : 50, Boster, Wuhan, China), anti-S-opsin (rabbit, 1 : 5000), and anti-L/M-opsin (rabbit, 1 : 5000), which were donated by Jeremy Nathans (Department of Molecular Biology and Genetics, Neuroscience, and Ophthalmology, the Johns Hopkins University School of Medicine).

    Techniques: Expressing, Staining, Marker

    Coimmunolabeling of Islet1 and CHX10 in human fetal retinal and retinal organoid. (a–c) CHX10 did not colocalize with Islet1 in the early stage, indicating CHX10 is a retinal progenitor marker during this period. (d, e) Layer of double-positive bipolar cells settled in the INL in the fetal retina. (f–i) CHX10-labeled retinal progenitors in the neuroblast layer in retinal organoids. (j) Some CHX10-positive cells colabeled with Islet1. (j, k) Another neural progenitor marker, MCM2, revealed undifferentiated cells in the fetal retina. (m–o) MCM2 and cell cycle marker, Ki67, confirmed that CHX10 was expressed in progenitors in this period rather than in bipolar cells. (p) To Dwk 26, CHX10-positive bipolar cells were MCM2 negative (scale bar = 100 μ m).

    Journal: Stem Cells International

    Article Title: Islet1 and Brn3 Expression Pattern Study in Human Retina and hiPSC-Derived Retinal Organoid

    doi: 10.1155/2019/8786396

    Figure Lengend Snippet: Coimmunolabeling of Islet1 and CHX10 in human fetal retinal and retinal organoid. (a–c) CHX10 did not colocalize with Islet1 in the early stage, indicating CHX10 is a retinal progenitor marker during this period. (d, e) Layer of double-positive bipolar cells settled in the INL in the fetal retina. (f–i) CHX10-labeled retinal progenitors in the neuroblast layer in retinal organoids. (j) Some CHX10-positive cells colabeled with Islet1. (j, k) Another neural progenitor marker, MCM2, revealed undifferentiated cells in the fetal retina. (m–o) MCM2 and cell cycle marker, Ki67, confirmed that CHX10 was expressed in progenitors in this period rather than in bipolar cells. (p) To Dwk 26, CHX10-positive bipolar cells were MCM2 negative (scale bar = 100 μ m).

    Article Snippet: The following primary antibodies were diluted in 2% (wt/vol) donkey serum, 0.04% (vol/vol) Triton X-100 in PBS: anti-Islet1 antibody (mouse, 1 : 200, Abcam, Cambridge, UK), anti-MCM2 antibody (rabbit, 1 : 200, Abcam), anti-VSX2 antibody (sheep, 1 : 500, Millipore, Billerica, MA, USA), anti-Brn3 antibody (goat, 1 : 200, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Brn3a antibody (mouse, 1 : 20, Santa Cruz Biotechnology, Dallas, TX, USA), anti-RPBMS antibody (rabbit, 1 : 200, Abcam), anti-HuD antibody (mouse, 1 : 100, Santa Cruz Biotechnology), anti-AP2α antibody (goat, 1 : 200, Abcam), anti-Recoverin antibody (rabbit, 1 : 500, Abcam), anti-Rhodopsin antibody (mouse, 1 : 200, Abcam), anti-Ki67 (rabbit, 1 : 50, Boster, Wuhan, China), anti-S-opsin (rabbit, 1 : 5000), and anti-L/M-opsin (rabbit, 1 : 5000), which were donated by Jeremy Nathans (Department of Molecular Biology and Genetics, Neuroscience, and Ophthalmology, the Johns Hopkins University School of Medicine).

    Techniques: Marker, Labeling

    Isl1 expression in developing photoreceptor cells. (a–d) A monolayer of Islet1 immunoreactive nuclei was observed from Fwk 15; the cells were Recoverin positive. However, as indicated by the white triangle, some Recoverin-positive cells were Islet1 negative, and their nuclei were located on the inner side to Islet1-positive cells. (e) In Dwk 15, Recoverin-positive developing photoreceptors were migrating from the basal side to the apical side. (f) Similar to in the fetal retina, some developing photoreceptors with inner arranged nuclei were Recoverin positive but Islet1 negative. (g–h) Developing photoreceptors in the retinal organoid. (i) In Fwk 27, cells with inner arranged nuclei were Rhodopsin-positive rods, and the Islet1-nuclei were between the soma and inner segment of rods as the schematic diagram (h). (j–m) Rod differentiation in retinal organoids (scale bar = 100 μ m).

    Journal: Stem Cells International

    Article Title: Islet1 and Brn3 Expression Pattern Study in Human Retina and hiPSC-Derived Retinal Organoid

    doi: 10.1155/2019/8786396

    Figure Lengend Snippet: Isl1 expression in developing photoreceptor cells. (a–d) A monolayer of Islet1 immunoreactive nuclei was observed from Fwk 15; the cells were Recoverin positive. However, as indicated by the white triangle, some Recoverin-positive cells were Islet1 negative, and their nuclei were located on the inner side to Islet1-positive cells. (e) In Dwk 15, Recoverin-positive developing photoreceptors were migrating from the basal side to the apical side. (f) Similar to in the fetal retina, some developing photoreceptors with inner arranged nuclei were Recoverin positive but Islet1 negative. (g–h) Developing photoreceptors in the retinal organoid. (i) In Fwk 27, cells with inner arranged nuclei were Rhodopsin-positive rods, and the Islet1-nuclei were between the soma and inner segment of rods as the schematic diagram (h). (j–m) Rod differentiation in retinal organoids (scale bar = 100 μ m).

    Article Snippet: The following primary antibodies were diluted in 2% (wt/vol) donkey serum, 0.04% (vol/vol) Triton X-100 in PBS: anti-Islet1 antibody (mouse, 1 : 200, Abcam, Cambridge, UK), anti-MCM2 antibody (rabbit, 1 : 200, Abcam), anti-VSX2 antibody (sheep, 1 : 500, Millipore, Billerica, MA, USA), anti-Brn3 antibody (goat, 1 : 200, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Brn3a antibody (mouse, 1 : 20, Santa Cruz Biotechnology, Dallas, TX, USA), anti-RPBMS antibody (rabbit, 1 : 200, Abcam), anti-HuD antibody (mouse, 1 : 100, Santa Cruz Biotechnology), anti-AP2α antibody (goat, 1 : 200, Abcam), anti-Recoverin antibody (rabbit, 1 : 500, Abcam), anti-Rhodopsin antibody (mouse, 1 : 200, Abcam), anti-Ki67 (rabbit, 1 : 50, Boster, Wuhan, China), anti-S-opsin (rabbit, 1 : 5000), and anti-L/M-opsin (rabbit, 1 : 5000), which were donated by Jeremy Nathans (Department of Molecular Biology and Genetics, Neuroscience, and Ophthalmology, the Johns Hopkins University School of Medicine).

    Techniques: Expressing

    Double immunolabeling of Islet1 and HuD in human fetal retinal and retinal organoid. (a–f) HuD coexpressed with Islet1 in GCL during the early stage of fetal retina development. From Fwk 16, a subpopulation of HuD-positive and Islet1-negative amacrine cells appeared in the INL. (g–l) HuD showed a similar expression pattern in retinal organoids. From Dwk 24, double-positive cells disappeared, and only HuD-single-positive amacrine cells organized as rosettes in the retinal organoid (scale bar = 100 μ m).

    Journal: Stem Cells International

    Article Title: Islet1 and Brn3 Expression Pattern Study in Human Retina and hiPSC-Derived Retinal Organoid

    doi: 10.1155/2019/8786396

    Figure Lengend Snippet: Double immunolabeling of Islet1 and HuD in human fetal retinal and retinal organoid. (a–f) HuD coexpressed with Islet1 in GCL during the early stage of fetal retina development. From Fwk 16, a subpopulation of HuD-positive and Islet1-negative amacrine cells appeared in the INL. (g–l) HuD showed a similar expression pattern in retinal organoids. From Dwk 24, double-positive cells disappeared, and only HuD-single-positive amacrine cells organized as rosettes in the retinal organoid (scale bar = 100 μ m).

    Article Snippet: The following primary antibodies were diluted in 2% (wt/vol) donkey serum, 0.04% (vol/vol) Triton X-100 in PBS: anti-Islet1 antibody (mouse, 1 : 200, Abcam, Cambridge, UK), anti-MCM2 antibody (rabbit, 1 : 200, Abcam), anti-VSX2 antibody (sheep, 1 : 500, Millipore, Billerica, MA, USA), anti-Brn3 antibody (goat, 1 : 200, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Brn3a antibody (mouse, 1 : 20, Santa Cruz Biotechnology, Dallas, TX, USA), anti-RPBMS antibody (rabbit, 1 : 200, Abcam), anti-HuD antibody (mouse, 1 : 100, Santa Cruz Biotechnology), anti-AP2α antibody (goat, 1 : 200, Abcam), anti-Recoverin antibody (rabbit, 1 : 500, Abcam), anti-Rhodopsin antibody (mouse, 1 : 200, Abcam), anti-Ki67 (rabbit, 1 : 50, Boster, Wuhan, China), anti-S-opsin (rabbit, 1 : 5000), and anti-L/M-opsin (rabbit, 1 : 5000), which were donated by Jeremy Nathans (Department of Molecular Biology and Genetics, Neuroscience, and Ophthalmology, the Johns Hopkins University School of Medicine).

    Techniques: Immunolabeling, Expressing

    Lamination of human fetal retina and Islet1 dynamic expression in human fetal retina and hiPSC-derived retinal organoid. (a–f) Ganglion cell layer (GCL) first developed in the human fetal retina. Next, the neuroblast layer gradually thickened and divided into an inner nucleus layer (INL) and outer nucleus layer (ONL). (a′–f′) Islet1 was expressed in the GCL in the early stage and then appeared as a monolayer nucleus on the most outer side. Later, sporadic positive nuclei were observed in the thickening neuroblast layer and finally collected on the outer side of the INL. (a ″ –f ″ ) Consistent with the human fetal retina, Islet1 was expressed in the GCL first in the retinal organoid. A monolayer of Islet1-positive cells was located on the apical side. In the midperiod, rosettes were detected in the organoid, disturbing lamination. Until late stages, retinal organoid was laminated and Islet1-positive cells were collected in the INL.

    Journal: Stem Cells International

    Article Title: Islet1 and Brn3 Expression Pattern Study in Human Retina and hiPSC-Derived Retinal Organoid

    doi: 10.1155/2019/8786396

    Figure Lengend Snippet: Lamination of human fetal retina and Islet1 dynamic expression in human fetal retina and hiPSC-derived retinal organoid. (a–f) Ganglion cell layer (GCL) first developed in the human fetal retina. Next, the neuroblast layer gradually thickened and divided into an inner nucleus layer (INL) and outer nucleus layer (ONL). (a′–f′) Islet1 was expressed in the GCL in the early stage and then appeared as a monolayer nucleus on the most outer side. Later, sporadic positive nuclei were observed in the thickening neuroblast layer and finally collected on the outer side of the INL. (a ″ –f ″ ) Consistent with the human fetal retina, Islet1 was expressed in the GCL first in the retinal organoid. A monolayer of Islet1-positive cells was located on the apical side. In the midperiod, rosettes were detected in the organoid, disturbing lamination. Until late stages, retinal organoid was laminated and Islet1-positive cells were collected in the INL.

    Article Snippet: The following primary antibodies were diluted in 2% (wt/vol) donkey serum, 0.04% (vol/vol) Triton X-100 in PBS: anti-Islet1 antibody (mouse, 1 : 200, Abcam, Cambridge, UK), anti-MCM2 antibody (rabbit, 1 : 200, Abcam), anti-VSX2 antibody (sheep, 1 : 500, Millipore, Billerica, MA, USA), anti-Brn3 antibody (goat, 1 : 200, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Brn3a antibody (mouse, 1 : 20, Santa Cruz Biotechnology, Dallas, TX, USA), anti-RPBMS antibody (rabbit, 1 : 200, Abcam), anti-HuD antibody (mouse, 1 : 100, Santa Cruz Biotechnology), anti-AP2α antibody (goat, 1 : 200, Abcam), anti-Recoverin antibody (rabbit, 1 : 500, Abcam), anti-Rhodopsin antibody (mouse, 1 : 200, Abcam), anti-Ki67 (rabbit, 1 : 50, Boster, Wuhan, China), anti-S-opsin (rabbit, 1 : 5000), and anti-L/M-opsin (rabbit, 1 : 5000), which were donated by Jeremy Nathans (Department of Molecular Biology and Genetics, Neuroscience, and Ophthalmology, the Johns Hopkins University School of Medicine).

    Techniques: Expressing, Derivative Assay

    Islet1 expression and postmitotic amacrine marker, AP2 α . (a–f) Amacrine cell developed as early as Fwk 9 and then organized in the inner side of the INL, without coexpression of Islet1. (g–l) In the retinal organoid, AP2 α -positive amacrine cell was observed at Dwk 13, later than in the fetal retina. Until Dwk 16, the number of amacrine cells obviously increased but was organized as rosettes. Until Dwk 31, the AP2 α -positive cells showed a disorderly arrangement (scale bar = 100 μ m).

