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  • 94
    Abcam anti pax3
    Ectopic expression of miR-193b induces C2C12 to form brown adipocytes under adipogenic differentiation conditions (a) , C2C12 cells ectopically expressing miR-193b or control were exposed to pro-adipogenic conditions (described in Methods) for 5 days. ORO staining was used to assess lipid accumulation in cells. Representative micrographs of these cells are depicted (bottom row). (b) . Western blot for myogenic markers <t>Pax3</t> , MyoD and brown fat marker Ucp1 . n=3. (c) , Real-time PCR analysis for expression of common adipogenesis markers (upper row) and brown fat selective markers (bottom row). n=3. (d) , C2C12 cells expressing miR-193b (day 6) were stimulated with 500uM dibutyrul cAMP for 4h, and the expression of thermogenic markers, Pgc-1α and Ucp1 was examined by real-time PCR. n=3. (e) , The metabolic profile of C2C12 cells expressing miR-193b (day 6) was assessed using the Seahorse XF24 Extracellular Flux Analyzer. A representative curve of the oxygen consumption rates (OCR) of control and miR-193b expressing cells at their basal states and upon treatment with drugs used to dissect the multiple components of the respiration process is plotted in the top-left panel. The parameters analyzed are represented by different colors in the upper panel and quantitated in other panels. In vitro differentiated primary brown adipocytes (BAT) were used as a reference. n=8. * P
    Anti Pax3, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Developmental Studies Hybridoma Bank anti pax3
    Enhanced Shh signaling in Ptch1 Wig/Wig mutants affects cranial placode development. (A and D) Immunostaining of <t>PAX3</t> (green), SOX10 (red), and DAPI (blue) in wild type (A) and Ptch1 Wig mutants (D). PAX3-positive cells (placodal cells) and PAX3/SOX10 double-positive cells (neural crest cells) are identified by green and white arrowheads respectively. (B, C, E and F) Pax3 mRNA expression in control (B and C) vs. Ptch1 Wig mutants (E and F). Sections in (C and F) correspond to the dotted line in (B and E). Red arrowheads indicate reduced Pax3 expression in Ptch1 Wig/Wig mutant placodal primordia (E, F vs. B, C). (G-L) Whole mount in situ hybridization of indicated placodal markers Ngn1 (G and J), Ngn2 (H and K), and NeuroD1 (I and L) on E9.5 wild type (G-I) vs. Ptch1 Wig mutants (J-L). Red and white arrowheads identify the trigeminal and epibranchial placodes respectively. Scale bars: 20μm (A and D); 100μm (B,E and G-L); 50μm (C and F).
    Anti Pax3, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 91/100, based on 253 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Developmental Studies Hybridoma Bank pax3
    <t>Pax3/Pax7</t> and MRF expression identify distinct cell populations in the somite. ( A ) Pax3 and Pax7 are coexpressed in almost all dermomyotomal (DM), but not myotomal (My) cells at E9.75; transverse section of interlimb somites. Pax3 also marks dorsal root
    Pax3, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 91/100, based on 221 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti pax3
    <t>PAX3-FKHR</t> substantially down-regulates PAI-1 and MHC class I protein expression in mouse and human RMS cells. (A) FACS analysis of MHC class I surface expression. The dotted line represents the secondary antibody alone, and the continuous line represents staining with anti–MHC class I. The staining is representative of four separate experiments. (B) Western blot analysis examining PAI-1 protein expression in RD-CMV and RD-P3F clones and in several human RMS cell lines. In a longer exposure, very faint bands can be detected in the lanes corresponding to the RD-P3F clones 6, 2, and 18. The RMS type and the expression of endogenous PAX3 and PAX3-FKHR RNA (determined by Northern blot; not depicted) for each cell line is indicated. A, ARMS; E, ERMS. (C) FACS analysis of MHC class I surface expression on cells cultured in the presence of 20 ng/ml IFN-γ for 48 h. The dotted line represents the secondary antibody alone, the continuous line represents MHC class I expression in the absence of IFN-γ, and the shaded histogram represents MHC class I in the presence of 20 ng/ml IFN-γ. FACS staining is representative of three separate experiments. Mean fluorescence intensities from the FACS plots are also indicated. Error bars = SEM. (D) Western blot of PAI-1 expression after treatment with 20 ng/ml IFN-γ for 48 h is representative of two independent experiments. PAI-1 was detected at a molecular mass of ∼47 kD, and α-tubulin expression was used as a loading control. (E) PAI-1 expression determined by quantitative RT-PCR. Expression is normalized to GAPDH and expressed relative to RH30 expression. Error bars = SEM of triplicate estimations. (F) FACS histograms of CXCR4 surface expression. The dotted line represents isotype control antibody, and the continuous line represents anti-CXCR4 antibody.
    Anti Pax3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti pax3 mouse monoclonal antibody
    The arrangement of HBMs pathways and pharyngeal arches in shark embryos. <t>Pax3</t> , Dlx5 and Tbx1 expressions in sharks at stage 27 ( a ), 28 ( b ) and 29 ( c ). ( a , b ) Lateral and ( c ) ventrolateral views. Section in situ hybridization of Pax3 , Dlx5 and Tbx1 at stage 27 ( d – f ), 28 ( g – i ) and 29 ( k – m ). Transverse section levels are at the fourth branchial arch ( d – f ), the second branchial arch ( g – i ) and the hyoid arch ( j – m ), as indicated in ( a ), ( b ) and ( c ). Arrowheads indicate HBM precursors. Pharyngeal arches and the pericardium were in the same transverse plane. Note that HBM precursors passed through the lateral side of pericardium, and then extended medially at the hyoid arch. Adp, anterodorsal lateral line placode; AER, apical ectodermal ridge; ba, branchial arch mesoderm; bbm, basibranchial mesenchyme; dm, dermomyotome; drg, dorsal root ganglion; eb, ectomesenchyme of branchial arches; ed., endolymphatic duct; eh, ectomesenchyme of hyoid arch; em, ectomesenchyme of mandibular arch; gb, gill bud; ha, hyoid arch mesoderm; he, heart; hg, hatching gland; ih interhyoideus; ma, mandibular arch mesoderm; ne, nasal epithelium; np, nasal prominence; nt, neural tube; ot, otic vesicle; pcc, pericardial cavity; pcm, pericardial mesoderm; pe, pharyngeal endoderm; pfm, pectoral fin muscle; pohm, postotic paraxial head mesoderm; scl, sclerotome; thy thyroid gland. Scale bars on whole embryos, 200 μm. Scale bars on sections, 50 μm
    Anti Pax3 Mouse Monoclonal Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher pax3
    Abnormal gene expression in the patches. A–R , In situ hybridization of E12.5 and E16.5 Otx1 cre/+ ; Otx2 +/− and conditional mutants with Gli1 ( A , F ), Gli2 ( B , G ), Gli3 ( C , H ), Ptch1 ( D , I ), Smo ( E , J ), Lim1 ( K , O ), <t>Pax3</t> ( L , P ), Pax7 ( M , Q ), and Gbx2 ( N , R ) probes. Note that Gbx2 is lost in the patches in which the expression of Lim1 , Pax3 , and Pax7 is activated. For control embryos, the sections at E12.5 from A–D and from E–N are two different groups of adjacent sections; similarly, also for the conditional mutant, the sections at E12.5 from A–D and from E–N are two groups of adjacent sections belonging to two different embryos. Th, Thalamus.
    Pax3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    R&D Systems anti pax3
    Ex vivo correction of dystrophic iPS cells (a) Scheme of the ex vivo gene therapy approach, which involves (1) reprogramming of dystrophic donor-derived fibroblasts into iPS cells, (2) genetic repair of iPS cells with the μ-Utrn transgene using the Sleeping Beauty transposon system, (3) generation of myogenic progenitors from corrected iPS cells through <t>Pax3</t> induction, and (4) transplantation of corrected myogenic precursors into dystrophic donor mice. (b) The Sleeping Beauty transposon system: the transposon contains the hEF1α-eIF4g promoter, the μUtrn gene, and an iresGFP. The whole transgene is flanked by the terminal inverted repeats (IR/DR, arrowheads) each containing two binding sites for the transposase (DR, yellow arrows). The transposase protein SB100X (red spheres) catalyzes integration of the transposon into the genome with high efficiency. (c) Left panel: Graphic bars represent percentage of GFP + cells obtained prior to each sorting. Green bars correspond to iPS cells nucleofected with pKT2/μUTRN-iresGFP/SB100X, while yellow bars represent controls, iPS cells transduced with pKT2/μUTRN-IresGFP only (no transposase). Data are mean ±S.E.M. of 3 independent samples. (d) FACS profile for GFP shows stable expression of μUTRN in corrected iPS cells (right panel), while control iPS cells are GFP − (left panel). (e) Flow cytometric analyses for Flk-1 and PDGFαR expression on day 5 EBs of Pax3-induced uncorrected- (upper panel) and corrected-iPS (lower panel) cells. The PDGFαR + Flk-1 − cell fraction was gated (red square on left panels) and analyzed for the expression of GFP, representing μUTRN + cells (middle panels), and mCherry, representing Pax3 + cells (right panels). (f) Phase contrast images of monolayers under proliferation (left) and differentiation (right) culture conditions. (g) Immunofluorescence staining for μUTRN (green) in proliferating iPS-derived myogenic progenitors (left) and their derivative myotubes (right). Cells are co-stained with DAPI (blue). Scale bar is 200μm. (h) qPCR analyses indicate relative expression of μ-utrophin in corrected iPS-derived myogenic cells under proliferation (P) and differentiation (D) conditions. Actin was used as house-keeping gene. Error bars represent S.E.M. from three replicates of three independent experiments. (i) Immunoblotting with anti-UTRN and anti-FLAG antibodies confirm the presence of 163-kDa μUTRN only in corrected cells.
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    85
    Santa Cruz Biotechnology anti pax3 7
    Ex vivo correction of dystrophic iPS cells (a) Scheme of the ex vivo gene therapy approach, which involves (1) reprogramming of dystrophic donor-derived fibroblasts into iPS cells, (2) genetic repair of iPS cells with the μ-Utrn transgene using the Sleeping Beauty transposon system, (3) generation of myogenic progenitors from corrected iPS cells through <t>Pax3</t> induction, and (4) transplantation of corrected myogenic precursors into dystrophic donor mice. (b) The Sleeping Beauty transposon system: the transposon contains the hEF1α-eIF4g promoter, the μUtrn gene, and an iresGFP. The whole transgene is flanked by the terminal inverted repeats (IR/DR, arrowheads) each containing two binding sites for the transposase (DR, yellow arrows). The transposase protein SB100X (red spheres) catalyzes integration of the transposon into the genome with high efficiency. (c) Left panel: Graphic bars represent percentage of GFP + cells obtained prior to each sorting. Green bars correspond to iPS cells nucleofected with pKT2/μUTRN-iresGFP/SB100X, while yellow bars represent controls, iPS cells transduced with pKT2/μUTRN-IresGFP only (no transposase). Data are mean ±S.E.M. of 3 independent samples. (d) FACS profile for GFP shows stable expression of μUTRN in corrected iPS cells (right panel), while control iPS cells are GFP − (left panel). (e) Flow cytometric analyses for Flk-1 and PDGFαR expression on day 5 EBs of Pax3-induced uncorrected- (upper panel) and corrected-iPS (lower panel) cells. The PDGFαR + Flk-1 − cell fraction was gated (red square on left panels) and analyzed for the expression of GFP, representing μUTRN + cells (middle panels), and mCherry, representing Pax3 + cells (right panels). (f) Phase contrast images of monolayers under proliferation (left) and differentiation (right) culture conditions. (g) Immunofluorescence staining for μUTRN (green) in proliferating iPS-derived myogenic progenitors (left) and their derivative myotubes (right). Cells are co-stained with DAPI (blue). Scale bar is 200μm. (h) qPCR analyses indicate relative expression of μ-utrophin in corrected iPS-derived myogenic cells under proliferation (P) and differentiation (D) conditions. Actin was used as house-keeping gene. Error bars represent S.E.M. from three replicates of three independent experiments. (i) Immunoblotting with anti-UTRN and anti-FLAG antibodies confirm the presence of 163-kDa μUTRN only in corrected cells.
    Anti Pax3 7, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher anti pax3 7
    Neuroprogenitor migration in Ntn1 , Dcc , and Robo3 knockouts. a Schematic of the migration of the dorsal spinal cord progenitors and interneurons. VZ, ventricular zone. b Cross sections of the spinal cord electroporated with Actb-gfp ( βactin-gfp) . The closeup images are of the boxed area. The embryos were cultured for 20 h. GFP+ neurons from all three KOs migrate out of the VZ (demarcated by <t>PAX3/7</t> staining) later than WT neurons. c Quantification of the ratio between GFP+ neurons within the VZ and the total GFP+ neurons. A higher percentage of neurons is seen within the VZ in all three KOs. Data are represented as the mean ± SEM (Student’s t -test; **, p
    Anti Pax3 7, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Developmental Studies Hybridoma Bank mouse anti pax3
    Nkx3.2, but not Sox9, inhibits the expression of <t>Pax3</t> and Pax7 in ovo A. Ectopic expression of Nkx3.2 in the dorsal somite cells. In situ hybridization (ISH) analysis of chicken embryos electroporated with pMES-Nkx3.2-GFP into the lumen of the newly formed somites two days after electroporation, using an Nkx3.2 RNA probe. 5 embryos were analyzed and all exhibited ectopic Nkx3.2 in the dorsal somitic cells. Left panel: whole mount ISH; right panel: a cross section of the whole mount ISH embryo. Arrows: exogenous Nkx3.2 expression. Arrowheads: endogenous Nkx3.2 expression. B. Ectopic expression of Sox9 in the dorsal somite cells. Immunocytochemistry analysis on sectioned embryos electroporated with pMES-Sox9-GFP two days after electroporation. 5 sections were analyzed and all cells that expressed GFP also expressed Sox9V5. GFP-positive Panel 1, V5 staining showing Sox9V5 expression in dorsal somite cells. A bright field view of the section was overlaid on the fluorescent image. Panel 2, magnified view of Sox9V5-positive cells. Panel 3, magnified view of GFP-positive cells. Panel 4, merged image (yellow) of Sox9V5 and GFP. C. Nkx3.2, but not Sox9, inhibited Pax3 and Pax7 expression. Embryos electroporated with either pMES-GFP (vector), pMES-Nkx3.2-GFP or pMES-Sox9V5-GFP into the lumen of the newly formed somites were whole mount fixed and serial sectioned. The sections were immunostained with antibodies against Pax3 and Pax7. Cells harboring the introduced DNA are GFP-positive, and thus are green. Only overlaid images are shown. Most of the cells that expressed the introduced plasmids were laterally located. However, the few medially-targeted cells exhibit the same phenotype as these laterally located cells. Panels 1-4, GFP electroporated embryos (Panels 1-2 are 10X views, and Panels 3-4 are 40X views). Panels 5-8, Nkx3.2 electroporated embryos (Panels 5-6 are 10X views, and Panels 7-8 are 40X views). Panels 9-12, Sox9V5 electroporated embryos (Panels 9-10 are 10X views, panels 11-12 are 40X views). Arrows: cells that express introduced DNA (GFP-positive), but are not Pax3 or Pax7 -positive (red). Arrowheads: cells that express introduced DNA (GFP-positive), and are also Pax3 or Pax7-positive (red), and are thus yellow. D. Quantification of the results of Fig.5C. Percentage of electroporated cells that express Pax3 and Pax7 was quantified. For each electroporated sample, a total number of 100-200 targeted cells from at least 3 embryos were analyzed under the confocal microscope. Only the target cells (GFP-positive) that were located in the normal Pax3 and Pax7 expression domains were used for counting. Pax3, blue. Pax7, orange. E. Nkx3.2 induced the expression of Sox9 in the dorsal somite cells. Embryos electroporated with pMES-Nkx3.2-GFP into the lumen of the newly formed somites were subjected to whole mount in situ hybridization and sectioning, using a Sox9 RNA probe. 5 embryos were analyzed. Panel 1, in situ hybridization of Sox9. Arrowhead: exogenous Sox9 expression. Arrow: endogenous Sox9 expression. A bright field view of the section was overlaid on the fluorescent image. Panel 2, Anti-GFP staining indicating the ectopic expression of the introduced DNA pMES-Nkx3.2-GFP (arrow). Panel 3, Overlay of the bright field ISH image with the GFP immunostaining image (arrow). Panel 4, merged bright field image of ISH and immunofluorescent staining of endogenous Pax3, showing ectopic Sox9 expression within the Pax3-expressing domain.
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    94
    Cell Signaling Technology Inc anti pax3
    Co-localization of <t>Pax3</t> and Foxo1 in primary mouse myoblasts. A. Percentages of Pax3 and Foxo1 co-localization in interphase nuclei of primary mouse myoblasts and MEFs as determined by FISH analysis using Pax3 and Foxo1 BAC probes. Myo = myoblasts; MEFs = mouse embryo fibroblasts. P2 and P3 represent the second or third passage of the myoblasts in culture, B. Compilation of eight independent Pax3 and Foxo1 co-localization experiments in interphase nuclei of primary fore limb and hind limb mouse myoblasts as determined by FISH analysis using Pax3 and Foxo1 BAC probes. Experiments1, 3, 5, 6, 7 and 8 were done with normal myoblasts, while experiments 2 and 4 were performed with Foxo1-inv+/+ myoblasts. This compilation includes the two fore limb and hind limb co-localization experiments shown in A.C. Confocal FISH micrographs showing co-localization of Pax3 (shown in green) and Foxo1 (shown in red) genes in interphase nuclei of mouse primary myoblasts. D. Q-RT-PCR analysis of Pax3 , Foxo1 , Pax7 expression in primary mouse fore limb and hind limb myoblasts.
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    Santa Cruz Biotechnology goat anti pax3
    <t>Pax3</t> flox/flox /P0-Cre conditional knockout mice do not exhibit congenital hydrocephalus, indicating that restricted loss of Pax3 within the migratory neural crest is insufficient to cause defects. ( A ) P0-Cre/R26r lineage mapping in E10.5 control embryos reveals that the neural crest-colonized tissues, such as cranial-facial (green arrow), pharyngeal arches (blue arrows), and dorsal root ganglia (red arrow) are all positively labeled (blue staining). However, as P0-Cre is only expressed in neural crest derivatives after emigration from the neural tube, P0-Cre/R26r lineage mapping verified that the neural tube (white arrow), hindbrain, and brain are not labeled; ( B ) lateral view of P12 Pax3 flox/flox /P0-Cre (upper) and control (lower) littermates. Their lateral cranial profile is indistinguishable (indicated by dotted lines and compared side-by-side in insert). Also note normal pigmentation of Pax3 flox/flox /P0-Cre neonate: ( C,D ) coronal views of neonates in B , verifying Pax3 flox/flox /P0-Cre neonate lateral ventricles (arrows in C) are unaffected.
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    Developmental Studies Hybridoma Bank monoclonal antibody anti pax3
    5f restored neural tube development and marker expression in chick embryos on EDD 5. Glucose concentration ( A ) and <t>Pax3</t> protein expression ( B ) were determined in chick embryos. The plasma glucose concentration was measured using a glucose oxidase-coupled spectrophotometric assay kit. Proteins were detected using the monoclonal antibody anti-Pax3 diluted 1:1000 (DSHB, USA) and visualized using anti-mouse IgG conjugated with horseradish peroxidase (HRP) and Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, USA) as the substrate of HRP. The abbreviations “CON”, “GLU”, “CL”, “CM”, “CH”, “EPA”, “EDA” mean “controlled”, “glucose treated”, “low concentration of 5f treated”, “mild concentration of 5f treated”, “high concentration of 5f treated”, “epalrestat treated”, “edaravone treated” groups, respectively. Values were expressed as mean ± SD in each group (n = 10). * P
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    Bioss rabbit anti pax3
    Analysis of relative hair color gene expression for evidence of a transcriptome profile, gp100 expression and MC4R protein expression in the skin of Cdk5 knockdown mice. ( A ) Western blot analysis of MITF, TYRP1, TYRP2, <t>PAX3,</t> MC1R, gp100 and MC4R protein expression in the skin of Cdk5 knockdown mice comp ompared to the wild-type mouse ared with wild-type mice. ( B ) The abundance of the MITF, TYRP1, TYRP2, PAX3 and MC1R proteins was quantified using Image-Pro Plus software and normalized relative to the abundance of β-actin. Bars represent the mean ± standard error (n = 3). ***P
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    Image Search Results


    Ectopic expression of miR-193b induces C2C12 to form brown adipocytes under adipogenic differentiation conditions (a) , C2C12 cells ectopically expressing miR-193b or control were exposed to pro-adipogenic conditions (described in Methods) for 5 days. ORO staining was used to assess lipid accumulation in cells. Representative micrographs of these cells are depicted (bottom row). (b) . Western blot for myogenic markers Pax3 , MyoD and brown fat marker Ucp1 . n=3. (c) , Real-time PCR analysis for expression of common adipogenesis markers (upper row) and brown fat selective markers (bottom row). n=3. (d) , C2C12 cells expressing miR-193b (day 6) were stimulated with 500uM dibutyrul cAMP for 4h, and the expression of thermogenic markers, Pgc-1α and Ucp1 was examined by real-time PCR. n=3. (e) , The metabolic profile of C2C12 cells expressing miR-193b (day 6) was assessed using the Seahorse XF24 Extracellular Flux Analyzer. A representative curve of the oxygen consumption rates (OCR) of control and miR-193b expressing cells at their basal states and upon treatment with drugs used to dissect the multiple components of the respiration process is plotted in the top-left panel. The parameters analyzed are represented by different colors in the upper panel and quantitated in other panels. In vitro differentiated primary brown adipocytes (BAT) were used as a reference. n=8. * P