    Journal: Stem Cells International

    Article Title: Islet1 and Brn3 Expression Pattern Study in Human Retina and hiPSC-Derived Retinal Organoid

    doi: 10.1155/2019/8786396

    Figure Lengend Snippet: Islet1 expression and postmitotic amacrine marker, AP2 α . (a–f) Amacrine cell developed as early as Fwk 9 and then organized in the inner side of the INL, without coexpression of Islet1. (g–l) In the retinal organoid, AP2 α -positive amacrine cell was observed at Dwk 13, later than in the fetal retina. Until Dwk 16, the number of amacrine cells obviously increased but was organized as rosettes. Until Dwk 31, the AP2 α -positive cells showed a disorderly arrangement (scale bar = 100 μ m).

    Article Snippet: The following primary antibodies were diluted in 2% (wt/vol) donkey serum, 0.04% (vol/vol) Triton X-100 in PBS: anti-Islet1 antibody (mouse, 1 : 200, Abcam, Cambridge, UK), anti-MCM2 antibody (rabbit, 1 : 200, Abcam), anti-VSX2 antibody (sheep, 1 : 500, Millipore, Billerica, MA, USA), anti-Brn3 antibody (goat, 1 : 200, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Brn3a antibody (mouse, 1 : 20, Santa Cruz Biotechnology, Dallas, TX, USA), anti-RPBMS antibody (rabbit, 1 : 200, Abcam), anti-HuD antibody (mouse, 1 : 100, Santa Cruz Biotechnology), anti-AP2α antibody (goat, 1 : 200, Abcam), anti-Recoverin antibody (rabbit, 1 : 500, Abcam), anti-Rhodopsin antibody (mouse, 1 : 200, Abcam), anti-Ki67 (rabbit, 1 : 50, Boster, Wuhan, China), anti-S-opsin (rabbit, 1 : 5000), and anti-L/M-opsin (rabbit, 1 : 5000), which were donated by Jeremy Nathans (Department of Molecular Biology and Genetics, Neuroscience, and Ophthalmology, the Johns Hopkins University School of Medicine).

    Techniques: Expressing, Marker

    Sox13 overexpression partly antagonizes Phox2b activity. (a-m

    Journal: Neural Development

    Article Title: Identification of Phox2b-regulated genes by expression profiling of cranial motoneuron precursors

    doi: 10.1186/1749-8104-3-14

    Figure Lengend Snippet: Sox13 overexpression partly antagonizes Phox2b activity. (a-m") Chicken neural tubes electroporated at HH 12–13 either separately with pCAGGS::Phox2b-IRES-EGFP (a-a", d-d", g-g", j-j") or pCAGGS::Sox13-IRES-EGFP (b-b", e-e", h-h", k-k") or with pCAGGS::Phox2b-IRES-EGFP plus pCAGGS::Sox13-IRES-EGFP (c-c", f-f", i-i", l-l", m-m") were analyzed 48 h after electroporation. Transverse sections were stained with anti-GFP antibodies in combination with anti-NeuN (a-c"), anti-Sox2 (g-i"), anti-Islet1/2 (j-l") or anti-Lhx1,5 immunohistochemistry (m-m") or in combination with anti-BrdU antibodies (d-f") after BrdU injection into the amniotic cavity 2 h before fixation. Phox2b induces and Sox13 represses NeuN (asterisks in (a', a", b')) in the ML. When co-expressed with Sox13 , Phox2b is still capable of inducing NeuN, which, however, is now also switched on in the VZ (c', c"). Cells electroporated with Phox2b are BrdU-negative whether Sox13 was co-transfected (f) or not (d). Cells electroporated with Sox13 whether co-transfected with Phox2b (i) or not (h) express Sox2, while cells electroporated with Phox2b alone are always Sox2-negative (g). Islet1,2 induction by Phox2b (j) is abolished by co-transfection of Sox13 (l). Repression of Lhx1,5 by Phox2b is not prevented by co-expressing Sox13 (m). Co-transfection of Sox13 together with Phox2b inhibits relocation to the ML (c, f, i, l, m) implemented by expression of Phox2b alone (a, d, g, j).

    Article Snippet: The following antibodies were used: rabbit anti-axonin-1 [ ], mouse monoclonal anti-BrdU (1/100; Sigma), mouse monoclonal (Roche, Basel, Switzerland) and rabbit (Chemicon, Temecula, CA, USA) anti-GFP (1/400), mouse anti-Islet1,2 (1/100) [ ], rabbit anti-Islet1 (1/500; Abcam, Paris, France), mouse anti-Lhx1,5 (Developmental Studies Hybridoma Bank), mouse anti-NeuN (1/500), rabbit anti-Sox2 (1/1,000; Abcam), rabbit anti-Sox13 [ ] and adequate fluorescent secondary antibodies.

    Techniques: Over Expression, Activity Assay, Electroporation, Staining, Immunohistochemistry, Injection, Transfection, Cotransfection, Expressing

    Sfrp1 partially restores a commissural axonal phenotype. (a-c) Chicken neural tubes electroporated at HH12-13 with pCAGGS::Sfrp1-IRES-EGFP (a), pCAGGS::Phox2b-IRES-EGFP (b) or pCAGGS::Sfrp1-IRES-EGFP plus pCAGGS::Phox2b-IRES-EGFP (c) were analyzed by anti-GFP immunohistochemistry. Expression of Sfrp1 did not grossly alter the morphology of neuroepithelial cells, neither did it prevent Phox2b from promoting relocation to the ML. However, Sfrp1 prevented Phox2b from repressing the growth of commissural axons (arrowheads). (d-d

    Journal: Neural Development

    Article Title: Identification of Phox2b-regulated genes by expression profiling of cranial motoneuron precursors

    doi: 10.1186/1749-8104-3-14

    Figure Lengend Snippet: Sfrp1 partially restores a commissural axonal phenotype. (a-c) Chicken neural tubes electroporated at HH12-13 with pCAGGS::Sfrp1-IRES-EGFP (a), pCAGGS::Phox2b-IRES-EGFP (b) or pCAGGS::Sfrp1-IRES-EGFP plus pCAGGS::Phox2b-IRES-EGFP (c) were analyzed by anti-GFP immunohistochemistry. Expression of Sfrp1 did not grossly alter the morphology of neuroepithelial cells, neither did it prevent Phox2b from promoting relocation to the ML. However, Sfrp1 prevented Phox2b from repressing the growth of commissural axons (arrowheads). (d-d") Chicken neural tubes electroporated with pCAGGS::Sfrp1-IRES-EGFP and a Phox2b expression vector were analyzed by anti-GFP and anti-Phox2b immunohistochemistry as indicated. Virtually all the transfected cells express both GFP and Phox2b. (e-e") Chicken neural tubes electroporated with pCAGGS::Sfrp1-IRES-EGFP plus pCAGGS::Phox2b-IRES-EGFP were analyzed by anti-GFP and anti-Islet1,2 immunohistochemistry as indicated. Co-expression of Sfrp1 did not prevent Phox2b from inducing Islet1,2 (bracket). ( f-h') Chicken neural tubes electroporated with pCAGGS::Phox2b-IRES-EGFP (f, f'), pCAGGS::Sfrp1-IRES-EGFP (g, g') or with pCAGGS::Sfrp1-IRES-EGFP plus pCAGGS::Phox2b-IRES-EGFP (h, h') were analyzed by anti-axonin-1 and anti-GFP immunohistochemistry. In (f, g, h), the anti-axonin-1 staining is shown alone, and in (f', g', h') it is merged with the anti-GFP immunofluorescence. The fascicle formed by the commissural fibers en route to the floor plate is marked by an asterisk; it is absent after Phox2b transfection and partially restored by co-expressing Sfrp1 .

    Article Snippet: The following antibodies were used: rabbit anti-axonin-1 [ ], mouse monoclonal anti-BrdU (1/100; Sigma), mouse monoclonal (Roche, Basel, Switzerland) and rabbit (Chemicon, Temecula, CA, USA) anti-GFP (1/400), mouse anti-Islet1,2 (1/100) [ ], rabbit anti-Islet1 (1/500; Abcam, Paris, France), mouse anti-Lhx1,5 (Developmental Studies Hybridoma Bank), mouse anti-NeuN (1/500), rabbit anti-Sox2 (1/1,000; Abcam), rabbit anti-Sox13 [ ] and adequate fluorescent secondary antibodies.

    Techniques: Immunohistochemistry, Expressing, Plasmid Preparation, Transfection, Staining, Immunofluorescence

    ISL1 bound to CCNB1 , CCNB2 and c-MYC (A) MatInspector software analysis of the consensus binding site (TAAT box) for ISL1 on the human CCNB1 promoter, the CCNB2 promoter, and the c-MYC enhancer. Sequences containing mutant base pairs (boxes) were used to construct the luciferase reporter constructs mutant-CCNB1-luc, mutant-CCNB2-luc, and mutant-c-MYC-luc. ( B, C ) Luciferase reporter assay of ISL1 transcriptional activity on the luciferase reporter constructs (-luc) of CCNB1 , CCNB2 and c-MYC in MKN28 cells (WT, wild-type plasmid; M, mutant plasmid). Luciferase activity on the reporter constructs was normalized to Renilla luciferase activity. (D) ChIP assay was performed with anti-ISL1 antibody using chromatin harvested from MKN28 cells. Extracted DNA was amplified using primers that cover the ISL1 binding sites on the CCNB1 and the CCNB2 promoters and the c-MYC enhancer by real-time PCR; normal IgG was used as the control. Data represent three independent experiments, each performed in triplicate. Bars represent the means ± SD (* p

    Journal: Oncotarget

    Article Title: ISL1, a novel regulator of CCNB1, CCNB2 and c-MYC genes, promotes gastric cancer cell proliferation and tumor growth

    doi: 10.18632/oncotarget.9269

    Figure Lengend Snippet: ISL1 bound to CCNB1 , CCNB2 and c-MYC (A) MatInspector software analysis of the consensus binding site (TAAT box) for ISL1 on the human CCNB1 promoter, the CCNB2 promoter, and the c-MYC enhancer. Sequences containing mutant base pairs (boxes) were used to construct the luciferase reporter constructs mutant-CCNB1-luc, mutant-CCNB2-luc, and mutant-c-MYC-luc. ( B, C ) Luciferase reporter assay of ISL1 transcriptional activity on the luciferase reporter constructs (-luc) of CCNB1 , CCNB2 and c-MYC in MKN28 cells (WT, wild-type plasmid; M, mutant plasmid). Luciferase activity on the reporter constructs was normalized to Renilla luciferase activity. (D) ChIP assay was performed with anti-ISL1 antibody using chromatin harvested from MKN28 cells. Extracted DNA was amplified using primers that cover the ISL1 binding sites on the CCNB1 and the CCNB2 promoters and the c-MYC enhancer by real-time PCR; normal IgG was used as the control. Data represent three independent experiments, each performed in triplicate. Bars represent the means ± SD (* p

    Article Snippet: The specimens were subjected to IHC analysis using an EnVision Detection Kit/DAB (GK500705, DAKO A/S, Glostrup, Denmark) according to the manufacturer's protocol with mouse monoclonal anti-ISL1 (ab86472; Abcam, Hong Kong, China).

    Techniques: Software, Binding Assay, Mutagenesis, Construct, Luciferase, Reporter Assay, Activity Assay, Plasmid Preparation, Chromatin Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction

    ISL1 promoted colony formation in vitro (A) Western blotting analysis of ISL1 levels in MKN28 and MGC803 cell lines with stable ISL1 overexpression (ISL1) or knockdown (si-ISL1) by transfection with pcDNA3.1-ISL1 and pLL3.7-ISL1-siRNA plasmids (cells transfected with pcDNA3.1 vector were used as the negative control), respectively. GAPDH levels served as the internal control. (B) PCF assay performed to determine the proliferation of MKN28 or MGC803 cells stably transfected with ISL1, si-ISL1, or the vector control (a–f). The quantification results are presented on the right. (C) SACF assay of MKN28 and MGC803 cells stably transfected with ISL1, si-ISL1, or vector (a–f). The quantification results are presented on the right. The data are the means ± SD from three independent experiments, each performed in triplicate. (** p

    Journal: Oncotarget

    Article Title: ISL1, a novel regulator of CCNB1, CCNB2 and c-MYC genes, promotes gastric cancer cell proliferation and tumor growth

    doi: 10.18632/oncotarget.9269

    Figure Lengend Snippet: ISL1 promoted colony formation in vitro (A) Western blotting analysis of ISL1 levels in MKN28 and MGC803 cell lines with stable ISL1 overexpression (ISL1) or knockdown (si-ISL1) by transfection with pcDNA3.1-ISL1 and pLL3.7-ISL1-siRNA plasmids (cells transfected with pcDNA3.1 vector were used as the negative control), respectively. GAPDH levels served as the internal control. (B) PCF assay performed to determine the proliferation of MKN28 or MGC803 cells stably transfected with ISL1, si-ISL1, or the vector control (a–f). The quantification results are presented on the right. (C) SACF assay of MKN28 and MGC803 cells stably transfected with ISL1, si-ISL1, or vector (a–f). The quantification results are presented on the right. The data are the means ± SD from three independent experiments, each performed in triplicate. (** p

    Article Snippet: The specimens were subjected to IHC analysis using an EnVision Detection Kit/DAB (GK500705, DAKO A/S, Glostrup, Denmark) according to the manufacturer's protocol with mouse monoclonal anti-ISL1 (ab86472; Abcam, Hong Kong, China).

    Techniques: In Vitro, Western Blot, Over Expression, Transfection, Plasmid Preparation, Negative Control, Stable Transfection

    ISL1 promoted tumor growth in nude mice ( A, B ) MGC803 cells stably transfected with pcDNA3.1 (control), pcDNA3.1-ISL1 (ISL1), ( C, D) MGC803 cells stably transfected with pLL3.7-Non-silencer (Non-silencer), or pLL3.7-ISL1-siRNA (si-ISL1). A total 1 × 10 7 appropriate cells suspended in 200 μl PBS were injected subcutaneously into the right oxter flank of BALB/c nude mice. Tumors isolated at day 21 (ISL1 and control) or day 24 (si-ISL1 and Non-silencer) are shown in A and C Tumor size was measured at the indicated days post-injection and the results are shown in B and D ( n = 3 at every time point in each group, * p

    Journal: Oncotarget

    Article Title: ISL1, a novel regulator of CCNB1, CCNB2 and c-MYC genes, promotes gastric cancer cell proliferation and tumor growth

    doi: 10.18632/oncotarget.9269

    Figure Lengend Snippet: ISL1 promoted tumor growth in nude mice ( A, B ) MGC803 cells stably transfected with pcDNA3.1 (control), pcDNA3.1-ISL1 (ISL1), ( C, D) MGC803 cells stably transfected with pLL3.7-Non-silencer (Non-silencer), or pLL3.7-ISL1-siRNA (si-ISL1). A total 1 × 10 7 appropriate cells suspended in 200 μl PBS were injected subcutaneously into the right oxter flank of BALB/c nude mice. Tumors isolated at day 21 (ISL1 and control) or day 24 (si-ISL1 and Non-silencer) are shown in A and C Tumor size was measured at the indicated days post-injection and the results are shown in B and D ( n = 3 at every time point in each group, * p

    Article Snippet: The specimens were subjected to IHC analysis using an EnVision Detection Kit/DAB (GK500705, DAKO A/S, Glostrup, Denmark) according to the manufacturer's protocol with mouse monoclonal anti-ISL1 (ab86472; Abcam, Hong Kong, China).

    Techniques: Mouse Assay, Stable Transfection, Transfection, Injection, Isolation

    ISL1 promoted GC cell proliferation ( A–C) The cell proliferation rate as determined using CCK-8 assay. The values at each time point are normalized to that of the relative cells at time 0, which was set as 1. The data are the means ± SD from three independent experiments, each performed in triplicate (** p

    Journal: Oncotarget

    Article Title: ISL1, a novel regulator of CCNB1, CCNB2 and c-MYC genes, promotes gastric cancer cell proliferation and tumor growth

    doi: 10.18632/oncotarget.9269

    Figure Lengend Snippet: ISL1 promoted GC cell proliferation ( A–C) The cell proliferation rate as determined using CCK-8 assay. The values at each time point are normalized to that of the relative cells at time 0, which was set as 1. The data are the means ± SD from three independent experiments, each performed in triplicate (** p

    Article Snippet: The specimens were subjected to IHC analysis using an EnVision Detection Kit/DAB (GK500705, DAKO A/S, Glostrup, Denmark) according to the manufacturer's protocol with mouse monoclonal anti-ISL1 (ab86472; Abcam, Hong Kong, China).

    Techniques: CCK-8 Assay

    ISL1 regulated CCNB1 , CCNB2 and c -MYC expression ( A, B) qRT-PCR analysis of CCNB1 , CCNB2 and c-MYC mRNA levels in MGC803 cells transfected with pcDNA3.1-ISL1 (ISL1) or pLL3.7-ISL1-siRNA (si-ISL1). The cells transfected with pcDNA3.1 (control) or pLL3.7-Non-silencer (Non-silencer, control) were used as the controls, whose values were set as 1. The data represent three independent experiments, each performed in triplicate. 18S rRNA levels served as the internal control. Bars represent the mean ± SD (* p

    Journal: Oncotarget

    Article Title: ISL1, a novel regulator of CCNB1, CCNB2 and c-MYC genes, promotes gastric cancer cell proliferation and tumor growth

    doi: 10.18632/oncotarget.9269

    Figure Lengend Snippet: ISL1 regulated CCNB1 , CCNB2 and c -MYC expression ( A, B) qRT-PCR analysis of CCNB1 , CCNB2 and c-MYC mRNA levels in MGC803 cells transfected with pcDNA3.1-ISL1 (ISL1) or pLL3.7-ISL1-siRNA (si-ISL1). The cells transfected with pcDNA3.1 (control) or pLL3.7-Non-silencer (Non-silencer, control) were used as the controls, whose values were set as 1. The data represent three independent experiments, each performed in triplicate. 18S rRNA levels served as the internal control. Bars represent the mean ± SD (* p

    Article Snippet: The specimens were subjected to IHC analysis using an EnVision Detection Kit/DAB (GK500705, DAKO A/S, Glostrup, Denmark) according to the manufacturer's protocol with mouse monoclonal anti-ISL1 (ab86472; Abcam, Hong Kong, China).

    Techniques: Expressing, Quantitative RT-PCR, Transfection

    Aberrant expression of ISL1 in GC tissues (A) Representative IHC stainings of ISL1 expression in normal gastric mucosa (top, n = 12) and GC samples (bottom, n = 24). (B) Grayscale scanning analysis results of ISL1 expression in the tested cell lines. Grayscale scanning was performed on the western blots of three independent experiments for quantitative analysis. Representative western blots of three experiments are shown at the bottom. β-Actin served as the internal control. GES1, normal gastric cell line; others, GC cell lines. Bars represent the means ± SD (** p

    Journal: Oncotarget

    Article Title: ISL1, a novel regulator of CCNB1, CCNB2 and c-MYC genes, promotes gastric cancer cell proliferation and tumor growth

    doi: 10.18632/oncotarget.9269

    Figure Lengend Snippet: Aberrant expression of ISL1 in GC tissues (A) Representative IHC stainings of ISL1 expression in normal gastric mucosa (top, n = 12) and GC samples (bottom, n = 24). (B) Grayscale scanning analysis results of ISL1 expression in the tested cell lines. Grayscale scanning was performed on the western blots of three independent experiments for quantitative analysis. Representative western blots of three experiments are shown at the bottom. β-Actin served as the internal control. GES1, normal gastric cell line; others, GC cell lines. Bars represent the means ± SD (** p

    Article Snippet: The specimens were subjected to IHC analysis using an EnVision Detection Kit/DAB (GK500705, DAKO A/S, Glostrup, Denmark) according to the manufacturer's protocol with mouse monoclonal anti-ISL1 (ab86472; Abcam, Hong Kong, China).

    Techniques: Expressing, Immunohistochemistry, Western Blot

    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

    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

    MED17.11 express the early neuronal markers FOX3 (NeuN), Isl1 and Tuj1, and can be transfected with GFP. Immunolabelling of MED17.11 cells cultured in permissive conditions for large T antigen expression. GFP transgene expression in MED17.11. Scale bar is 100 μm.

    Journal: PLoS ONE

    Article Title: Mouse DRG Cell Line with Properties of Nociceptors

    doi: 10.1371/journal.pone.0128670

    Figure Lengend Snippet: MED17.11 express the early neuronal markers FOX3 (NeuN), Isl1 and Tuj1, and can be transfected with GFP. Immunolabelling of MED17.11 cells cultured in permissive conditions for large T antigen expression. GFP transgene expression in MED17.11. Scale bar is 100 μm.

    Article Snippet: The polyclonal antibodies used were Isl1 (1:250, Abcam), FOX3 (NeuN, 1:250, Millipore), SOX10 (1:250, Abcam), Advillin (1:500, Abcam), TrkA, TrkB and TrkC (1:500, Alomone), Nav1.3 (1:500, Alomone), and SV40 (1:250, Santa Cruz).

    Techniques: Transfection, Cell Culture, Expressing

    Islet-1 (ISL1) overexpression increased human mesenchymal stem cell (hMSC) survival after transplantation of post-myocardial infection (MI) hearts. Representative images ( a ) and quantification ( b ) of surviving ISL1-hMSCs and control (Ctrl)-hMSCs in hearts at 7 days after transplantation. Red immunofluorescence of CM-Dil indicates surviving cells, and blue color of 4’6-diamidino-2-phenylindole (DAPI) indicates nuclei. Human nuclear antigen (HNA) staining is also shown. Representative flow cytometric images ( c ) with Annexin V/propidium iodide (PI) double staining assay of cells treated with or without H 2 O 2 . Quantification of Annexin-V-positive cells is shown as the apoptosis rate ( d ): (Annexin-V-positive cell amount / total cell amount) × 100%. Representative images and quantification ( e ) of cleaved caspase3 and full caspase3 in ISL1-hMSCs and Ctrl-hMSCs. Data are presented as the mean ± SD, n = 3; * p

    Journal: Stem Cell Research & Therapy

    Article Title: ISL1 overexpression enhances the survival of transplanted human mesenchymal stem cells in a murine myocardial infarction model

    doi: 10.1186/s13287-018-0803-7

    Figure Lengend Snippet: Islet-1 (ISL1) overexpression increased human mesenchymal stem cell (hMSC) survival after transplantation of post-myocardial infection (MI) hearts. Representative images ( a ) and quantification ( b ) of surviving ISL1-hMSCs and control (Ctrl)-hMSCs in hearts at 7 days after transplantation. Red immunofluorescence of CM-Dil indicates surviving cells, and blue color of 4’6-diamidino-2-phenylindole (DAPI) indicates nuclei. Human nuclear antigen (HNA) staining is also shown. Representative flow cytometric images ( c ) with Annexin V/propidium iodide (PI) double staining assay of cells treated with or without H 2 O 2 . Quantification of Annexin-V-positive cells is shown as the apoptosis rate ( d ): (Annexin-V-positive cell amount / total cell amount) × 100%. Representative images and quantification ( e ) of cleaved caspase3 and full caspase3 in ISL1-hMSCs and Ctrl-hMSCs. Data are presented as the mean ± SD, n = 3; * p

    Article Snippet: Cells were incubated overnight at 4 °C with primary antibodies against ISL1 (1:300, cat. no. ab178400, Abcam, Cambridge, UK), α-actinin (1:200, cat. no. ab50599, Abcam, Cambridge, UK) to detect cardiomyocytes, CD3 (1:300, cat. no. ab16669, Abcam, Cambridge, UK) to detect T lymphocytes, and CD68 (1:300, cat. no. ab125212, Abcam, Cambridge, UK) to detect macrophages.

    Techniques: Over Expression, Transplantation Assay, Infection, Immunofluorescence, Staining, Flow Cytometry, Double Staining

    The anti-apoptotic effect of insulin-like growth factor binding protein 3 (IGFBP3) in islet-1 human mesenchymal stem cells (ISL1-hMSCs) conditioned medium (CM) on cardiomyocytes subjected to oxidative injury. Representative images of TUNEL staining in cardiomyocytes ( a ). Cell nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI; blue) and TUNEL-positive nuclei (green). Quantification of TUNEL staining was shown. Apoptosis rate = (TUNEL positive nuclei / DAPI + nuclei) × 100% ( b ). Graphic of the mechanisms underlying the therapeutic effects of transplantation of ISL1-hMSCs ( c ). Data are presented as the mean ± SD, n = 3; * p

    Journal: Stem Cell Research & Therapy

    Article Title: ISL1 overexpression enhances the survival of transplanted human mesenchymal stem cells in a murine myocardial infarction model

    doi: 10.1186/s13287-018-0803-7

    Figure Lengend Snippet: The anti-apoptotic effect of insulin-like growth factor binding protein 3 (IGFBP3) in islet-1 human mesenchymal stem cells (ISL1-hMSCs) conditioned medium (CM) on cardiomyocytes subjected to oxidative injury. Representative images of TUNEL staining in cardiomyocytes ( a ). Cell nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI; blue) and TUNEL-positive nuclei (green). Quantification of TUNEL staining was shown. Apoptosis rate = (TUNEL positive nuclei / DAPI + nuclei) × 100% ( b ). Graphic of the mechanisms underlying the therapeutic effects of transplantation of ISL1-hMSCs ( c ). Data are presented as the mean ± SD, n = 3; * p

    Article Snippet: Cells were incubated overnight at 4 °C with primary antibodies against ISL1 (1:300, cat. no. ab178400, Abcam, Cambridge, UK), α-actinin (1:200, cat. no. ab50599, Abcam, Cambridge, UK) to detect cardiomyocytes, CD3 (1:300, cat. no. ab16669, Abcam, Cambridge, UK) to detect T lymphocytes, and CD68 (1:300, cat. no. ab125212, Abcam, Cambridge, UK) to detect macrophages.