    Journal: Nature cell biology

    Article Title: MiR-193b-365, a brown fat enriched microRNA cluster, is essential for brown fat differentiation

    doi: 10.1038/ncb2286

    Figure Lengend Snippet: Ectopic expression of miR-193b induces C2C12 to form brown adipocytes under adipogenic differentiation conditions (a) , C2C12 cells ectopically expressing miR-193b or control were exposed to pro-adipogenic conditions (described in Methods) for 5 days. ORO staining was used to assess lipid accumulation in cells. Representative micrographs of these cells are depicted (bottom row). (b) . Western blot for myogenic markers Pax3 , MyoD and brown fat marker Ucp1 . n=3. (c) , Real-time PCR analysis for expression of common adipogenesis markers (upper row) and brown fat selective markers (bottom row). n=3. (d) , C2C12 cells expressing miR-193b (day 6) were stimulated with 500uM dibutyrul cAMP for 4h, and the expression of thermogenic markers, Pgc-1α and Ucp1 was examined by real-time PCR. n=3. (e) , The metabolic profile of C2C12 cells expressing miR-193b (day 6) was assessed using the Seahorse XF24 Extracellular Flux Analyzer. A representative curve of the oxygen consumption rates (OCR) of control and miR-193b expressing cells at their basal states and upon treatment with drugs used to dissect the multiple components of the respiration process is plotted in the top-left panel. The parameters analyzed are represented by different colors in the upper panel and quantitated in other panels. In vitro differentiated primary brown adipocytes (BAT) were used as a reference. n=8. * P

    Article Snippet: Proteins were separated by SDS–PAGE, transferred to Nitrocellulose membrane (Millipore) and probed with anti-Cdon (AF2429, R & D system), anti-Igfbp5 (AF578, R & D system), Anti-Ucp1 (ab10983, Abcam), anti-MyoD (SC-760, Santa Cruz), anti-PAX3 (ab15717, Abcam), anti-Runx1t1 (SC-9737, Santa Cruz),and anti-Myosin (MF20, Developmental Studies Hybridoma Bank) antibodies.

    Techniques: Expressing, Staining, Western Blot, Marker, Real-time Polymerase Chain Reaction, Pyrolysis Gas Chromatography, In Vitro

    Expression of transcription factors and lncRNAs in tissues and cells ( A ) HOXD8, Lhx3, and Pax3 expression in the placenta tissues of both PE patients and healthy controls was measured using Western blot assays. ( B ) RP11-269F21.2, DIAPH2-AS1, and RP11-445K13.2 expression in the placenta tissue of PE patients and healthy controls was measured using PCR assays. ( C ) HOXD8 and Lhx3 expression in HTR-8/SVneo cells under normoxia and hypoxia was measured using Western blot assays. ( D ) RP11-269F21.2, DIAPH2-AS1, and RP11-445K13.2 expression in HTR-8/SVneo cells under normoxia and hypoxia was measured using PCR assays. * P

    Journal: Bioscience Reports

    Article Title: HOXD8/DIAPH2-AS1 epigenetically regulates PAX3 and impairs HTR-8/SVneo cell function under hypoxia

    doi: 10.1042/BSR20182022

    Figure Lengend Snippet: Expression of transcription factors and lncRNAs in tissues and cells ( A ) HOXD8, Lhx3, and Pax3 expression in the placenta tissues of both PE patients and healthy controls was measured using Western blot assays. ( B ) RP11-269F21.2, DIAPH2-AS1, and RP11-445K13.2 expression in the placenta tissue of PE patients and healthy controls was measured using PCR assays. ( C ) HOXD8 and Lhx3 expression in HTR-8/SVneo cells under normoxia and hypoxia was measured using Western blot assays. ( D ) RP11-269F21.2, DIAPH2-AS1, and RP11-445K13.2 expression in HTR-8/SVneo cells under normoxia and hypoxia was measured using PCR assays. * P

    Article Snippet: After blocking with non-fat dry milk (Yili Milk company, Inner Mongolia, China) at room temperature for 2 h, the membranes were hybridized with anti-Pax3 antibody (Dilution 1:1000, ab180754, Abcam, Cambridge, U.K.), anti-HOXD8 antibody (Dilution 1:200, ab229321, Abcam), anti-LHX3 antibody (Dilution 1:400, ab124697, Abcam), and anti-GAPDH antibody (Dilution 1:2000, SC-365062, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight.

    Techniques: Expressing, Western Blot, Polymerase Chain Reaction

    The regulatory effect of the HOXD8/DIAPH2-AS1/PAX3 network on cell proliferation and invasion Either HOXD8 or DIAPH2-AS1 were knocked down or PAX3 was overexpressed in HTR-8/SVneo cells under hypoxia. Afterward, HOXD8 and PAX3 expression was measured by Western blot assay ( A ). Cell viability was assessed using MTT testing agent ( B ). Cell migration and invasive capacities were evaluated by scratch ( C ) and transwell cell invasion ( D ) assays, respectively. * P

    Journal: Bioscience Reports

    Article Title: HOXD8/DIAPH2-AS1 epigenetically regulates PAX3 and impairs HTR-8/SVneo cell function under hypoxia

    doi: 10.1042/BSR20182022

    Figure Lengend Snippet: The regulatory effect of the HOXD8/DIAPH2-AS1/PAX3 network on cell proliferation and invasion Either HOXD8 or DIAPH2-AS1 were knocked down or PAX3 was overexpressed in HTR-8/SVneo cells under hypoxia. Afterward, HOXD8 and PAX3 expression was measured by Western blot assay ( A ). Cell viability was assessed using MTT testing agent ( B ). Cell migration and invasive capacities were evaluated by scratch ( C ) and transwell cell invasion ( D ) assays, respectively. * P

    Article Snippet: After blocking with non-fat dry milk (Yili Milk company, Inner Mongolia, China) at room temperature for 2 h, the membranes were hybridized with anti-Pax3 antibody (Dilution 1:1000, ab180754, Abcam, Cambridge, U.K.), anti-HOXD8 antibody (Dilution 1:200, ab229321, Abcam), anti-LHX3 antibody (Dilution 1:400, ab124697, Abcam), and anti-GAPDH antibody (Dilution 1:2000, SC-365062, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight.

    Techniques: Expressing, Western Blot, MTT Assay, Migration

    The regulatory effect of the HOXD8/DIAPH2-AS1 network on PAX3 expression ( A ) DIAPH2-AS1 and RP11-445K13.2 were knocked down in HTR-8/SVneo cells under hypoxia using RNA interference technology. ( B ) The expression of PAX3 in HTR-8/SVneo cells under hypoxia was measured by Western blot assay after DIAPH2-AS1 and RP11-445K13.2 knockdown. ( C ) The combination of LSD1 and DNMT1 with histone 3 was analyzed by chromatin immunoprecipitation assay after HOXD8 and DIAPH2-AS1 knockdown. ( D ) RIP assay was performed on HTR8/SVneo cells to investigate interactions between LSD1 and DIAPH2-AS1. The enrichment of DIAPH2-AS1 on LSD1 was determined by PCR after HOXD8 knockdown or not. ( E ) MeDIP-PCR assay was performed to evaluate the methylation levels at the promoter region of Pax3 gene after the knockdown of HOXD8 and DIAPH2-AS1. * P

    Journal: Bioscience Reports

    Article Title: HOXD8/DIAPH2-AS1 epigenetically regulates PAX3 and impairs HTR-8/SVneo cell function under hypoxia

    doi: 10.1042/BSR20182022

    Figure Lengend Snippet: The regulatory effect of the HOXD8/DIAPH2-AS1 network on PAX3 expression ( A ) DIAPH2-AS1 and RP11-445K13.2 were knocked down in HTR-8/SVneo cells under hypoxia using RNA interference technology. ( B ) The expression of PAX3 in HTR-8/SVneo cells under hypoxia was measured by Western blot assay after DIAPH2-AS1 and RP11-445K13.2 knockdown. ( C ) The combination of LSD1 and DNMT1 with histone 3 was analyzed by chromatin immunoprecipitation assay after HOXD8 and DIAPH2-AS1 knockdown. ( D ) RIP assay was performed on HTR8/SVneo cells to investigate interactions between LSD1 and DIAPH2-AS1. The enrichment of DIAPH2-AS1 on LSD1 was determined by PCR after HOXD8 knockdown or not. ( E ) MeDIP-PCR assay was performed to evaluate the methylation levels at the promoter region of Pax3 gene after the knockdown of HOXD8 and DIAPH2-AS1. * P

    Article Snippet: After blocking with non-fat dry milk (Yili Milk company, Inner Mongolia, China) at room temperature for 2 h, the membranes were hybridized with anti-Pax3 antibody (Dilution 1:1000, ab180754, Abcam, Cambridge, U.K.), anti-HOXD8 antibody (Dilution 1:200, ab229321, Abcam), anti-LHX3 antibody (Dilution 1:400, ab124697, Abcam), and anti-GAPDH antibody (Dilution 1:2000, SC-365062, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight.

    Techniques: Expressing, Western Blot, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Methylated DNA Immunoprecipitation, Methylation

    FKHR-PAX3 selectively increased cell adhesion over cell movement and invasion. ( A ) FKHR-PAX3 increased attachment of RD (top panel) and RH30 (bottom panel) cells to the indicated extracellular matrix components. ( B ) FKHR-PAX3 expression in RD (left panel) and RH30 (right panel) increased the strength of cell adhesion to culture dishes as indicated by a reduced sensitivity to trypsin. ( C ) Western blot analysis of the effect of ectopic FKHR-PAX3 expression on FAK phosphorylation in RD and RH30 cells. A total of 30 μg of whole cell extracts were analyzed. ( D ) FKHR-PAX3 reduced the migratory function in RD (top panel) and RH30 (bottom panel) cells as measured by scratch wound assay. Left panel: quantification of migratory index; right panel: representative micrographs of the scratched wound assays. ( E ) FKHR-PAX3 decreased the invasive potential of RD (left panel) and RH30 (right panel) as determined by Matrigel assay. ND: statistically no difference. ( A - B , D - E ) Assays were conducted as described in Materials and Methods. Representative data from FKHR-PAX3 isoform c is shown.