    Techniques: Binding Assay, TUNEL Assay, Staining, Transplantation Assay

    The anti-apoptotic effect of islet-1 human mesenchymal stem cells (ISL1-hMSCs) conditioned medium (CM) on cardiomyocyte cell line H9c2 subjected to oxidative injury ( a ) and quantification ( b ) of TUNEL staining in cardiomyocytes subjected to 200 μM H 2 O 2 for 6 h. Cell nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI; blue) and TUNEL-positive nuclei (green). Apoptosis rate = (TUNEL-positive nuclei / DAPI-positive nuclei) × 100%. Data are presented as the mean ± SD, n = 3; * p

    Journal: Stem Cell Research & Therapy

    Article Title: ISL1 overexpression enhances the survival of transplanted human mesenchymal stem cells in a murine myocardial infarction model

    doi: 10.1186/s13287-018-0803-7

    Figure Lengend Snippet: The anti-apoptotic effect of islet-1 human mesenchymal stem cells (ISL1-hMSCs) conditioned medium (CM) on cardiomyocyte cell line H9c2 subjected to oxidative injury ( a ) and quantification ( b ) of TUNEL staining in cardiomyocytes subjected to 200 μM H 2 O 2 for 6 h. Cell nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI; blue) and TUNEL-positive nuclei (green). Apoptosis rate = (TUNEL-positive nuclei / DAPI-positive nuclei) × 100%. Data are presented as the mean ± SD, n = 3; * p

    Article Snippet: Cells were incubated overnight at 4 °C with primary antibodies against ISL1 (1:300, cat. no. ab178400, Abcam, Cambridge, UK), α-actinin (1:200, cat. no. ab50599, Abcam, Cambridge, UK) to detect cardiomyocytes, CD3 (1:300, cat. no. ab16669, Abcam, Cambridge, UK) to detect T lymphocytes, and CD68 (1:300, cat. no. ab125212, Abcam, Cambridge, UK) to detect macrophages.

    Techniques: TUNEL Assay, Staining

    Islet-1 (ISL1) overexpression enhanced the human mesenchymal stem cell (hMSC) paracrine effect by increasing insulin-like growth factor binding protein 3 (IGFBP3) secretion. A heat map display of apoptosis-related genes between ISL1-hMSCs and control (Ctrl)-hMSCs ( a ). GDF6, IGFBP3, TGFB1, GAS6, and INHBA gene expression in Ctrl-hMSCs and ISL1-hMSCs were determined by qPCR ( b ). IGFBP3 in Ctrl-hMSC conditioned medium (CM) and ISL1-hMSCs-CM determined by ELISA assay ( c ). Data are presented as the mean ± SD, n = 3; * p

    Journal: Stem Cell Research & Therapy

    Article Title: ISL1 overexpression enhances the survival of transplanted human mesenchymal stem cells in a murine myocardial infarction model

    doi: 10.1186/s13287-018-0803-7

    Figure Lengend Snippet: Islet-1 (ISL1) overexpression enhanced the human mesenchymal stem cell (hMSC) paracrine effect by increasing insulin-like growth factor binding protein 3 (IGFBP3) secretion. A heat map display of apoptosis-related genes between ISL1-hMSCs and control (Ctrl)-hMSCs ( a ). GDF6, IGFBP3, TGFB1, GAS6, and INHBA gene expression in Ctrl-hMSCs and ISL1-hMSCs were determined by qPCR ( b ). IGFBP3 in Ctrl-hMSC conditioned medium (CM) and ISL1-hMSCs-CM determined by ELISA assay ( c ). Data are presented as the mean ± SD, n = 3; * p

    Article Snippet: Cells were incubated overnight at 4 °C with primary antibodies against ISL1 (1:300, cat. no. ab178400, Abcam, Cambridge, UK), α-actinin (1:200, cat. no. ab50599, Abcam, Cambridge, UK) to detect cardiomyocytes, CD3 (1:300, cat. no. ab16669, Abcam, Cambridge, UK) to detect T lymphocytes, and CD68 (1:300, cat. no. ab125212, Abcam, Cambridge, UK) to detect macrophages.

    Techniques: Over Expression, Binding Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Characterization of islet-1 human mesenchymal stem cells (ISL1-hMSCs). Flow cytometric analysis ( a ) showed that ISL1-hMSCs were positive for CD29, CD44, CD73, and CD90, and negative for CD14, CD31, CD34, and CD45. qPCR ( b ) showed that lentiviral transduction of hMSCs with ISL1 specifically increased the expression of ISL1. Flow cytometry ( c ) and immunofluorescence staining ( d ) further confirmed the overexpression of ISL1. Adipogenic differentiation of ISL1-hMSCs was determined by histochemical staining of adipocytes (Oil-Red O), osteogenic differentiation of ISL1-hMSCs was determined by histochemical staining of osteocytes (Alizarin Red), and chondrogenic differentiation was detected by Alcian Blue ( e ). Data are presented as the mean ± SD, n = 3; * p

    Journal: Stem Cell Research & Therapy

    Article Title: ISL1 overexpression enhances the survival of transplanted human mesenchymal stem cells in a murine myocardial infarction model

    doi: 10.1186/s13287-018-0803-7

    Figure Lengend Snippet: Characterization of islet-1 human mesenchymal stem cells (ISL1-hMSCs). Flow cytometric analysis ( a ) showed that ISL1-hMSCs were positive for CD29, CD44, CD73, and CD90, and negative for CD14, CD31, CD34, and CD45. qPCR ( b ) showed that lentiviral transduction of hMSCs with ISL1 specifically increased the expression of ISL1. Flow cytometry ( c ) and immunofluorescence staining ( d ) further confirmed the overexpression of ISL1. Adipogenic differentiation of ISL1-hMSCs was determined by histochemical staining of adipocytes (Oil-Red O), osteogenic differentiation of ISL1-hMSCs was determined by histochemical staining of osteocytes (Alizarin Red), and chondrogenic differentiation was detected by Alcian Blue ( e ). Data are presented as the mean ± SD, n = 3; * p

    Article Snippet: Cells were incubated overnight at 4 °C with primary antibodies against ISL1 (1:300, cat. no. ab178400, Abcam, Cambridge, UK), α-actinin (1:200, cat. no. ab50599, Abcam, Cambridge, UK) to detect cardiomyocytes, CD3 (1:300, cat. no. ab16669, Abcam, Cambridge, UK) to detect T lymphocytes, and CD68 (1:300, cat. no. ab125212, Abcam, Cambridge, UK) to detect macrophages.

    Techniques: Flow Cytometry, Real-time Polymerase Chain Reaction, Transduction, Expressing, Cytometry, Immunofluorescence, Staining, Over Expression

    Transplantation of islet-1 human mesenchymal stem cells (ISL1-hMSCs) improved cardiac function in a murine myocardial infarction (MI) model. Representative M-mode images ( a ) of hearts with sham surgery or MI at 4 weeks after phosphate-buffered saline (PBS), control (Ctrl)-hMSC, or ISL1-hMSC injection. Ejection fraction ( b ) and fractional shorting ( c ) at 4 weeks after ISL1-hMSC or Ctrl-hMSC transplantation were detected. Data are presented as the mean ± SD, n = 8; * p

    Journal: Stem Cell Research & Therapy

    Article Title: ISL1 overexpression enhances the survival of transplanted human mesenchymal stem cells in a murine myocardial infarction model

    doi: 10.1186/s13287-018-0803-7

    Figure Lengend Snippet: Transplantation of islet-1 human mesenchymal stem cells (ISL1-hMSCs) improved cardiac function in a murine myocardial infarction (MI) model. Representative M-mode images ( a ) of hearts with sham surgery or MI at 4 weeks after phosphate-buffered saline (PBS), control (Ctrl)-hMSC, or ISL1-hMSC injection. Ejection fraction ( b ) and fractional shorting ( c ) at 4 weeks after ISL1-hMSC or Ctrl-hMSC transplantation were detected. Data are presented as the mean ± SD, n = 8; * p

    Article Snippet: Cells were incubated overnight at 4 °C with primary antibodies against ISL1 (1:300, cat. no. ab178400, Abcam, Cambridge, UK), α-actinin (1:200, cat. no. ab50599, Abcam, Cambridge, UK) to detect cardiomyocytes, CD3 (1:300, cat. no. ab16669, Abcam, Cambridge, UK) to detect T lymphocytes, and CD68 (1:300, cat. no. ab125212, Abcam, Cambridge, UK) to detect macrophages.

    Techniques: Transplantation Assay, Injection

    Transplantation of islet-1 human mesenchymal stem cells (ISL1-hMSCs) increased angiogenesis and reduced apoptosis. Representative images ( a ) and quantification ( b ) of capillary density in the infarct border zone at 4 weeks post-myocardial infarct (MI) detected by CD31 staining. Representative images ( c ) and quantification ( d ) of TUNEL-positive cells in the infarct border. Data are presented as the mean ± SD, n = 8; * p

    Journal: Stem Cell Research & Therapy

    Article Title: ISL1 overexpression enhances the survival of transplanted human mesenchymal stem cells in a murine myocardial infarction model

    doi: 10.1186/s13287-018-0803-7

    Figure Lengend Snippet: Transplantation of islet-1 human mesenchymal stem cells (ISL1-hMSCs) increased angiogenesis and reduced apoptosis. Representative images ( a ) and quantification ( b ) of capillary density in the infarct border zone at 4 weeks post-myocardial infarct (MI) detected by CD31 staining. Representative images ( c ) and quantification ( d ) of TUNEL-positive cells in the infarct border. Data are presented as the mean ± SD, n = 8; * p

    Article Snippet: Cells were incubated overnight at 4 °C with primary antibodies against ISL1 (1:300, cat. no. ab178400, Abcam, Cambridge, UK), α-actinin (1:200, cat. no. ab50599, Abcam, Cambridge, UK) to detect cardiomyocytes, CD3 (1:300, cat. no. ab16669, Abcam, Cambridge, UK) to detect T lymphocytes, and CD68 (1:300, cat. no. ab125212, Abcam, Cambridge, UK) to detect macrophages.

    Techniques: Transplantation Assay, Staining, TUNEL Assay

    Transplantation of islet-1 human mesenchymal stem cells (ISL1-hMSCs) reduced cardiac infarct size and fibrotic size in a myocardial infarction (MI) model. ( a ) Representative images of infarct size. Representative images ( b ) and quantification ( c ) of the fibrotic area in the infarct border zone by Masson’s trichrome staining at 4 weeks post-MI. Data are presented as the mean ± SD, n = 8; * p

    Journal: Stem Cell Research & Therapy

    Article Title: ISL1 overexpression enhances the survival of transplanted human mesenchymal stem cells in a murine myocardial infarction model

    doi: 10.1186/s13287-018-0803-7

    Figure Lengend Snippet: Transplantation of islet-1 human mesenchymal stem cells (ISL1-hMSCs) reduced cardiac infarct size and fibrotic size in a myocardial infarction (MI) model. ( a ) Representative images of infarct size. Representative images ( b ) and quantification ( c ) of the fibrotic area in the infarct border zone by Masson’s trichrome staining at 4 weeks post-MI. Data are presented as the mean ± SD, n = 8; * p

    Article Snippet: Cells were incubated overnight at 4 °C with primary antibodies against ISL1 (1:300, cat. no. ab178400, Abcam, Cambridge, UK), α-actinin (1:200, cat. no. ab50599, Abcam, Cambridge, UK) to detect cardiomyocytes, CD3 (1:300, cat. no. ab16669, Abcam, Cambridge, UK) to detect T lymphocytes, and CD68 (1:300, cat. no. ab125212, Abcam, Cambridge, UK) to detect macrophages.

    Techniques: Transplantation Assay, Staining

    TDP-43 localization in patient iPSC-derived motor neurons (A) iPSC-MN stained for TDP-43 shows TDP-43 is ubiquitously expressed and nuclear in all cells including in ISLET1-positive neurons from control (left panel) and ALS (right panel) patients. ISLET1-positive cells from sporadic ALS iPSC-MN have nuclear staining as well as nuclear aggregates that stain with higher intensity for TDP-43 (arrowheads, right panel) but aggregates are not present in healthy controls (left panel). Fibroblasts from healthy control and sALS patients do not show nuclear aggregates. Scale bar: 30 µm. (B) Sporadic ALS patient-derived iPSC-MN cells stained with nuclear envelop marker LAMIN-A (green) and TDP-43 (red) shows TDP-43 aggregates are inside the nuclear envelope; scale bar: 20µm. (C) Quantification of healthy control IPRN.0013 and sALS IPRN.0048 clone 1 iPSC-MN cultures shows TDP-43 aggregation is present in higher fraction of ISLET1 or HB9-positive cells compared to negative cells. (30.7% of ISLET1/HB9-positive motor neurons as compared to 16.2% of ISLET1/HB9-negative cells in sALS iPSC-MN cultures. Bars are standard deviation 9% and 8% respectively; P

    Journal: Molecular and cellular neurosciences

    Article Title: A cellular model for sporadic ALS using patient-derived induced pluripotent stem cells

    doi: 10.1016/j.mcn.2013.07.007

    Figure Lengend Snippet: TDP-43 localization in patient iPSC-derived motor neurons (A) iPSC-MN stained for TDP-43 shows TDP-43 is ubiquitously expressed and nuclear in all cells including in ISLET1-positive neurons from control (left panel) and ALS (right panel) patients. ISLET1-positive cells from sporadic ALS iPSC-MN have nuclear staining as well as nuclear aggregates that stain with higher intensity for TDP-43 (arrowheads, right panel) but aggregates are not present in healthy controls (left panel). Fibroblasts from healthy control and sALS patients do not show nuclear aggregates. Scale bar: 30 µm. (B) Sporadic ALS patient-derived iPSC-MN cells stained with nuclear envelop marker LAMIN-A (green) and TDP-43 (red) shows TDP-43 aggregates are inside the nuclear envelope; scale bar: 20µm. (C) Quantification of healthy control IPRN.0013 and sALS IPRN.0048 clone 1 iPSC-MN cultures shows TDP-43 aggregation is present in higher fraction of ISLET1 or HB9-positive cells compared to negative cells. (30.7% of ISLET1/HB9-positive motor neurons as compared to 16.2% of ISLET1/HB9-negative cells in sALS iPSC-MN cultures. Bars are standard deviation 9% and 8% respectively; P

    Article Snippet: The following antibodies were used ISLET1 at 1:1000 (Abcam, ab86501), phospho-S409/S410-TDP-43 (Sigma), LAMIN-A at 1:200 (Cell Signaling, 4777), MNR2/HB9 at 1:100 (Developmental Studies Hybridoma Bank, 81.5C10), SMI31 at 1:1000 (Covance, SMI-31R) and/or TARDBP at 1:500 (ProteinTech, 10782-2-AP), CTIP2 at 1:500 (Abcam).

    Techniques: Derivative Assay, Staining, Marker, Standard Deviation

    Patient-specific iPSC and iPSC-derived motor neurons Representative images of: (A) iPSC colonies from one control and one ALS patient in phase-contrast. (B) iPSC colonies stained with antibodies for pluripotency markers NANOG and TRA-1-60. (C) iPSC-MN cultures stained with nuclear marker DAPI (blue) and antibodies to motor neuron markers ISLET1 (red) and HB9 (green). (D) iPSC-MN cultures stained with antibody to axonal marker Neurofilament (SMI31). iPSC and iPSC-MN shown are from healthy control IPRN.0013 and fALS patient IPRN.0028. (Scale bars a-b: 200 µm, c-d: 75 µm). (E) (i-ii), Representative trace showing electrical activity of iPS-MN from a healthy individual (Scale bars: 100 mV; 100 ms. 8 neurons recorded from fired robust and repetitive action potentials (bursts) to depolarizing current steps. (Iii) Example trace showing action potential elicited by the rebound depolarization following 300 pA hyperpolarizing step for 100 ms (Scales bars: 100 mV; 100 ms) and example trace of spontaneous bursts of action potentials and spontaneous post-synaptic potentials from iPS derived motor neurons (50 mV; 3 Sec). (Insets) Examples of EPSP (scales: 3 mV; 250 ms), and IPSP (scales: 30 mV, 100 ms). (F) (i) Sporadic ALS iPSC-MN responses −10, 0, and 10 pA current steps for 300 msec (scale: 25 mV). (ii) APs elicited by rebound depolarization following hyperpolarizing current step (300 pA; 300 msec). (iii) Spontaneous activity of sALS iPSC-MN (scale bars; 50 mV; 500 msec) (insets example of sEPSP and sIPSP (scale bars: 5 mV; 200 msec))( n = 7).

    Journal: Molecular and cellular neurosciences

    Article Title: A cellular model for sporadic ALS using patient-derived induced pluripotent stem cells

    doi: 10.1016/j.mcn.2013.07.007

    Figure Lengend Snippet: Patient-specific iPSC and iPSC-derived motor neurons Representative images of: (A) iPSC colonies from one control and one ALS patient in phase-contrast. (B) iPSC colonies stained with antibodies for pluripotency markers NANOG and TRA-1-60. (C) iPSC-MN cultures stained with nuclear marker DAPI (blue) and antibodies to motor neuron markers ISLET1 (red) and HB9 (green). (D) iPSC-MN cultures stained with antibody to axonal marker Neurofilament (SMI31). iPSC and iPSC-MN shown are from healthy control IPRN.0013 and fALS patient IPRN.0028. (Scale bars a-b: 200 µm, c-d: 75 µm). (E) (i-ii), Representative trace showing electrical activity of iPS-MN from a healthy individual (Scale bars: 100 mV; 100 ms. 8 neurons recorded from fired robust and repetitive action potentials (bursts) to depolarizing current steps. (Iii) Example trace showing action potential elicited by the rebound depolarization following 300 pA hyperpolarizing step for 100 ms (Scales bars: 100 mV; 100 ms) and example trace of spontaneous bursts of action potentials and spontaneous post-synaptic potentials from iPS derived motor neurons (50 mV; 3 Sec). (Insets) Examples of EPSP (scales: 3 mV; 250 ms), and IPSP (scales: 30 mV, 100 ms). (F) (i) Sporadic ALS iPSC-MN responses −10, 0, and 10 pA current steps for 300 msec (scale: 25 mV). (ii) APs elicited by rebound depolarization following hyperpolarizing current step (300 pA; 300 msec). (iii) Spontaneous activity of sALS iPSC-MN (scale bars; 50 mV; 500 msec) (insets example of sEPSP and sIPSP (scale bars: 5 mV; 200 msec))( n = 7).

    Article Snippet: The following antibodies were used ISLET1 at 1:1000 (Abcam, ab86501), phospho-S409/S410-TDP-43 (Sigma), LAMIN-A at 1:200 (Cell Signaling, 4777), MNR2/HB9 at 1:100 (Developmental Studies Hybridoma Bank, 81.5C10), SMI31 at 1:1000 (Covance, SMI-31R) and/or TARDBP at 1:500 (ProteinTech, 10782-2-AP), CTIP2 at 1:500 (Abcam).

    Techniques: Derivative Assay, Staining, Marker, Activity Assay, Mass Spectrometry, Size-exclusion Chromatography

    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 bound to CCNB1 , CCNB2 and c-MYC (A) MatInspector software analysis of the consensus binding site (TAAT box) for ISL1 on the human CCNB1 promoter, the CCNB2 promoter, and the c-MYC enhancer. Sequences containing mutant base pairs (boxes) were used to construct the luciferase reporter constructs mutant-CCNB1-luc, mutant-CCNB2-luc, and mutant-c-MYC-luc. ( B, C ) Luciferase reporter assay of ISL1 transcriptional activity on the luciferase reporter constructs (-luc) of CCNB1 , CCNB2 and c-MYC in MKN28 cells (WT, wild-type plasmid; M, mutant plasmid). Luciferase activity on the reporter constructs was normalized to Renilla luciferase activity. (D) ChIP assay was performed with anti-ISL1 antibody using chromatin harvested from MKN28 cells. Extracted DNA was amplified using primers that cover the ISL1 binding sites on the CCNB1 and the CCNB2 promoters and the c-MYC enhancer by real-time PCR; normal IgG was used as the control. Data represent three independent experiments, each performed in triplicate. Bars represent the means ± SD (* p

    Journal: Oncotarget

    Article Title: ISL1, a novel regulator of CCNB1, CCNB2 and c-MYC genes, promotes gastric cancer cell proliferation and tumor growth

    doi: 10.18632/oncotarget.9269

    Figure Lengend Snippet: ISL1 bound to CCNB1 , CCNB2 and c-MYC (A) MatInspector software analysis of the consensus binding site (TAAT box) for ISL1 on the human CCNB1 promoter, the CCNB2 promoter, and the c-MYC enhancer. Sequences containing mutant base pairs (boxes) were used to construct the luciferase reporter constructs mutant-CCNB1-luc, mutant-CCNB2-luc, and mutant-c-MYC-luc. ( B, C ) Luciferase reporter assay of ISL1 transcriptional activity on the luciferase reporter constructs (-luc) of CCNB1 , CCNB2 and c-MYC in MKN28 cells (WT, wild-type plasmid; M, mutant plasmid). Luciferase activity on the reporter constructs was normalized to Renilla luciferase activity. (D) ChIP assay was performed with anti-ISL1 antibody using chromatin harvested from MKN28 cells. Extracted DNA was amplified using primers that cover the ISL1 binding sites on the CCNB1 and the CCNB2 promoters and the c-MYC enhancer by real-time PCR; normal IgG was used as the control. Data represent three independent experiments, each performed in triplicate. Bars represent the means ± SD (* p

    Article Snippet: Rabbit monoclonal anti-ISL1 antibody (ab109517) was purchased from Abcam.

    Techniques: Software, Binding Assay, Mutagenesis, Construct, Luciferase, Reporter Assay, Activity Assay, Plasmid Preparation, Chromatin Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction

    ISL1 promoted colony formation in vitro (A) Western blotting analysis of ISL1 levels in MKN28 and MGC803 cell lines with stable ISL1 overexpression (ISL1) or knockdown (si-ISL1) by transfection with pcDNA3.1-ISL1 and pLL3.7-ISL1-siRNA plasmids (cells transfected with pcDNA3.1 vector were used as the negative control), respectively. GAPDH levels served as the internal control. (B) PCF assay performed to determine the proliferation of MKN28 or MGC803 cells stably transfected with ISL1, si-ISL1, or the vector control (a–f). The quantification results are presented on the right. (C) SACF assay of MKN28 and MGC803 cells stably transfected with ISL1, si-ISL1, or vector (a–f). The quantification results are presented on the right. The data are the means ± SD from three independent experiments, each performed in triplicate. (** p

    Journal: Oncotarget

    Article Title: ISL1, a novel regulator of CCNB1, CCNB2 and c-MYC genes, promotes gastric cancer cell proliferation and tumor growth

    doi: 10.18632/oncotarget.9269

    Figure Lengend Snippet: ISL1 promoted colony formation in vitro (A) Western blotting analysis of ISL1 levels in MKN28 and MGC803 cell lines with stable ISL1 overexpression (ISL1) or knockdown (si-ISL1) by transfection with pcDNA3.1-ISL1 and pLL3.7-ISL1-siRNA plasmids (cells transfected with pcDNA3.1 vector were used as the negative control), respectively. GAPDH levels served as the internal control. (B) PCF assay performed to determine the proliferation of MKN28 or MGC803 cells stably transfected with ISL1, si-ISL1, or the vector control (a–f). The quantification results are presented on the right. (C) SACF assay of MKN28 and MGC803 cells stably transfected with ISL1, si-ISL1, or vector (a–f). The quantification results are presented on the right. The data are the means ± SD from three independent experiments, each performed in triplicate. (** p

    Article Snippet: Rabbit monoclonal anti-ISL1 antibody (ab109517) was purchased from Abcam.

    Techniques: In Vitro, Western Blot, Over Expression, Transfection, Plasmid Preparation, Negative Control, Stable Transfection

    ISL1 promoted tumor growth in nude mice ( A, B ) MGC803 cells stably transfected with pcDNA3.1 (control), pcDNA3.1-ISL1 (ISL1), ( C, D) MGC803 cells stably transfected with pLL3.7-Non-silencer (Non-silencer), or pLL3.7-ISL1-siRNA (si-ISL1). A total 1 × 10 7 appropriate cells suspended in 200 μl PBS were injected subcutaneously into the right oxter flank of BALB/c nude mice. Tumors isolated at day 21 (ISL1 and control) or day 24 (si-ISL1 and Non-silencer) are shown in A and C Tumor size was measured at the indicated days post-injection and the results are shown in B and D ( n = 3 at every time point in each group, * p

    Journal: Oncotarget

    Article Title: ISL1, a novel regulator of CCNB1, CCNB2 and c-MYC genes, promotes gastric cancer cell proliferation and tumor growth

    doi: 10.18632/oncotarget.9269

    Figure Lengend Snippet: ISL1 promoted tumor growth in nude mice ( A, B ) MGC803 cells stably transfected with pcDNA3.1 (control), pcDNA3.1-ISL1 (ISL1), ( C, D) MGC803 cells stably transfected with pLL3.7-Non-silencer (Non-silencer), or pLL3.7-ISL1-siRNA (si-ISL1). A total 1 × 10 7 appropriate cells suspended in 200 μl PBS were injected subcutaneously into the right oxter flank of BALB/c nude mice. Tumors isolated at day 21 (ISL1 and control) or day 24 (si-ISL1 and Non-silencer) are shown in A and C Tumor size was measured at the indicated days post-injection and the results are shown in B and D ( n = 3 at every time point in each group, * p

    Article Snippet: Rabbit monoclonal anti-ISL1 antibody (ab109517) was purchased from Abcam.