    Journal: PLoS ONE

    Article Title: Structural and Functional Studies of FKHR-PAX3, a Reciprocal Fusion Gene of the t(2;13) Chromosomal Translocation in Alveolar Rhabdomyosarcoma

    doi: 10.1371/journal.pone.0068065

    Figure Lengend Snippet: FKHR-PAX3 selectively increased cell adhesion over cell movement and invasion. ( A ) FKHR-PAX3 increased attachment of RD (top panel) and RH30 (bottom panel) cells to the indicated extracellular matrix components. ( B ) FKHR-PAX3 expression in RD (left panel) and RH30 (right panel) increased the strength of cell adhesion to culture dishes as indicated by a reduced sensitivity to trypsin. ( C ) Western blot analysis of the effect of ectopic FKHR-PAX3 expression on FAK phosphorylation in RD and RH30 cells. A total of 30 μg of whole cell extracts were analyzed. ( D ) FKHR-PAX3 reduced the migratory function in RD (top panel) and RH30 (bottom panel) cells as measured by scratch wound assay. Left panel: quantification of migratory index; right panel: representative micrographs of the scratched wound assays. ( E ) FKHR-PAX3 decreased the invasive potential of RD (left panel) and RH30 (right panel) as determined by Matrigel assay. ND: statistically no difference. ( A - B , D - E ) Assays were conducted as described in Materials and Methods. Representative data from FKHR-PAX3 isoform c is shown.

    Article Snippet: Antibodies were purchased for MyoD and MyoG (BD Biosciences), myosin heavy chain and α-tubulin (Developmental Studies Hybridoma Bank), PAX3 (abcam), FAK (Santa Cruz), and FKHR, p-Y397, p-Y576/577, and p-Y925-FAK (Cell signaling).

    Techniques: Expressing, Western Blot, Scratch Wound Assay Assay, Matrigel Assay

    FKHR-PAX3 promoted low-density cell proliferation and blocked terminal myogenic differentiation. ( A ) Comparison of cell proliferation in NIH3T3, RD, RH4, and RH30 cultures with or without FKHR-PAX3 (FP3) expression. Cells were seeded in triplicate at 1 x 10 4 cells/well into a 24 well-plate. Proliferation was measured daily by counting the number of live cells (trypan blue-negative) over five days beginning a day after the initial seeding. Cell death was minimal in all experiments. ( B ) The effect of FKHR-PAX3 expression on low-density RD and RH30 growth. Cells were seeded in triplicate at 2 x 10 3 cells/well into a 24 well-plate. Cell growth was quantified daily using the WST-1 cell proliferation kit. ( C ) Top panel: Immunodetection of MyoG and MHC expression in proliferating (GM) and differentiated (five days, DM) C2C12 cells with or without FKHR-PAX3. Bottom panel: light (left panel, 100X magnification) and fluorescent (middle and right panels, (250X magnification) microscopic images of day-5 differentiated cells stained with MF20 antibody against MHC (middle) or with DAPI (right). ( D ) The effect of PAX3-FKHR knockdown on MyoG and MHC expression in control and FKHR-PAX3 expressing RH30 cells. Cell extracts were analyzed by western blot as in Figure 2D. ( C - D ) Alpha-tubulin was used to normalize sample loading. Representative data from FKHR-PAX3 isoform c is shown.

    Journal: PLoS ONE

    Article Title: Structural and Functional Studies of FKHR-PAX3, a Reciprocal Fusion Gene of the t(2;13) Chromosomal Translocation in Alveolar Rhabdomyosarcoma

    doi: 10.1371/journal.pone.0068065

    Figure Lengend Snippet: FKHR-PAX3 promoted low-density cell proliferation and blocked terminal myogenic differentiation. ( A ) Comparison of cell proliferation in NIH3T3, RD, RH4, and RH30 cultures with or without FKHR-PAX3 (FP3) expression. Cells were seeded in triplicate at 1 x 10 4 cells/well into a 24 well-plate. Proliferation was measured daily by counting the number of live cells (trypan blue-negative) over five days beginning a day after the initial seeding. Cell death was minimal in all experiments. ( B ) The effect of FKHR-PAX3 expression on low-density RD and RH30 growth. Cells were seeded in triplicate at 2 x 10 3 cells/well into a 24 well-plate. Cell growth was quantified daily using the WST-1 cell proliferation kit. ( C ) Top panel: Immunodetection of MyoG and MHC expression in proliferating (GM) and differentiated (five days, DM) C2C12 cells with or without FKHR-PAX3. Bottom panel: light (left panel, 100X magnification) and fluorescent (middle and right panels, (250X magnification) microscopic images of day-5 differentiated cells stained with MF20 antibody against MHC (middle) or with DAPI (right). ( D ) The effect of PAX3-FKHR knockdown on MyoG and MHC expression in control and FKHR-PAX3 expressing RH30 cells. Cell extracts were analyzed by western blot as in Figure 2D. ( C - D ) Alpha-tubulin was used to normalize sample loading. Representative data from FKHR-PAX3 isoform c is shown.

    Article Snippet: Antibodies were purchased for MyoD and MyoG (BD Biosciences), myosin heavy chain and α-tubulin (Developmental Studies Hybridoma Bank), PAX3 (abcam), FAK (Santa Cruz), and FKHR, p-Y397, p-Y576/577, and p-Y925-FAK (Cell signaling).

    Techniques: Expressing, Immunodetection, Staining, Western Blot

    FKHR-PAX3 did not transactivate promoters that were responsive to FKHR (IGFBP1, A) and PAX3-FKHR (e5, B; MyoG, C; PDGFαR, D). C2C12 cells were transiently transfected with a total of 2 μg of DNA including 0.1 μg of LacZ, 0.5 μg of promoter-reporter (Luc or CAT as indicated), and 0.2 μg of the pcDNA3 vector or vector expressing the transcription factor by lipofection. After 48 hours, cells were harvested for LacZ, Luc, and CAT assays. Fold increase was calculated as the ratio of reporter activity from cells expressing the indicated transcription factor to the activity in cells transfected with the empty expression vector. Results were normalized to LacZ activity. The reporter activity in the presence of empty expression vector was assigned a value of 1. ND: statistically no difference. Representative data from FKHR-PAX3 isoform c is shown.

    Journal: PLoS ONE

    Article Title: Structural and Functional Studies of FKHR-PAX3, a Reciprocal Fusion Gene of the t(2;13) Chromosomal Translocation in Alveolar Rhabdomyosarcoma

    doi: 10.1371/journal.pone.0068065

    Figure Lengend Snippet: FKHR-PAX3 did not transactivate promoters that were responsive to FKHR (IGFBP1, A) and PAX3-FKHR (e5, B; MyoG, C; PDGFαR, D). C2C12 cells were transiently transfected with a total of 2 μg of DNA including 0.1 μg of LacZ, 0.5 μg of promoter-reporter (Luc or CAT as indicated), and 0.2 μg of the pcDNA3 vector or vector expressing the transcription factor by lipofection. After 48 hours, cells were harvested for LacZ, Luc, and CAT assays. Fold increase was calculated as the ratio of reporter activity from cells expressing the indicated transcription factor to the activity in cells transfected with the empty expression vector. Results were normalized to LacZ activity. The reporter activity in the presence of empty expression vector was assigned a value of 1. ND: statistically no difference. Representative data from FKHR-PAX3 isoform c is shown.

    Article Snippet: Antibodies were purchased for MyoD and MyoG (BD Biosciences), myosin heavy chain and α-tubulin (Developmental Studies Hybridoma Bank), PAX3 (abcam), FAK (Santa Cruz), and FKHR, p-Y397, p-Y576/577, and p-Y925-FAK (Cell signaling).

    Techniques: Transfection, Plasmid Preparation, Expressing, Activity Assay

    FKHR-PAX3 preserved high level of PAX3-FKHR expression in myogenic cells. (A) RH4 cells that do not express endogenous FKHR-PAX3 were transfected with empty vector (lane 1) or FKHR-PAX3 expression vector (lane 2). After continuous culture for 35 passages, cells were assayed for PAX3, FKHR, PAX3-FKHR, and FKHR-PAX3 expression by western blot. (B) Western blot analysis on the ability of FKHR-PAX3 to sustain high PAX3-FKHR expression. Stable clones of C2C12 cells expressing high levels of PAX3-FKHR were subjected to a second round of transfection to select for FKHR-PAX3 expression as described in Materials and Methods. The first confluent plate was designated as passage zero and used to generate early (

    Journal: PLoS ONE

    Article Title: Structural and Functional Studies of FKHR-PAX3, a Reciprocal Fusion Gene of the t(2;13) Chromosomal Translocation in Alveolar Rhabdomyosarcoma

    doi: 10.1371/journal.pone.0068065

    Figure Lengend Snippet: FKHR-PAX3 preserved high level of PAX3-FKHR expression in myogenic cells. (A) RH4 cells that do not express endogenous FKHR-PAX3 were transfected with empty vector (lane 1) or FKHR-PAX3 expression vector (lane 2). After continuous culture for 35 passages, cells were assayed for PAX3, FKHR, PAX3-FKHR, and FKHR-PAX3 expression by western blot. (B) Western blot analysis on the ability of FKHR-PAX3 to sustain high PAX3-FKHR expression. Stable clones of C2C12 cells expressing high levels of PAX3-FKHR were subjected to a second round of transfection to select for FKHR-PAX3 expression as described in Materials and Methods. The first confluent plate was designated as passage zero and used to generate early (

    Article Snippet: Antibodies were purchased for MyoD and MyoG (BD Biosciences), myosin heavy chain and α-tubulin (Developmental Studies Hybridoma Bank), PAX3 (abcam), FAK (Santa Cruz), and FKHR, p-Y397, p-Y576/577, and p-Y925-FAK (Cell signaling).

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Clone Assay

    Immunodetection of in vitro and in vivo expressed FKHR-PAX3 protein. ( A ) Left panel: schematic of deduced sizes and amino acid sequence variations for FKHR-PAX3 isoforms c, d, and e. Right panel: Autoradiographic image of S 35 -methionine labeled in vitro translated FKHR-PAX3 protein isoforms. ( B ) Verification of PAX3-specific C2 and FKHR-specific L27 antibodies in detecting in vivo expressed FKHR-PAX3 proteins. Top panel: diagrammatic illustration of the epitope locations within the FKHR-PAX3 protein recognized by L27 and C2 antibodies. Bottom panel: western blot detection of FKHR-PAX3 in whole cell extracts (30 μg) prepared from RD cells that were transiently transfected with control expression vector (lane1), FKHR-PAX3 isoform c (lane 2), and FKHR-PAX3 isoform d (lane 3) using C2 (left panel) and L27 (right panel) antibodies. ( C ) Western blot detection of the endogenously expressed FKHR-PAX3 in RH28 and RH30 cells by L27 and C2 antibodies. Protein extract from FKHR-PAX3 negative RH4 cells was included as negative control. n.s.: non-specific bands resulting from high amount of protein extracts used and long film exposure. ( D ) Effect of PAX3-FKHR knockdown on the endogenous level of FKHR-PAX3 in RH28 and RH30 cells. Whole cell extracts were prepared from cells that stably expressed the inducible PAX3-FKHR shRNA treated with DMSO or DOX for 48 hours, and analyzed for FKHR and FKHR-PAX3 expression. ( E ) Effect of MG132 (10 μM for 12 hours) and 5’-Aza-C (1 μM for 48 hours) on endogenous FKHR-PAX3 expression levels in RH28 and RH30 cells. ( C - E ) A total of 400 μg of protein extracts were used for the analyses. ( D - E ) Alpha-tubulin was used to normalize sample loading.

    Journal: PLoS ONE

    Article Title: Structural and Functional Studies of FKHR-PAX3, a Reciprocal Fusion Gene of the t(2;13) Chromosomal Translocation in Alveolar Rhabdomyosarcoma

    doi: 10.1371/journal.pone.0068065

    Figure Lengend Snippet: Immunodetection of in vitro and in vivo expressed FKHR-PAX3 protein. ( A ) Left panel: schematic of deduced sizes and amino acid sequence variations for FKHR-PAX3 isoforms c, d, and e. Right panel: Autoradiographic image of S 35 -methionine labeled in vitro translated FKHR-PAX3 protein isoforms. ( B ) Verification of PAX3-specific C2 and FKHR-specific L27 antibodies in detecting in vivo expressed FKHR-PAX3 proteins. Top panel: diagrammatic illustration of the epitope locations within the FKHR-PAX3 protein recognized by L27 and C2 antibodies. Bottom panel: western blot detection of FKHR-PAX3 in whole cell extracts (30 μg) prepared from RD cells that were transiently transfected with control expression vector (lane1), FKHR-PAX3 isoform c (lane 2), and FKHR-PAX3 isoform d (lane 3) using C2 (left panel) and L27 (right panel) antibodies. ( C ) Western blot detection of the endogenously expressed FKHR-PAX3 in RH28 and RH30 cells by L27 and C2 antibodies. Protein extract from FKHR-PAX3 negative RH4 cells was included as negative control. n.s.: non-specific bands resulting from high amount of protein extracts used and long film exposure. ( D ) Effect of PAX3-FKHR knockdown on the endogenous level of FKHR-PAX3 in RH28 and RH30 cells. Whole cell extracts were prepared from cells that stably expressed the inducible PAX3-FKHR shRNA treated with DMSO or DOX for 48 hours, and analyzed for FKHR and FKHR-PAX3 expression. ( E ) Effect of MG132 (10 μM for 12 hours) and 5’-Aza-C (1 μM for 48 hours) on endogenous FKHR-PAX3 expression levels in RH28 and RH30 cells. ( C - E ) A total of 400 μg of protein extracts were used for the analyses. ( D - E ) Alpha-tubulin was used to normalize sample loading.

    Article Snippet: Antibodies were purchased for MyoD and MyoG (BD Biosciences), myosin heavy chain and α-tubulin (Developmental Studies Hybridoma Bank), PAX3 (abcam), FAK (Santa Cruz), and FKHR, p-Y397, p-Y576/577, and p-Y925-FAK (Cell signaling).

    Techniques: Immunodetection, In Vitro, In Vivo, Sequencing, Labeling, Western Blot, Transfection, Expressing, Plasmid Preparation, Negative Control, Stable Transfection, shRNA

    FKHR-PAX3 induced early onset of tumor formation in nude mice xenograft model. ( A ) RD (top panel) and RH30 (bottom panel) cells expressing empty vector or FKHR-PAX3 were injected intramuscularly into the hind legs of nude mice as described in Materials and Methods. Data points represent mean ± s.d. of tumor volume (mm 3 ) of all injected mice at the indicted time points. Inset: an expanded view over the early tumor development period. ( B ) Dissecting microscopic images (magnification 25X) of lung organs of two representative mice from the control (left) and FKHR-PAX3 (right) group, showing clear evidence of more extensive tumor mass and infiltration metastasis in the control group (red dashed lines: margins surrounding the tumor mass; blue arrow heads: light-reflective artifacts). Representative data from FKHR-PAX3 isoform c is shown.

    Journal: PLoS ONE

    Article Title: Structural and Functional Studies of FKHR-PAX3, a Reciprocal Fusion Gene of the t(2;13) Chromosomal Translocation in Alveolar Rhabdomyosarcoma

    doi: 10.1371/journal.pone.0068065

    Figure Lengend Snippet: FKHR-PAX3 induced early onset of tumor formation in nude mice xenograft model. ( A ) RD (top panel) and RH30 (bottom panel) cells expressing empty vector or FKHR-PAX3 were injected intramuscularly into the hind legs of nude mice as described in Materials and Methods. Data points represent mean ± s.d. of tumor volume (mm 3 ) of all injected mice at the indicted time points. Inset: an expanded view over the early tumor development period. ( B ) Dissecting microscopic images (magnification 25X) of lung organs of two representative mice from the control (left) and FKHR-PAX3 (right) group, showing clear evidence of more extensive tumor mass and infiltration metastasis in the control group (red dashed lines: margins surrounding the tumor mass; blue arrow heads: light-reflective artifacts). Representative data from FKHR-PAX3 isoform c is shown.

    Article Snippet: Antibodies were purchased for MyoD and MyoG (BD Biosciences), myosin heavy chain and α-tubulin (Developmental Studies Hybridoma Bank), PAX3 (abcam), FAK (Santa Cruz), and FKHR, p-Y397, p-Y576/577, and p-Y925-FAK (Cell signaling).

    Techniques: Mouse Assay, Expressing, Plasmid Preparation, Injection

    FKHR-PAX3 localized predominantly in the cytoplasm of cells. ( A ) NIH3T3 and RH30 cells were transfected with vectors expressing wild-type FKHR-GFP, triple-mutant (T24A/S256A/S319A) FKHR-GFP, or FKHR-PAX3-GFP (FP3-GFP) by lipofection. Cells were maintained in low serum medium (0.5% FBS) overnight prior to the addition of DMSO or LepB (2 μM) for six hours. At the end of treatment, fluorescent microscopy (magnification: 200X) was used to visualize the GFP-tagged proteins as indicated. ( B ) Western analysis confirmed the cytoplasmic localization of the FKHR-PAX3 protein in cells. RH30 cells were transfected with FKHR-PAX3 and treated with or without LepB as described in ( 4A ). MyoG and α-tubulin served as nuclear and cytoplasmic specific controls, respectively, to evaluate the fractionation protocol. L27 antibody was used to detect FKHR-PAX3. Representative data from FKHR-PAX3 isoform c is shown.

    Journal: PLoS ONE

    Article Title: Structural and Functional Studies of FKHR-PAX3, a Reciprocal Fusion Gene of the t(2;13) Chromosomal Translocation in Alveolar Rhabdomyosarcoma

    doi: 10.1371/journal.pone.0068065

    Figure Lengend Snippet: FKHR-PAX3 localized predominantly in the cytoplasm of cells. ( A ) NIH3T3 and RH30 cells were transfected with vectors expressing wild-type FKHR-GFP, triple-mutant (T24A/S256A/S319A) FKHR-GFP, or FKHR-PAX3-GFP (FP3-GFP) by lipofection. Cells were maintained in low serum medium (0.5% FBS) overnight prior to the addition of DMSO or LepB (2 μM) for six hours. At the end of treatment, fluorescent microscopy (magnification: 200X) was used to visualize the GFP-tagged proteins as indicated. ( B ) Western analysis confirmed the cytoplasmic localization of the FKHR-PAX3 protein in cells. RH30 cells were transfected with FKHR-PAX3 and treated with or without LepB as described in ( 4A ). MyoG and α-tubulin served as nuclear and cytoplasmic specific controls, respectively, to evaluate the fractionation protocol. L27 antibody was used to detect FKHR-PAX3. Representative data from FKHR-PAX3 isoform c is shown.

    Article Snippet: Antibodies were purchased for MyoD and MyoG (BD Biosciences), myosin heavy chain and α-tubulin (Developmental Studies Hybridoma Bank), PAX3 (abcam), FAK (Santa Cruz), and FKHR, p-Y397, p-Y576/577, and p-Y925-FAK (Cell signaling).

    Techniques: Transfection, Expressing, Mutagenesis, Microscopy, Western Blot, Fractionation

    Cloning of FKHR-PAX3 cDNA. ( A ) Schematic of PAX3, FKHR, PAX3-FKHR and the predicted FKHR-PAX3 protein structures indicating the known functional domains. R: repressor; DBD: DNA binding domain; AD: activation domain. ( B ) Diagrammatic illustration of the exon-intron organization of human PAX3 gene, and the five alternatively spliced mRNAs that could result from processing of the FKHR-PAX3 primary transcript. PAX3c, PAX3d, and PAX3e use stop codons in intron 8, intron 9, and exon 10. PAX3g and PAX3h are truncated isoforms of PAX3d and PAX3e, respectively, that splice out exon 8. ( C ) Expression of FKHR-PAX3 transcript isoforms c, d, and e in ERMS (RD) and ARMS (RH4, RH28, RH30) cell lines as detected by RT-PCR and confirmed by Southern hybridization. Top panel: schematic indicates the positions of the FKHR-specific primer (F4) and the isoform-specific PAX3 PCR primer pairs, and the DNA probe spanning the FKHR-PAX3 fusion site used in the Southern analysis are indicated (not to scale). ( D ) Quantitative RT-PCR analysis of PAX3, FKHR, PAX3-FKHR, and FKHR-PAX3 expression in ARMS cell lines. The relative expression data are presented at two different scales on the Y-axis, high (left panel) and low (right panel) to compensate for the high levels of PAX3-FKHR expression. The relative expression level of PAX3/GAPDH in RH4 cells was assigned an arbitrary value of 1, and used as the reference to calculate fold change. ( E ) Nucleotide sequences of the cloned FKHR-PAX3 isoforms c, d, and e cDNAs. Top panel: schematic of the FKHR and the isoform-specific PAX3 primer pairs used to clone the full-length protein coding cDNA from RH30 cells. Primer location is approximate for illustrative purposes only. Sequence data is annotated by text in: Plain: 5’ FKHR-UTR; plain/italic: 3’ PAX3-UTR; Bold: protein coding sequences; bold/capital/underline: translation start codon; bold/capital/italic/underline: translational stop codons; underline: isoform-specific sequences.