    Techniques: Mouse Assay, Stable Transfection, Transfection, Injection, Isolation

    ISL1 promoted GC cell proliferation ( A–C) The cell proliferation rate as determined using CCK-8 assay. The values at each time point are normalized to that of the relative cells at time 0, which was set as 1. The data are the means ± SD from three independent experiments, each performed in triplicate (** p

    Journal: Oncotarget

    Article Title: ISL1, a novel regulator of CCNB1, CCNB2 and c-MYC genes, promotes gastric cancer cell proliferation and tumor growth

    doi: 10.18632/oncotarget.9269

    Figure Lengend Snippet: ISL1 promoted GC cell proliferation ( A–C) The cell proliferation rate as determined using CCK-8 assay. The values at each time point are normalized to that of the relative cells at time 0, which was set as 1. The data are the means ± SD from three independent experiments, each performed in triplicate (** p

    Article Snippet: Rabbit monoclonal anti-ISL1 antibody (ab109517) was purchased from Abcam.

    Techniques: CCK-8 Assay

    ISL1 regulated CCNB1 , CCNB2 and c -MYC expression ( A, B) qRT-PCR analysis of CCNB1 , CCNB2 and c-MYC mRNA levels in MGC803 cells transfected with pcDNA3.1-ISL1 (ISL1) or pLL3.7-ISL1-siRNA (si-ISL1). The cells transfected with pcDNA3.1 (control) or pLL3.7-Non-silencer (Non-silencer, control) were used as the controls, whose values were set as 1. The data represent three independent experiments, each performed in triplicate. 18S rRNA levels served as the internal control. Bars represent the mean ± SD (* p

    Journal: Oncotarget

    Article Title: ISL1, a novel regulator of CCNB1, CCNB2 and c-MYC genes, promotes gastric cancer cell proliferation and tumor growth

    doi: 10.18632/oncotarget.9269

    Figure Lengend Snippet: ISL1 regulated CCNB1 , CCNB2 and c -MYC expression ( A, B) qRT-PCR analysis of CCNB1 , CCNB2 and c-MYC mRNA levels in MGC803 cells transfected with pcDNA3.1-ISL1 (ISL1) or pLL3.7-ISL1-siRNA (si-ISL1). The cells transfected with pcDNA3.1 (control) or pLL3.7-Non-silencer (Non-silencer, control) were used as the controls, whose values were set as 1. The data represent three independent experiments, each performed in triplicate. 18S rRNA levels served as the internal control. Bars represent the mean ± SD (* p

    Article Snippet: Rabbit monoclonal anti-ISL1 antibody (ab109517) was purchased from Abcam.

    Techniques: Expressing, Quantitative RT-PCR, Transfection

    Aberrant expression of ISL1 in GC tissues (A) Representative IHC stainings of ISL1 expression in normal gastric mucosa (top, n = 12) and GC samples (bottom, n = 24). (B) Grayscale scanning analysis results of ISL1 expression in the tested cell lines. Grayscale scanning was performed on the western blots of three independent experiments for quantitative analysis. Representative western blots of three experiments are shown at the bottom. β-Actin served as the internal control. GES1, normal gastric cell line; others, GC cell lines. Bars represent the means ± SD (** p

    Journal: Oncotarget

    Article Title: ISL1, a novel regulator of CCNB1, CCNB2 and c-MYC genes, promotes gastric cancer cell proliferation and tumor growth

    doi: 10.18632/oncotarget.9269

    Figure Lengend Snippet: Aberrant expression of ISL1 in GC tissues (A) Representative IHC stainings of ISL1 expression in normal gastric mucosa (top, n = 12) and GC samples (bottom, n = 24). (B) Grayscale scanning analysis results of ISL1 expression in the tested cell lines. Grayscale scanning was performed on the western blots of three independent experiments for quantitative analysis. Representative western blots of three experiments are shown at the bottom. β-Actin served as the internal control. GES1, normal gastric cell line; others, GC cell lines. Bars represent the means ± SD (** p

    Article Snippet: Rabbit monoclonal anti-ISL1 antibody (ab109517) was purchased from Abcam.

    Techniques: Expressing, Immunohistochemistry, Western Blot

    Pallial boundaries during development . Micrographs of transverse sections through the telencephalon of Xenopus laevis at embryonic (A–D) , premetamorphic (E–G) and prometamorphic (H–N) stages. In each panel the developmental stage and the color code for the used markers are indicated. In the developing telencephalon, the combined immunohistochemical detection of Tbr1, expressed in the pallium, and Isl1, a subpallial marker, clearly allowed the identification of the boundary between both regions throughout the rostrocaudal extent (A–G,J,L–N) . The localization of Tbr1 (H) at rostral level, in comparison with GABA (I) , highlights the olfactory bulb, where GABA was very abundant, in contrast to the pallium, where the Tbr1 expression was observed (H,I) . Simultaneous labeling for GABA and Isl1 discerns the SPa from the pallium (K) . Scale bars = 50 μm (A–G) , 100 μm (H–N) . See list for abbreviations.

    Journal: Frontiers in Neuroanatomy

    Article Title: Pattern of Neurogenesis and Identification of Neuronal Progenitor Subtypes during Pallial Development in Xenopus laevis

    doi: 10.3389/fnana.2017.00024

    Figure Lengend Snippet: Pallial boundaries during development . Micrographs of transverse sections through the telencephalon of Xenopus laevis at embryonic (A–D) , premetamorphic (E–G) and prometamorphic (H–N) stages. In each panel the developmental stage and the color code for the used markers are indicated. In the developing telencephalon, the combined immunohistochemical detection of Tbr1, expressed in the pallium, and Isl1, a subpallial marker, clearly allowed the identification of the boundary between both regions throughout the rostrocaudal extent (A–G,J,L–N) . The localization of Tbr1 (H) at rostral level, in comparison with GABA (I) , highlights the olfactory bulb, where GABA was very abundant, in contrast to the pallium, where the Tbr1 expression was observed (H,I) . Simultaneous labeling for GABA and Isl1 discerns the SPa from the pallium (K) . Scale bars = 50 μm (A–G) , 100 μm (H–N) . See list for abbreviations.

    Article Snippet: The anti-Isl1 (39.4D5), BrdU (G3G4), and Lhx2 (2C10) monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at the University of Iowa, Department of Biology, Iowa City, IA 52242.

    Techniques: Immunohistochemistry, Marker, Expressing, Labeling

    Green fluorescent protein (GFP) expression detected under a fluorescence microscope. The lentiviral vectors carried the GFP gene; thus, a fluorescence microscope was used to detected GFP expression in the C3H10T1/2 cells transfected with Lenti-N and the C3H10T1/2 cells transfected with Lenti-Islet-1 at 3 days after transfection. (A) Cell morphology of the C3H10T1/2 cells transfected with Lenti-N observed under a microscope (magnification, ×20). (B) GFP expression of the C3H10T1/2 cells transfected with Lenti-N observed udner a fluorescence microscope (magnification, ×20). (C) Cell morphology of the C3H10T1/2 cells transfected with Lenti-Islet-1 observed under a microscope (magnification, ×20). (D) Cell morphology of the C3H10T1/2 cells transfected with Lenti-Islet-1 observed under a microscope (magnification, ×20). Scale bar, 20 μm. (E and F) Transfection efficiency of teh C3H10T1/2 cells transfected with Lenti-N and the C3H10T1/2 cells transfected with Lenti-Islet-1 detected by flow cytometry (FCM). The transfection efficiency of the C3H10T1/2 cells transduced with lentiviral vectors with pWPI-GFP plasmid or lentiviral vectors with pWPI-GFP-Islet-1 plasmid was detected by FCM. (E) The transfection efficiency of the C3H10T1/2 cells transfected with Lenti-N was 90.12%. (F) The transfection efficiency of the C3H10T1/2 cells transfected with Lenti-Islet-1 was 88.82%.

    Journal: International Journal of Molecular Medicine

    Article Title: Islet-1 promotes the cardiac-specific differentiation of mesenchymal stem cells through the regulation of histone acetylation

    doi: 10.3892/ijmm.2014.1687

    Figure Lengend Snippet: Green fluorescent protein (GFP) expression detected under a fluorescence microscope. The lentiviral vectors carried the GFP gene; thus, a fluorescence microscope was used to detected GFP expression in the C3H10T1/2 cells transfected with Lenti-N and the C3H10T1/2 cells transfected with Lenti-Islet-1 at 3 days after transfection. (A) Cell morphology of the C3H10T1/2 cells transfected with Lenti-N observed under a microscope (magnification, ×20). (B) GFP expression of the C3H10T1/2 cells transfected with Lenti-N observed udner a fluorescence microscope (magnification, ×20). (C) Cell morphology of the C3H10T1/2 cells transfected with Lenti-Islet-1 observed under a microscope (magnification, ×20). (D) Cell morphology of the C3H10T1/2 cells transfected with Lenti-Islet-1 observed under a microscope (magnification, ×20). Scale bar, 20 μm. (E and F) Transfection efficiency of teh C3H10T1/2 cells transfected with Lenti-N and the C3H10T1/2 cells transfected with Lenti-Islet-1 detected by flow cytometry (FCM). The transfection efficiency of the C3H10T1/2 cells transduced with lentiviral vectors with pWPI-GFP plasmid or lentiviral vectors with pWPI-GFP-Islet-1 plasmid was detected by FCM. (E) The transfection efficiency of the C3H10T1/2 cells transfected with Lenti-N was 90.12%. (F) The transfection efficiency of the C3H10T1/2 cells transfected with Lenti-Islet-1 was 88.82%.

    Article Snippet: The antibodies used were as follows: anti-Islet-1 (sc-23590; Santa Cruz Biotechnology, Inc.), mouse monoclonal to cardiac troponin T (cTnT; ab33589), albumin (ALB), bone-specific alkaline phosphatase (BALP), glial fibrillary acidic protein (GFAP) and rabbit polyclonal to histone H3-ChIP Grade (ab1791) (all from Abcam, Cambridge, UK).

    Techniques: Expressing, Fluorescence, Microscopy, Transfection, Flow Cytometry, Cytometry, Transduction, Plasmid Preparation

    Differences in histone acetylation levels in the untransfected C3H10T1/2 cells, the C3H10T1/2 cells transfected with Lenti-N and the C3H10T1/2 cells transfected with Lenti-Islet-1. (A) Acetylated histone H3 (AcH3) was detected by western blot analysis in the untranstected C3H10T1/2 cells, the C3H10T1/2 cells transfected with Lenti-N and the C3H10T1/2 cells transfected with Lenti-Islet-1. The AcH3 relative amount in the C3H10T1/2 cells transfected with Lenti-Islet-1 was higher than that in the untransfected C3H10T1/2 cells and the C3H10T1/2 cells transfected with Lenti-N ( * P

    Journal: International Journal of Molecular Medicine

    Article Title: Islet-1 promotes the cardiac-specific differentiation of mesenchymal stem cells through the regulation of histone acetylation

    doi: 10.3892/ijmm.2014.1687

    Figure Lengend Snippet: Differences in histone acetylation levels in the untransfected C3H10T1/2 cells, the C3H10T1/2 cells transfected with Lenti-N and the C3H10T1/2 cells transfected with Lenti-Islet-1. (A) Acetylated histone H3 (AcH3) was detected by western blot analysis in the untranstected C3H10T1/2 cells, the C3H10T1/2 cells transfected with Lenti-N and the C3H10T1/2 cells transfected with Lenti-Islet-1. The AcH3 relative amount in the C3H10T1/2 cells transfected with Lenti-Islet-1 was higher than that in the untransfected C3H10T1/2 cells and the C3H10T1/2 cells transfected with Lenti-N ( * P

    Article Snippet: The antibodies used were as follows: anti-Islet-1 (sc-23590; Santa Cruz Biotechnology, Inc.), mouse monoclonal to cardiac troponin T (cTnT; ab33589), albumin (ALB), bone-specific alkaline phosphatase (BALP), glial fibrillary acidic protein (GFAP) and rabbit polyclonal to histone H3-ChIP Grade (ab1791) (all from Abcam, Cambridge, UK).