    Journal: PLoS ONE

    Article Title: Structural and Functional Studies of FKHR-PAX3, a Reciprocal Fusion Gene of the t(2;13) Chromosomal Translocation in Alveolar Rhabdomyosarcoma

    doi: 10.1371/journal.pone.0068065

    Figure Lengend Snippet: Cloning of FKHR-PAX3 cDNA. ( A ) Schematic of PAX3, FKHR, PAX3-FKHR and the predicted FKHR-PAX3 protein structures indicating the known functional domains. R: repressor; DBD: DNA binding domain; AD: activation domain. ( B ) Diagrammatic illustration of the exon-intron organization of human PAX3 gene, and the five alternatively spliced mRNAs that could result from processing of the FKHR-PAX3 primary transcript. PAX3c, PAX3d, and PAX3e use stop codons in intron 8, intron 9, and exon 10. PAX3g and PAX3h are truncated isoforms of PAX3d and PAX3e, respectively, that splice out exon 8. ( C ) Expression of FKHR-PAX3 transcript isoforms c, d, and e in ERMS (RD) and ARMS (RH4, RH28, RH30) cell lines as detected by RT-PCR and confirmed by Southern hybridization. Top panel: schematic indicates the positions of the FKHR-specific primer (F4) and the isoform-specific PAX3 PCR primer pairs, and the DNA probe spanning the FKHR-PAX3 fusion site used in the Southern analysis are indicated (not to scale). ( D ) Quantitative RT-PCR analysis of PAX3, FKHR, PAX3-FKHR, and FKHR-PAX3 expression in ARMS cell lines. The relative expression data are presented at two different scales on the Y-axis, high (left panel) and low (right panel) to compensate for the high levels of PAX3-FKHR expression. The relative expression level of PAX3/GAPDH in RH4 cells was assigned an arbitrary value of 1, and used as the reference to calculate fold change. ( E ) Nucleotide sequences of the cloned FKHR-PAX3 isoforms c, d, and e cDNAs. Top panel: schematic of the FKHR and the isoform-specific PAX3 primer pairs used to clone the full-length protein coding cDNA from RH30 cells. Primer location is approximate for illustrative purposes only. Sequence data is annotated by text in: Plain: 5’ FKHR-UTR; plain/italic: 3’ PAX3-UTR; Bold: protein coding sequences; bold/capital/underline: translation start codon; bold/capital/italic/underline: translational stop codons; underline: isoform-specific sequences.

    Article Snippet: Antibodies were purchased for MyoD and MyoG (BD Biosciences), myosin heavy chain and α-tubulin (Developmental Studies Hybridoma Bank), PAX3 (abcam), FAK (Santa Cruz), and FKHR, p-Y397, p-Y576/577, and p-Y925-FAK (Cell signaling).

    Techniques: Clone Assay, Functional Assay, Binding Assay, Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Hybridization, Polymerase Chain Reaction, Quantitative RT-PCR, Sequencing

    FKHR-PAX3 enhanced anchorage-dependent and anchorage-independent colony growth. ( A ) Photographic images of crystal violet blue stained cell colonies in the clonogenicity assay at the end of a 15-day growth (10% FBS). ( B ) Quantitative analysis of anchorage-independent soft agar colony formation under growth (10% FBS, top panel) and differentiation (2% HS, bottom panel) conditions. Inset: representative micrographic images of RD cells showing that FKHR-PAX3 (isoform c) enhanced both number and size of the colonies. ( A - B ) Assays were conducted as described in Materials and Methods. Representative quantitative data from FKHR-PAX3 isoform d is shown.

    Journal: PLoS ONE

    Article Title: Structural and Functional Studies of FKHR-PAX3, a Reciprocal Fusion Gene of the t(2;13) Chromosomal Translocation in Alveolar Rhabdomyosarcoma

    doi: 10.1371/journal.pone.0068065

    Figure Lengend Snippet: FKHR-PAX3 enhanced anchorage-dependent and anchorage-independent colony growth. ( A ) Photographic images of crystal violet blue stained cell colonies in the clonogenicity assay at the end of a 15-day growth (10% FBS). ( B ) Quantitative analysis of anchorage-independent soft agar colony formation under growth (10% FBS, top panel) and differentiation (2% HS, bottom panel) conditions. Inset: representative micrographic images of RD cells showing that FKHR-PAX3 (isoform c) enhanced both number and size of the colonies. ( A - B ) Assays were conducted as described in Materials and Methods. Representative quantitative data from FKHR-PAX3 isoform d is shown.

    Article Snippet: Antibodies were purchased for MyoD and MyoG (BD Biosciences), myosin heavy chain and α-tubulin (Developmental Studies Hybridoma Bank), PAX3 (abcam), FAK (Santa Cruz), and FKHR, p-Y397, p-Y576/577, and p-Y925-FAK (Cell signaling).

    Techniques: Staining

    Evaluation of cell proliferation and cell cycle after transfection of siPAX3 and NC-siRNA (10 nM) in the osteosarcoma cell line. A: qRTPCR analyses of PAX3 expression at 48 h. B: Western blot analysis of PAX3 expression at 48 h. C: Cell proliferation at 24, 48, and 72 h. D: Distribution of cells in each phase of the cell cycle at 48 h. E: qRT-PCR analysis of p21 expression levels and F: western blot analysis of p21 expression levels. *p

    Journal: Cancer Genomics & Proteomics

    Article Title: MiR-1 Suppresses Proliferation of Osteosarcoma Cells by Up-regulating p21 via PAX3

    doi: 10.21873/cgp.20113

    Figure Lengend Snippet: Evaluation of cell proliferation and cell cycle after transfection of siPAX3 and NC-siRNA (10 nM) in the osteosarcoma cell line. A: qRTPCR analyses of PAX3 expression at 48 h. B: Western blot analysis of PAX3 expression at 48 h. C: Cell proliferation at 24, 48, and 72 h. D: Distribution of cells in each phase of the cell cycle at 48 h. E: qRT-PCR analysis of p21 expression levels and F: western blot analysis of p21 expression levels. *p

    Article Snippet: The diluted concentrations of each antibody were as follows: Rabbit anti-p53 antibody (DO-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phosphorylated p53 antibody (Cell Signaling Technology), anti-p73 antibody (Cell Signaling Technology), anti-p21 antibody (Santa Cruz Biotechnologies, CA, USA) at 1:1,000; mouse anti-PAX3 antibody (Abcam, Bristol, England) at 1:1,000; and anti-GAPDH rabbit polyclonal antibody (Abcam) as the endogenous control at 1:2,500.

    Techniques: Transfection, Expressing, Western Blot, Quantitative RT-PCR

    Enhanced Shh signaling in Ptch1 Wig/Wig mutants affects cranial placode development. (A and D) Immunostaining of PAX3 (green), SOX10 (red), and DAPI (blue) in wild type (A) and Ptch1 Wig mutants (D). PAX3-positive cells (placodal cells) and PAX3/SOX10 double-positive cells (neural crest cells) are identified by green and white arrowheads respectively. (B, C, E and F) Pax3 mRNA expression in control (B and C) vs. Ptch1 Wig mutants (E and F). Sections in (C and F) correspond to the dotted line in (B and E). Red arrowheads indicate reduced Pax3 expression in Ptch1 Wig/Wig mutant placodal primordia (E, F vs. B, C). (G-L) Whole mount in situ hybridization of indicated placodal markers Ngn1 (G and J), Ngn2 (H and K), and NeuroD1 (I and L) on E9.5 wild type (G-I) vs. Ptch1 Wig mutants (J-L). Red and white arrowheads identify the trigeminal and epibranchial placodes respectively. Scale bars: 20μm (A and D); 100μm (B,E and G-L); 50μm (C and F).

    Journal: PLoS ONE

    Article Title: Cranial Nerve Development Requires Co-Ordinated Shh and Canonical Wnt Signaling

    doi: 10.1371/journal.pone.0120821

    Figure Lengend Snippet: Enhanced Shh signaling in Ptch1 Wig/Wig mutants affects cranial placode development. (A and D) Immunostaining of PAX3 (green), SOX10 (red), and DAPI (blue) in wild type (A) and Ptch1 Wig mutants (D). PAX3-positive cells (placodal cells) and PAX3/SOX10 double-positive cells (neural crest cells) are identified by green and white arrowheads respectively. (B, C, E and F) Pax3 mRNA expression in control (B and C) vs. Ptch1 Wig mutants (E and F). Sections in (C and F) correspond to the dotted line in (B and E). Red arrowheads indicate reduced Pax3 expression in Ptch1 Wig/Wig mutant placodal primordia (E, F vs. B, C). (G-L) Whole mount in situ hybridization of indicated placodal markers Ngn1 (G and J), Ngn2 (H and K), and NeuroD1 (I and L) on E9.5 wild type (G-I) vs. Ptch1 Wig mutants (J-L). Red and white arrowheads identify the trigeminal and epibranchial placodes respectively. Scale bars: 20μm (A and D); 100μm (B,E and G-L); 50μm (C and F).

    Article Snippet: Slides were incubated with anti-SOX10 (R & D systems, 1:50), anti-PAX3 (Developmental Studies Hybridoma Bank, 1:100), anti-TUJ1 (Covance, 1:1000), anti-GFP (Invitrogen, 1:500) with 1% BSA in TBST for overnight at 4°C.

    Techniques: Immunostaining, Expressing, Mutagenesis, In Situ Hybridization

    Pax3-3′UTR is a target for miR-1 and miR-206. (A) CMV promoter driving Luc reporter gene was used for creating Luc-3′UTR, which contains the longer Pax3-3′UTR fragment downstream of the Luc reporter gene. Two putative miR-1/miR-206–binding sites (M1 and M2) were mutated in Luc-3′UTRM. (B) Relative Luc activity was measured in wild-type and MyoD −/− myoblasts after transfection with control Luc reporter gene + empty expression vector, Luc-3′UTR + empty expression vector, Luc-3′UTR + MyoD expression vector, and Luc-3′UTRM + MyoD expression vector. (C) Relative Luc activity was measured in wild-type and MyoD −/− myoblasts after transfection with Luc-3′UTR + control pre-miRNA, Luc-3′UTR + pre–miR-1, Luc-3′UTR + pre–miR-206, Luc-3′UTRM + control pre-miRNA, Luc-3′UTRM + pre–miR-1, and Luc-3′UTRM + pre–miR-206. (D) Under differentiation conditions from day 0 to 3 or after UV exposure or treatment with thapsigargin (Thap) for 1 d, cell death was compared between wild-type myoblasts transfected with the control pre-miRNA, pre–miR-1, and pre–miR-206. (E) After UV exposure or treatment with thapsigargin for 1 d, cell death was compared between wild-type myoblasts or MyoD −/− myoblasts expressing ectopic MyoD transfected with the control miRNA or anti–miR-1 + anti–miR-206. *, P

    Journal: The Journal of Cell Biology

    Article Title: MyoD regulates apoptosis of myoblasts through microRNA-mediated down-regulation of Pax3

    doi: 10.1083/jcb.201006025

    Figure Lengend Snippet: Pax3-3′UTR is a target for miR-1 and miR-206. (A) CMV promoter driving Luc reporter gene was used for creating Luc-3′UTR, which contains the longer Pax3-3′UTR fragment downstream of the Luc reporter gene. Two putative miR-1/miR-206–binding sites (M1 and M2) were mutated in Luc-3′UTRM. (B) Relative Luc activity was measured in wild-type and MyoD −/− myoblasts after transfection with control Luc reporter gene + empty expression vector, Luc-3′UTR + empty expression vector, Luc-3′UTR + MyoD expression vector, and Luc-3′UTRM + MyoD expression vector. (C) Relative Luc activity was measured in wild-type and MyoD −/− myoblasts after transfection with Luc-3′UTR + control pre-miRNA, Luc-3′UTR + pre–miR-1, Luc-3′UTR + pre–miR-206, Luc-3′UTRM + control pre-miRNA, Luc-3′UTRM + pre–miR-1, and Luc-3′UTRM + pre–miR-206. (D) Under differentiation conditions from day 0 to 3 or after UV exposure or treatment with thapsigargin (Thap) for 1 d, cell death was compared between wild-type myoblasts transfected with the control pre-miRNA, pre–miR-1, and pre–miR-206. (E) After UV exposure or treatment with thapsigargin for 1 d, cell death was compared between wild-type myoblasts or MyoD −/− myoblasts expressing ectopic MyoD transfected with the control miRNA or anti–miR-1 + anti–miR-206. *, P

    Article Snippet: Immunological staining for cell culture Immunostaining was performed with an anti-sarcomeric MHC (MF20; Developmental Studies Hybridoma Bank), anti-MyoD antibody (Ab-1; Thermo Fisher Scientific), anti-Pax3 antibodies (Developmental Studies Hybridoma Bank and EMD), anti-Pax7 antibody, and anti–c-met antibody (Santa Cruz Biotechnology, Inc.) followed by Alexa Fluor 488–conjugated anti–mouse IgG antibody (Invitrogen).

    Techniques: Binding Assay, Activity Assay, Transfection, Expressing, Plasmid Preparation

    Pax3 expression is regulated by dicer and Pax3-3′UTR. (A) Pax3 expression was compared between wild-type myoblasts infected with a retrovirus vector expressing Pax3, Pax3-3′UTR, Pax3-3′UTR with mutations at miR-1/miR-206–binding sites (Pax3-3′UTRM), and a control empty vector by immunostaining (green). Nuclei were counterstained with DAPI (blue). (B) Pax3, Pax7, Bcl-2, and Bcl-xL gene expression were compared between wild-type myoblasts transfected with Pax3, Pax3-3′UTR, Pax3-3′UTRM, and a control empty vector by RT-PCR. (C) Relative Pax3 expression levels shown in B normalized by β-actin expression were measured. (D) After treatment with thapsigargin for 1 d, caspase-3 activity was compared between wild-type myoblasts infected with a retrovirus vector expressing Pax3, Pax3-3′UTR and Pax3-3′UTRM, and a control empty vector. (E) Myoblasts isolated from floxed dicer ( dicer fl/fl ) mice were infected with adenovirus lacZ expression vector (control [Cont]) or adenovirus Cre/EGFP expression vector. GFP is only detected in myoblasts infected with adenovirus Cre/EGFP expression vector. (F) Control myoblasts infected with adenovirus lacZ expression vector shows the loxP band but not excised band after PCR. Infection with more adenovirus Cre/EGFP expression vector (10 and 100 µl) increased in amount of the excised band (Cre10 and Cre 100, respectively). (G) Cre, dicer , myogenic marker, and miRNA expression were compared between control dicer fl/fl myoblasts infected with adenovirus lacZ expression vector and infected with adenovirus Cre/EGFP expression vector by RT-PCR. For miRNAs, all PCR products are ∼90-bp long. (H) ChIP assay with antibody against RNA polymerase II was performed. Input denotes each PCR product from naked myoblast genomic DNA. Pax3 and Pax7 but not Ig heavy chain (IgH) were detected by ChIP assay in wild-type (wt) and MyoD −/− myoblasts. (B and G) β-Actin was monitored as a loading control. *, P

    Journal: The Journal of Cell Biology

    Article Title: MyoD regulates apoptosis of myoblasts through microRNA-mediated down-regulation of Pax3

    doi: 10.1083/jcb.201006025

    Figure Lengend Snippet: Pax3 expression is regulated by dicer and Pax3-3′UTR. (A) Pax3 expression was compared between wild-type myoblasts infected with a retrovirus vector expressing Pax3, Pax3-3′UTR, Pax3-3′UTR with mutations at miR-1/miR-206–binding sites (Pax3-3′UTRM), and a control empty vector by immunostaining (green). Nuclei were counterstained with DAPI (blue). (B) Pax3, Pax7, Bcl-2, and Bcl-xL gene expression were compared between wild-type myoblasts transfected with Pax3, Pax3-3′UTR, Pax3-3′UTRM, and a control empty vector by RT-PCR. (C) Relative Pax3 expression levels shown in B normalized by β-actin expression were measured. (D) After treatment with thapsigargin for 1 d, caspase-3 activity was compared between wild-type myoblasts infected with a retrovirus vector expressing Pax3, Pax3-3′UTR and Pax3-3′UTRM, and a control empty vector. (E) Myoblasts isolated from floxed dicer ( dicer fl/fl ) mice were infected with adenovirus lacZ expression vector (control [Cont]) or adenovirus Cre/EGFP expression vector. GFP is only detected in myoblasts infected with adenovirus Cre/EGFP expression vector. (F) Control myoblasts infected with adenovirus lacZ expression vector shows the loxP band but not excised band after PCR. Infection with more adenovirus Cre/EGFP expression vector (10 and 100 µl) increased in amount of the excised band (Cre10 and Cre 100, respectively). (G) Cre, dicer , myogenic marker, and miRNA expression were compared between control dicer fl/fl myoblasts infected with adenovirus lacZ expression vector and infected with adenovirus Cre/EGFP expression vector by RT-PCR. For miRNAs, all PCR products are ∼90-bp long. (H) ChIP assay with antibody against RNA polymerase II was performed. Input denotes each PCR product from naked myoblast genomic DNA. Pax3 and Pax7 but not Ig heavy chain (IgH) were detected by ChIP assay in wild-type (wt) and MyoD −/− myoblasts. (B and G) β-Actin was monitored as a loading control. *, P

    Article Snippet: Immunological staining for cell culture Immunostaining was performed with an anti-sarcomeric MHC (MF20; Developmental Studies Hybridoma Bank), anti-MyoD antibody (Ab-1; Thermo Fisher Scientific), anti-Pax3 antibodies (Developmental Studies Hybridoma Bank and EMD), anti-Pax7 antibody, and anti–c-met antibody (Santa Cruz Biotechnology, Inc.) followed by Alexa Fluor 488–conjugated anti–mouse IgG antibody (Invitrogen).

    Techniques: Expressing, Infection, Plasmid Preparation, Binding Assay, Immunostaining, Transfection, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Isolation, Mouse Assay, Polymerase Chain Reaction, Marker, Chromatin Immunoprecipitation

    Pax3-3′UTR contains conserved miR-1/miR-206–binding sites. (A) Mouse Pax3 gene structure. Numbered boxes denote each exon. White boxes denote the 5′UTR and the shorter 3′UTR. Black boxes denote coding regions. The gray box denotes the longer 3′UTR containing two putative miR-1/miR-206–binding sites (M1 and M2). There are two stop codons and two polyA signal sequences (polyA1 and polyA2) in the mouse Pax3 gene. (B) Sequences of two putative miR-1/miR-206–binding sites (M1 and M2) and their flanking regions. Core sequences for miR-1/miR-206 and consensus sequences are denoted by bold letters. (C) Similar levels of expression of the Pax3 coding region and the longer 3′UTR were detected in MyoD −/− myoblasts by RT-PCR. β-Actin was monitored as a loading control. (D) MyoD-regulated miRNA expression levels were compared between wild-type (wt) and MyoD −/− myoblasts by RT-PCR. miR-24 was monitored as a loading control. (E) miR-1 and -206 expression were compared between wild-type myoblasts infected with a lentivirus vector expressing shRNA for MyoD and control shRNA (Cont) vector or MyoD −/− myoblasts infected with a lentivirus vector expressing MyoD (Ect-MyoD) and a control empty vector by RT-PCR. miR-24 was monitored as a loading control. (F) Pax3, Pax7, Bcl-2, and Bcl-xL gene expression were compared between MyoD −/− myoblasts transfected with pre–miR-1, pre–miR-206, and the control pre-miRNA by RT-PCR. β-Actin was monitored as a loading control. (G) Pax3, Pax7, Bcl-2, and Bcl-xL gene expression were compared between wild-type myoblasts transfected with anti–miR-1 (anti-1), anti–miR-206 (anti-206), anti–miR-1 + anti–miR-206 (anti-1 + 206), and the control RNA by RT-PCR. β-Actin was monitored as a loading control. (D and E) All PCR products are ∼90-bp long.

    Journal: The Journal of Cell Biology

    Article Title: MyoD regulates apoptosis of myoblasts through microRNA-mediated down-regulation of Pax3

    doi: 10.1083/jcb.201006025

    Figure Lengend Snippet: Pax3-3′UTR contains conserved miR-1/miR-206–binding sites. (A) Mouse Pax3 gene structure. Numbered boxes denote each exon. White boxes denote the 5′UTR and the shorter 3′UTR. Black boxes denote coding regions. The gray box denotes the longer 3′UTR containing two putative miR-1/miR-206–binding sites (M1 and M2). There are two stop codons and two polyA signal sequences (polyA1 and polyA2) in the mouse Pax3 gene. (B) Sequences of two putative miR-1/miR-206–binding sites (M1 and M2) and their flanking regions. Core sequences for miR-1/miR-206 and consensus sequences are denoted by bold letters. (C) Similar levels of expression of the Pax3 coding region and the longer 3′UTR were detected in MyoD −/− myoblasts by RT-PCR. β-Actin was monitored as a loading control. (D) MyoD-regulated miRNA expression levels were compared between wild-type (wt) and MyoD −/− myoblasts by RT-PCR. miR-24 was monitored as a loading control. (E) miR-1 and -206 expression were compared between wild-type myoblasts infected with a lentivirus vector expressing shRNA for MyoD and control shRNA (Cont) vector or MyoD −/− myoblasts infected with a lentivirus vector expressing MyoD (Ect-MyoD) and a control empty vector by RT-PCR. miR-24 was monitored as a loading control. (F) Pax3, Pax7, Bcl-2, and Bcl-xL gene expression were compared between MyoD −/− myoblasts transfected with pre–miR-1, pre–miR-206, and the control pre-miRNA by RT-PCR. β-Actin was monitored as a loading control. (G) Pax3, Pax7, Bcl-2, and Bcl-xL gene expression were compared between wild-type myoblasts transfected with anti–miR-1 (anti-1), anti–miR-206 (anti-206), anti–miR-1 + anti–miR-206 (anti-1 + 206), and the control RNA by RT-PCR. β-Actin was monitored as a loading control. (D and E) All PCR products are ∼90-bp long.