    Techniques: Transfection, Western Blot

    (A) The expression of Gata4, Nkx2.5 and Mef2c gene at different time points in C3H10T1/2 cells transfected with Lenti-Islet-1. 2d, 2nd day; 1w, 1st week; 2w, 2nd week; 3w, 3rd week. The peak expression of cardiac-specific genes in the C3H10T1/2 cells transfected with Lenti-Islet-1 was significantly higher than that in the untransfected C3H10T1/2 cells and the C3H10T1/2 cells transfected with Lenti-N at 2 weeks after transfection ( * P

    Journal: International Journal of Molecular Medicine

    Article Title: Islet-1 promotes the cardiac-specific differentiation of mesenchymal stem cells through the regulation of histone acetylation

    doi: 10.3892/ijmm.2014.1687

    Figure Lengend Snippet: (A) The expression of Gata4, Nkx2.5 and Mef2c gene at different time points in C3H10T1/2 cells transfected with Lenti-Islet-1. 2d, 2nd day; 1w, 1st week; 2w, 2nd week; 3w, 3rd week. The peak expression of cardiac-specific genes in the C3H10T1/2 cells transfected with Lenti-Islet-1 was significantly higher than that in the untransfected C3H10T1/2 cells and the C3H10T1/2 cells transfected with Lenti-N at 2 weeks after transfection ( * P

    Article Snippet: The antibodies used were as follows: anti-Islet-1 (sc-23590; Santa Cruz Biotechnology, Inc.), mouse monoclonal to cardiac troponin T (cTnT; ab33589), albumin (ALB), bone-specific alkaline phosphatase (BALP), glial fibrillary acidic protein (GFAP) and rabbit polyclonal to histone H3-ChIP Grade (ab1791) (all from Abcam, Cambridge, UK).

    Techniques: Expressing, Transfection

    (A) Islet-1 gene expression in the C3H10T1/2 cells transfected with Lenti-Islet-1 was higher ( * P

    Journal: International Journal of Molecular Medicine

    Article Title: Islet-1 promotes the cardiac-specific differentiation of mesenchymal stem cells through the regulation of histone acetylation

    doi: 10.3892/ijmm.2014.1687

    Figure Lengend Snippet: (A) Islet-1 gene expression in the C3H10T1/2 cells transfected with Lenti-Islet-1 was higher ( * P

    Article Snippet: The antibodies used were as follows: anti-Islet-1 (sc-23590; Santa Cruz Biotechnology, Inc.), mouse monoclonal to cardiac troponin T (cTnT; ab33589), albumin (ALB), bone-specific alkaline phosphatase (BALP), glial fibrillary acidic protein (GFAP) and rabbit polyclonal to histone H3-ChIP Grade (ab1791) (all from Abcam, Cambridge, UK).

    Techniques: Expressing, Transfection

    (A) Detection of lentiviral vector. PCR of random clones. Lane M, DL15000 DNA marker; lanes 1–9, clones that we selected. The 7th lane shows the positive clone. (B and C) Part of the Lenti-Islet-1 plasmid sequencing result. (D) Detection of green fluorescent protein (GFP) expression in 293T cells following transfection with Lenti-Islet-1 vectors (magnification, ×10). Scale bar, 100 μm.

    Journal: International Journal of Molecular Medicine

    Article Title: Islet-1 promotes the cardiac-specific differentiation of mesenchymal stem cells through the regulation of histone acetylation

    doi: 10.3892/ijmm.2014.1687

    Figure Lengend Snippet: (A) Detection of lentiviral vector. PCR of random clones. Lane M, DL15000 DNA marker; lanes 1–9, clones that we selected. The 7th lane shows the positive clone. (B and C) Part of the Lenti-Islet-1 plasmid sequencing result. (D) Detection of green fluorescent protein (GFP) expression in 293T cells following transfection with Lenti-Islet-1 vectors (magnification, ×10). Scale bar, 100 μm.

    Article Snippet: The antibodies used were as follows: anti-Islet-1 (sc-23590; Santa Cruz Biotechnology, Inc.), mouse monoclonal to cardiac troponin T (cTnT; ab33589), albumin (ALB), bone-specific alkaline phosphatase (BALP), glial fibrillary acidic protein (GFAP) and rabbit polyclonal to histone H3-ChIP Grade (ab1791) (all from Abcam, Cambridge, UK).

    Techniques: Plasmid Preparation, Polymerase Chain Reaction, Clone Assay, Marker, Sequencing, Expressing, Transfection

    DNA methylation levels and acetylation levels of the histone H3K9 site in the GATA4 and Nkx2.5 promoter regions during the differentiation process promoted by Islet-1. (A) The detection of methylation levels on the GATA4 promoter (1329–1489 bp) by MSP assay. (B) The detection of the methylation levels at the Nkx2.5 promoter (51–219 bp) by MSP assay. (C) ChIP results demonstrated the levels of histone acetylation on the promoter regions of GATA4 and Nkx2.5. *P

    Journal: Molecular Medicine Reports

    Article Title: Islet-1 induces the differentiation of mesenchymal stem cells into cardiomyocyte-like cells through the regulation of Gcn5 and DNMT-1

    doi: 10.3892/mmr.2017.6343

    Figure Lengend Snippet: DNA methylation levels and acetylation levels of the histone H3K9 site in the GATA4 and Nkx2.5 promoter regions during the differentiation process promoted by Islet-1. (A) The detection of methylation levels on the GATA4 promoter (1329–1489 bp) by MSP assay. (B) The detection of the methylation levels at the Nkx2.5 promoter (51–219 bp) by MSP assay. (C) ChIP results demonstrated the levels of histone acetylation on the promoter regions of GATA4 and Nkx2.5. *P

    Article Snippet: The primary antibodies used in the present study were as follows: Anti-Islet-1 (EP4182; 1:2,000; Epitomics, Burlingame, CA, USA), anti-Gcn5 (ab18381; 1:1,000; Abcam), anti-P300 (ab14984; 1:1,000; Abcam), anti-DNMT1 (ab87656; 1:1,000; Abcam), anti-DNMT3a (ab2850; 1:1,000; Abcam) and anti-DNMT3b (ab2851; 1:1,000; Abcam).

    Techniques: DNA Methylation Assay, Methylation, MSP Assay, Chromatin Immunoprecipitation

    Islet-1 induces the differentiation of C3H10T1/2 cells into cardiomyocytes. (A) The morphological alterations in C3H10T1/2 cells transfected with Lv-GFP or Lv-islet-1 were observed under a microscope. Scale bar=100 µm. (B) Expression of cTnT detected by immunofluorescence microscopy. Scale bar=100 µm. (C) Reverse transcription-quantitative polymerase chain reaction detected variations in mRNA expression levels of cardiac-specific transcription factors in C3H10T1/2 cells infected with lentiviral vectors containing Islet-1. *P

    Journal: Molecular Medicine Reports

    Article Title: Islet-1 induces the differentiation of mesenchymal stem cells into cardiomyocyte-like cells through the regulation of Gcn5 and DNMT-1

    doi: 10.3892/mmr.2017.6343

    Figure Lengend Snippet: Islet-1 induces the differentiation of C3H10T1/2 cells into cardiomyocytes. (A) The morphological alterations in C3H10T1/2 cells transfected with Lv-GFP or Lv-islet-1 were observed under a microscope. Scale bar=100 µm. (B) Expression of cTnT detected by immunofluorescence microscopy. Scale bar=100 µm. (C) Reverse transcription-quantitative polymerase chain reaction detected variations in mRNA expression levels of cardiac-specific transcription factors in C3H10T1/2 cells infected with lentiviral vectors containing Islet-1. *P

    Article Snippet: The primary antibodies used in the present study were as follows: Anti-Islet-1 (EP4182; 1:2,000; Epitomics, Burlingame, CA, USA), anti-Gcn5 (ab18381; 1:1,000; Abcam), anti-P300 (ab14984; 1:1,000; Abcam), anti-DNMT1 (ab87656; 1:1,000; Abcam), anti-DNMT3a (ab2850; 1:1,000; Abcam) and anti-DNMT3b (ab2851; 1:1,000; Abcam).

    Techniques: Transfection, Microscopy, Expressing, Immunofluorescence, Real-time Polymerase Chain Reaction, Infection

    Successful establishment of Islet-1 overexpression model in C3H10T1/2 cells. (A) Fluorescence microscopy. Scale bar=100 µm. (B) Infection efficiency, as GFP detected by flow cytometry, was 91.7%. (C) Islet-1 protein expression detected by western blotting, with β-actin as a loading control. Islet-1, insulin gene enhancer binding protein ISL-1; GFP, green fluorescent protein.

    Journal: Molecular Medicine Reports

    Article Title: Islet-1 induces the differentiation of mesenchymal stem cells into cardiomyocyte-like cells through the regulation of Gcn5 and DNMT-1

    doi: 10.3892/mmr.2017.6343

    Figure Lengend Snippet: Successful establishment of Islet-1 overexpression model in C3H10T1/2 cells. (A) Fluorescence microscopy. Scale bar=100 µm. (B) Infection efficiency, as GFP detected by flow cytometry, was 91.7%. (C) Islet-1 protein expression detected by western blotting, with β-actin as a loading control. Islet-1, insulin gene enhancer binding protein ISL-1; GFP, green fluorescent protein.

    Article Snippet: The primary antibodies used in the present study were as follows: Anti-Islet-1 (EP4182; 1:2,000; Epitomics, Burlingame, CA, USA), anti-Gcn5 (ab18381; 1:1,000; Abcam), anti-P300 (ab14984; 1:1,000; Abcam), anti-DNMT1 (ab87656; 1:1,000; Abcam), anti-DNMT3a (ab2850; 1:1,000; Abcam) and anti-DNMT3b (ab2851; 1:1,000; Abcam).

    Techniques: Over Expression, Fluorescence, Microscopy, Infection, Flow Cytometry, Cytometry, Expressing, Western Blot, Binding Assay

    tbx1+ cells contribute to LPM-derived cardiac lineages. a – p Representative maximum intensity projections of whole-mount tbx1 :EGFP-expressing embryos counterstained for anti-EGFP and anti-MHC ( n = 3) ( a – h ) or anti-Isl1 ( n = 11) ( i – p ) at 26 hpf; lateral views, anterior to the left. a – d tbx1 reporter expression can be detected in the MHC-positive linear heart tube and in the MHC-negative poles at the cardiac inflow and outflow tracts (arrowheads); e – h depicts a 2.25x magnification of the framed area in a – d . tbx1 :EGFP also marks the pharyngeal arches (pa) and endothelial cells of the cranial vasculature (cv) ( d ). i – p tbx1 reporter-expressing cells at the IFT co-express the SHF marker Isl1 (asterisks n , o ); m – p depicts a 3x magnification of the framed area in i – l . q Quantification of tbx1 :EGFP/Isl1 double- compared to Isl1 single-positive cells at the IFT of the linear heart tube, n = 11 individual embryos analyzed, means ± s.d. r Lineage tracing of tbx1 and drl reporter-expressing cells, shown in representative embryos. tbx1:creERT2 ( n = 11) or drl:creERT2 ( n = 3) transgenics, respectively, were crossed to the ubiquitous hsp70l:Switch loxP tracer line, embryos were 4-OHT-induced at 90% epiboly, and heat-shocked at 3 dpf. s-a’ Live SPIM imaging of still hearts of representative lineage-traced and control embryos; maximum intensity projections of ventral views, anterior to the top, dashed outlines mark the heart with the bulbus arteriosus (BA), atrium (A), and ventricle (V). s – u tbx1 : creERT2 lineage tracing ( tbx1 > EGFP) at late gastrulation labels myl7 :DsRed2-expressing cardiomyocytes (arrowheads) in the ventricle and inflow tract of the atrium, and DAR-4M-stained cells (arrowhead) in the BA. v – x drl :creERT2 -mediated lineage tracing ( drl > EGFP) at 90% epiboly marks all cardiomyocytes (arrowheads) in the ventricle and atrium, BA cells (arrowhead), and the endocardium (arrows). y - a ’ tbx1:creERT2;hsp70l:Switch transgenics without 4-OHT treatment and heat-shocked at 3 dpf show no specific EGFP expression (asterisks mark the autofluorescent pigment cell). Scale bars 20 μm ( e – h , m – p ), 100 μm ( a – d , i – l , s – a ’)

    Journal: Nature Communications

    Article Title: Continuous addition of progenitors forms the cardiac ventricle in zebrafish

    doi: 10.1038/s41467-018-04402-6

    Figure Lengend Snippet: tbx1+ cells contribute to LPM-derived cardiac lineages. a – p Representative maximum intensity projections of whole-mount tbx1 :EGFP-expressing embryos counterstained for anti-EGFP and anti-MHC ( n = 3) ( a – h ) or anti-Isl1 ( n = 11) ( i – p ) at 26 hpf; lateral views, anterior to the left. a – d tbx1 reporter expression can be detected in the MHC-positive linear heart tube and in the MHC-negative poles at the cardiac inflow and outflow tracts (arrowheads); e – h depicts a 2.25x magnification of the framed area in a – d . tbx1 :EGFP also marks the pharyngeal arches (pa) and endothelial cells of the cranial vasculature (cv) ( d ). i – p tbx1 reporter-expressing cells at the IFT co-express the SHF marker Isl1 (asterisks n , o ); m – p depicts a 3x magnification of the framed area in i – l . q Quantification of tbx1 :EGFP/Isl1 double- compared to Isl1 single-positive cells at the IFT of the linear heart tube, n = 11 individual embryos analyzed, means ± s.d. r Lineage tracing of tbx1 and drl reporter-expressing cells, shown in representative embryos. tbx1:creERT2 ( n = 11) or drl:creERT2 ( n = 3) transgenics, respectively, were crossed to the ubiquitous hsp70l:Switch loxP tracer line, embryos were 4-OHT-induced at 90% epiboly, and heat-shocked at 3 dpf. s-a’ Live SPIM imaging of still hearts of representative lineage-traced and control embryos; maximum intensity projections of ventral views, anterior to the top, dashed outlines mark the heart with the bulbus arteriosus (BA), atrium (A), and ventricle (V). s – u tbx1 : creERT2 lineage tracing ( tbx1 > EGFP) at late gastrulation labels myl7 :DsRed2-expressing cardiomyocytes (arrowheads) in the ventricle and inflow tract of the atrium, and DAR-4M-stained cells (arrowhead) in the BA. v – x drl :creERT2 -mediated lineage tracing ( drl > EGFP) at 90% epiboly marks all cardiomyocytes (arrowheads) in the ventricle and atrium, BA cells (arrowhead), and the endocardium (arrows). y - a ’ tbx1:creERT2;hsp70l:Switch transgenics without 4-OHT treatment and heat-shocked at 3 dpf show no specific EGFP expression (asterisks mark the autofluorescent pigment cell). Scale bars 20 μm ( e – h , m – p ), 100 μm ( a – d , i – l , s – a ’)

    Article Snippet: Primary antibodies used were anti-MHC (MF20 supernatant, DSHB, 1:50), anti-GFP (Abcam, ab13970, 1:500 or Sigma, G1544, 1:100), and anti-Isl1 (GeneTex, GTX128201, 1:50).

    Techniques: Derivative Assay, Expressing, Marker, Imaging, Staining

    Evf-2  and Dlx-2 form a complex in vivo. ( a ) Nuclear extracts made from C17 neural cells transfected with Flag-tagged Emx-1, Flag-tagged Dlx-2, or pcDNA control were analyzed for the presence of  Evf-2 –Dlx-2 complexes by immunoprecipitation with anti-Flag antibody, followed by RT–PCR against  Evf-2 -specific primers and S17 control primers. Western analysis shows that both Flag-Emx-1 and Flag-Dlx-2 are present in nuclear extracts transfected with constructs expressing these proteins. ( b ) Nuclear extracts made from rat E11.5 BAs were analyzed for the presence of  Evf-2 /Dlx-2 complexes by immunoprecipitation with anti- dll,  anti-Islet 1/2, or anti-IgG antibody, followed by RT–PCR against  Evf-2 -specific primers or GAPDH. ( c ) Single-cell suspensions made from mouse E12.5 dorsal and ventral telencephalon were dissected as shown in the schematic, centrifuged onto slides, and processed for fluorescent in situ/immunolocalization using  Evf-2  antisense RNA probe and anti- dll  antibody. DAPI staining (blue) reveals nuclei.  Evf-2  RNA (Alexa fluor 568) is in red, Dlx (Alexa fluor 488) is in green, and regions of overlap are in yellow. ( d ) A model proposing that a complex of  Evf-2  and Dlx-2 affects ei activity, ultimately affecting transcription of the Dlx-5 and Dlx-6 genes.

    Journal: Genes & Development

    Article Title: The Evf-2 noncoding RNA is transcribed from the Dlx-5/6 ultraconserved region and functions as a Dlx-2 transcriptional coactivator

    doi: 10.1101/gad.1416106

    Figure Lengend Snippet: Evf-2 and Dlx-2 form a complex in vivo. ( a ) Nuclear extracts made from C17 neural cells transfected with Flag-tagged Emx-1, Flag-tagged Dlx-2, or pcDNA control were analyzed for the presence of Evf-2 –Dlx-2 complexes by immunoprecipitation with anti-Flag antibody, followed by RT–PCR against Evf-2 -specific primers and S17 control primers. Western analysis shows that both Flag-Emx-1 and Flag-Dlx-2 are present in nuclear extracts transfected with constructs expressing these proteins. ( b ) Nuclear extracts made from rat E11.5 BAs were analyzed for the presence of Evf-2 /Dlx-2 complexes by immunoprecipitation with anti- dll, anti-Islet 1/2, or anti-IgG antibody, followed by RT–PCR against Evf-2 -specific primers or GAPDH. ( c ) Single-cell suspensions made from mouse E12.5 dorsal and ventral telencephalon were dissected as shown in the schematic, centrifuged onto slides, and processed for fluorescent in situ/immunolocalization using Evf-2 antisense RNA probe and anti- dll antibody. DAPI staining (blue) reveals nuclei. Evf-2 RNA (Alexa fluor 568) is in red, Dlx (Alexa fluor 488) is in green, and regions of overlap are in yellow. ( d ) A model proposing that a complex of Evf-2 and Dlx-2 affects ei activity, ultimately affecting transcription of the Dlx-5 and Dlx-6 genes.

    Article Snippet: The C17 nuclear extracts were then incubated with mouse anti-Flag (Sigma) prebound to protein G-agarose, whereas the BA nuclear extracts were incubated with anti- dll (affinity-purified as described by ), anti-Islet 1/2 (40.2D6, Developmental Hybridoma Studies Bank), or anti-rabbit IgG prebound to protein G-agarose overnight at 4°C.

    Techniques: In Vivo, Transfection, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Construct, Expressing, In Situ, Staining, Activity Assay

    Hb9::Cre-derived INs do not overlap with the Shox2 non-V2a population. ( A ) Co-expression of YFP (green) and Isl1 antibody (red) in the Hb9 :: Cre;Rosa26-YFP mouse spinal cord at E11.5. Motor neurons are also labeled by Isl1 antibody (blue box). Rightmost pictures are magnifications of the white boxed area. Arrowheads indicate overlap between Isl1 (red) and Hb9::Cre-derived INs (green). Scale bars: 100 μm and 50 μm. ( B ) Co-expression of YFP (green), Shox2 antibody (red) and/or Chx10 antibody (blue) in the Hb9 :: Cre;Rosa26-YFP mouse ventral spinal cord at E11.5. Rightmost pictures are magnifications of the white boxed area. Arrowheads indicate overlap between Hb9::Cre-derived INs (green) and Shox2 + Chx10 − (red), Shox2 − Chx10 + (blue) or Shox2 + Chx10 + (pink). Scale bars: 100 μm and 50 μm. ( C ) Quantification of overlap in (A) and (B). Bar graph showing percent of overlap between Hb9::Cre-derived INs (YFP + ) and Shox2 V2a (Shox2 + Chx10 + , 4% ± 1%), Shox2 OFF V2a (Shox2 − Chx10 + , 2% ± 0.1%), Shox2 non-V2a (Shox2 + Chx10 − , 1.3% ± 0.2%), and Isl1 (Isl1 + , 6% ± 0.2%) INs. Error bars represent ± SEM. ( D ) Percent of the Shox2 non-V2a IN population (Shox2 + Chx10 − ) that overlaps with Hb9::Cre-derived INs (YFP + ) in the Hb9 :: Cre;Rosa26-YFP mouse spinal cord at E11.5. Shox2 non-V2a INs rarely co-express YFP (Shox2 + YFP + , darker grey) (12% ± 2%). Error bars represent ± SEM.

    Journal: Scientific Reports

    Article Title: Spinal Hb9::Cre-derived excitatory interneurons contribute to rhythm generation in the mouse

    doi: 10.1038/srep41369

    Figure Lengend Snippet: Hb9::Cre-derived INs do not overlap with the Shox2 non-V2a population. ( A ) Co-expression of YFP (green) and Isl1 antibody (red) in the Hb9 :: Cre;Rosa26-YFP mouse spinal cord at E11.5. Motor neurons are also labeled by Isl1 antibody (blue box). Rightmost pictures are magnifications of the white boxed area. Arrowheads indicate overlap between Isl1 (red) and Hb9::Cre-derived INs (green). Scale bars: 100 μm and 50 μm. ( B ) Co-expression of YFP (green), Shox2 antibody (red) and/or Chx10 antibody (blue) in the Hb9 :: Cre;Rosa26-YFP mouse ventral spinal cord at E11.5. Rightmost pictures are magnifications of the white boxed area. Arrowheads indicate overlap between Hb9::Cre-derived INs (green) and Shox2 + Chx10 − (red), Shox2 − Chx10 + (blue) or Shox2 + Chx10 + (pink). Scale bars: 100 μm and 50 μm. ( C ) Quantification of overlap in (A) and (B). Bar graph showing percent of overlap between Hb9::Cre-derived INs (YFP + ) and Shox2 V2a (Shox2 + Chx10 + , 4% ± 1%), Shox2 OFF V2a (Shox2 − Chx10 + , 2% ± 0.1%), Shox2 non-V2a (Shox2 + Chx10 − , 1.3% ± 0.2%), and Isl1 (Isl1 + , 6% ± 0.2%) INs. Error bars represent ± SEM. ( D ) Percent of the Shox2 non-V2a IN population (Shox2 + Chx10 − ) that overlaps with Hb9::Cre-derived INs (YFP + ) in the Hb9 :: Cre;Rosa26-YFP mouse spinal cord at E11.5. Shox2 non-V2a INs rarely co-express YFP (Shox2 + YFP + , darker grey) (12% ± 2%). Error bars represent ± SEM.

    Article Snippet: Sections were incubated for 24 hours with one or several of the following primary antibodies: rabbit anti-Shox2 #860 (1:32,000, generated against the peptide CKTTSKNSSIADLR), sheep anti-Chx10 (1:400, Chemicon), mouse anti-Isl1 (40.2D6 and 39.4D5) (1:250, DSHB), rabbit anti-Hb9 (1:16000, DSHB).

    Techniques: Derivative Assay, Expressing, Labeling

    OTX2 is transiently expressed by differentiating RGCs and by cells differentiating into phenotypes other than RGCs. Confocal microscopic images of frontal cryostat sections of chick retinas immunostained with antiserum against OTX2 ( red ) at E3 ( A , B ), E5 ( C, D ), and E7 ( E, F ) double stained with the RA4 mAb ( green in A, C ), the anti-islet-1 mAb ( blue in B, D ), the 3A10 mAb ( green in E ), or the 3CB2 mAb ( green in F ). The majority of OTX2-positive cells could be identified as RGCs with the RA4 mAb; only a few cells ( arrows ) were stained only for OTX2 ( A, C ). Islet-1-positive RGCs in the mantle do not express OTX2 ( C, D ). At E7 the majority of OTX2-positive cells are neurons expressing the 3A10 antigen ( E ). A few OTX2-positive cells can be identified as Müller cells ( arrows ) by their expression of the 3CB2 antigen ( F ). fl, Fiber layer; gcl, ganglion cell layer; pe, pigment epithelium. Scale bar, A–D, 30 μm; E, F, 20 μm.

    Journal: The Journal of Neuroscience

    Article Title: Implication of OTX2 in Pigment Epithelium Determination and Neural Retina Differentiation

    doi: 10.1523/JNEUROSCI.17-11-04243.1997

    Figure Lengend Snippet: OTX2 is transiently expressed by differentiating RGCs and by cells differentiating into phenotypes other than RGCs. Confocal microscopic images of frontal cryostat sections of chick retinas immunostained with antiserum against OTX2 ( red ) at E3 ( A , B ), E5 ( C, D ), and E7 ( E, F ) double stained with the RA4 mAb ( green in A, C ), the anti-islet-1 mAb ( blue in B, D ), the 3A10 mAb ( green in E ), or the 3CB2 mAb ( green in F ). The majority of OTX2-positive cells could be identified as RGCs with the RA4 mAb; only a few cells ( arrows ) were stained only for OTX2 ( A, C ). Islet-1-positive RGCs in the mantle do not express OTX2 ( C, D ). At E7 the majority of OTX2-positive cells are neurons expressing the 3A10 antigen ( E ). A few OTX2-positive cells can be identified as Müller cells ( arrows ) by their expression of the 3CB2 antigen ( F ). fl, Fiber layer; gcl, ganglion cell layer; pe, pigment epithelium. Scale bar, A–D, 30 μm; E, F, 20 μm.

    Article Snippet: The mAb anti-Islet-1, developed by Prof. T. M. Jessell, was obtained by the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University, School of Medicine (Baltimore, MD), and the Department of Biological Sciences, University of Iowa (Iowa City, IA), under contract N01-HD-2-3144 from the National Institute of Child Health and Human Development.

    Techniques: Staining, Expressing