    Article Snippet: Immunological staining for cell culture Immunostaining was performed with an anti-sarcomeric MHC (MF20; Developmental Studies Hybridoma Bank), anti-MyoD antibody (Ab-1; Thermo Fisher Scientific), anti-Pax3 antibodies (Developmental Studies Hybridoma Bank and EMD), anti-Pax7 antibody, and anti–c-met antibody (Santa Cruz Biotechnology, Inc.) followed by Alexa Fluor 488–conjugated anti–mouse IgG antibody (Invitrogen).

    Techniques: Binding Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Infection, Plasmid Preparation, shRNA, Transfection, miRNA RT, Polymerase Chain Reaction

    MyoD negatively regulates Pax3, Bcl-2, and Bcl-xL expression. (A) MyoD, Bcl-2, Bcl-xL, Pax3, and Pax7 gene expression were compared between wild-type myoblasts infected with a lentivirus vector expressing shRNA vector for MyoD and a control shRNA vector (Cont) or MyoD −/− myoblasts infected with a lentivirus vector expressing MyoD (Ect-MyoD) and a control empty vector by RT-PCR. (B) Bcl-2, Bcl-xL, and Pax3 protein expression were compared between wild-type myoblasts infected with a lentivirus vector expressing shRNA vector for MyoD and a control shRNA vector or MyoD −/− myoblasts infected with a lentivirus vector expressing MyoD and a control empty vector by Western blotting. (A and B) β-Actin was monitored as a loading control. (C) Relative Pax3, Bcl-2, and Bcl-xL protein expression levels normalized by β-actin expression were measured by Western blotting. *, P

    Journal: The Journal of Cell Biology

    Article Title: MyoD regulates apoptosis of myoblasts through microRNA-mediated down-regulation of Pax3

    doi: 10.1083/jcb.201006025

    Figure Lengend Snippet: MyoD negatively regulates Pax3, Bcl-2, and Bcl-xL expression. (A) MyoD, Bcl-2, Bcl-xL, Pax3, and Pax7 gene expression were compared between wild-type myoblasts infected with a lentivirus vector expressing shRNA vector for MyoD and a control shRNA vector (Cont) or MyoD −/− myoblasts infected with a lentivirus vector expressing MyoD (Ect-MyoD) and a control empty vector by RT-PCR. (B) Bcl-2, Bcl-xL, and Pax3 protein expression were compared between wild-type myoblasts infected with a lentivirus vector expressing shRNA vector for MyoD and a control shRNA vector or MyoD −/− myoblasts infected with a lentivirus vector expressing MyoD and a control empty vector by Western blotting. (A and B) β-Actin was monitored as a loading control. (C) Relative Pax3, Bcl-2, and Bcl-xL protein expression levels normalized by β-actin expression were measured by Western blotting. *, P

    Article Snippet: Immunological staining for cell culture Immunostaining was performed with an anti-sarcomeric MHC (MF20; Developmental Studies Hybridoma Bank), anti-MyoD antibody (Ab-1; Thermo Fisher Scientific), anti-Pax3 antibodies (Developmental Studies Hybridoma Bank and EMD), anti-Pax7 antibody, and anti–c-met antibody (Santa Cruz Biotechnology, Inc.) followed by Alexa Fluor 488–conjugated anti–mouse IgG antibody (Invitrogen).

    Techniques: Expressing, Infection, Plasmid Preparation, shRNA, Reverse Transcription Polymerase Chain Reaction, Western Blot

    MyoD −/− myoblasts up-regulate Pax3 expression. (A) Under growth conditions (control [Cont]), differentiation conditions (Diff), UV exposure, or treatment with thapsigargin (Thap), Pax3, Pax7, Bcl-2, and Bcl-xL gene expression were compared between wild-type and MyoD −/− myoblasts by RT-PCR. (B) Pax3 and Pax7 expression were compared between wild-type and MyoD −/− myoblasts by immunostaining (green). Nuclei were counterstained with DAPI (blue). Bar, 40 µm. (C) Pax3 protein expression was compared between wild-type (wt) and MyoD −/− myoblasts by Western blotting. (D) Relative Pax3 expression levels normalized by β-actin expression shown in C were measured. (A and C) β-Actin was monitored as a loading control. **, P

    Journal: The Journal of Cell Biology

    Article Title: MyoD regulates apoptosis of myoblasts through microRNA-mediated down-regulation of Pax3

    doi: 10.1083/jcb.201006025

    Figure Lengend Snippet: MyoD −/− myoblasts up-regulate Pax3 expression. (A) Under growth conditions (control [Cont]), differentiation conditions (Diff), UV exposure, or treatment with thapsigargin (Thap), Pax3, Pax7, Bcl-2, and Bcl-xL gene expression were compared between wild-type and MyoD −/− myoblasts by RT-PCR. (B) Pax3 and Pax7 expression were compared between wild-type and MyoD −/− myoblasts by immunostaining (green). Nuclei were counterstained with DAPI (blue). Bar, 40 µm. (C) Pax3 protein expression was compared between wild-type (wt) and MyoD −/− myoblasts by Western blotting. (D) Relative Pax3 expression levels normalized by β-actin expression shown in C were measured. (A and C) β-Actin was monitored as a loading control. **, P

    Article Snippet: Immunological staining for cell culture Immunostaining was performed with an anti-sarcomeric MHC (MF20; Developmental Studies Hybridoma Bank), anti-MyoD antibody (Ab-1; Thermo Fisher Scientific), anti-Pax3 antibodies (Developmental Studies Hybridoma Bank and EMD), anti-Pax7 antibody, and anti–c-met antibody (Santa Cruz Biotechnology, Inc.) followed by Alexa Fluor 488–conjugated anti–mouse IgG antibody (Invitrogen).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunostaining, Western Blot

    Pax3 expression is down-regulated during satellite cell activation. (A) MyoD, Pax3, Pax7, Bcl-2, Bcl-xL, miR-1, and miR-206 gene expression were compared between freshly isolated (day 0) and cultured satellite cells (days 1, 2, and 4) derived from wild-type and MyoD −/− mice by RT-PCR. 18S rRNA and miR-24 were monitored as loading controls. For miRNAs, all PCR products are ∼90-bp long. (B) Relative Pax3, Bcl-2, and Bcl-xL gene expression levels shown in A normalized by 18S rRNA expression were measured. (C) MyoD and Pax3 gene expression were compared between wild-type satellite cells transfected with siRNA for MyoD and a control siRNA (Cont) or MyoD −/− satellite cells infected with a lentivirus vector expressing MyoD (Ect-MyoD) and a control empty vector by RT-PCR. (D) Relative Pax3 gene expression shown in C normalized by 18S rRNA expression was measured. (E) The model for apoptotic cascade regulated by MyoD. MyoD is a master regulator for muscle differentiation. Under differentiation or stressful conditions, MyoD transcriptionally activates miR-1 and miR-206 gene expression, which suppresses Pax3 expression. Down-regulation of Pax3 results in down-regulation of Bcl-2 and Bcl-xL, which causes apoptosis. In addition, down-regulation of Pax3 by MyoD also induces muscle differentiation. *, P

    Journal: The Journal of Cell Biology

    Article Title: MyoD regulates apoptosis of myoblasts through microRNA-mediated down-regulation of Pax3

    doi: 10.1083/jcb.201006025

    Figure Lengend Snippet: Pax3 expression is down-regulated during satellite cell activation. (A) MyoD, Pax3, Pax7, Bcl-2, Bcl-xL, miR-1, and miR-206 gene expression were compared between freshly isolated (day 0) and cultured satellite cells (days 1, 2, and 4) derived from wild-type and MyoD −/− mice by RT-PCR. 18S rRNA and miR-24 were monitored as loading controls. For miRNAs, all PCR products are ∼90-bp long. (B) Relative Pax3, Bcl-2, and Bcl-xL gene expression levels shown in A normalized by 18S rRNA expression were measured. (C) MyoD and Pax3 gene expression were compared between wild-type satellite cells transfected with siRNA for MyoD and a control siRNA (Cont) or MyoD −/− satellite cells infected with a lentivirus vector expressing MyoD (Ect-MyoD) and a control empty vector by RT-PCR. (D) Relative Pax3 gene expression shown in C normalized by 18S rRNA expression was measured. (E) The model for apoptotic cascade regulated by MyoD. MyoD is a master regulator for muscle differentiation. Under differentiation or stressful conditions, MyoD transcriptionally activates miR-1 and miR-206 gene expression, which suppresses Pax3 expression. Down-regulation of Pax3 results in down-regulation of Bcl-2 and Bcl-xL, which causes apoptosis. In addition, down-regulation of Pax3 by MyoD also induces muscle differentiation. *, P

    Article Snippet: Immunological staining for cell culture Immunostaining was performed with an anti-sarcomeric MHC (MF20; Developmental Studies Hybridoma Bank), anti-MyoD antibody (Ab-1; Thermo Fisher Scientific), anti-Pax3 antibodies (Developmental Studies Hybridoma Bank and EMD), anti-Pax7 antibody, and anti–c-met antibody (Santa Cruz Biotechnology, Inc.) followed by Alexa Fluor 488–conjugated anti–mouse IgG antibody (Invitrogen).

    Techniques: Expressing, Activation Assay, Isolation, Cell Culture, Derivative Assay, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Transfection, Infection, Plasmid Preparation

    Pax3 positively regulates Bcl-2 and Bcl-xL expression. (A) Pax3, Bcl-2, and Bcl-xL gene expression were compared between wild-type myoblasts infected with a retrovirus vector expressing Pax3 (Ect-Pax3) and a control empty vector or MyoD −/− myoblasts transfected with siRNA for Pax3 and a control siRNA (Cont) by RT-PCR. (B) Pax3, Bcl-2, and Bcl-xL protein expression were compared between wild-type myoblasts infected with a retrovirus vector expressing Pax3 and a control empty vector or MyoD −/− myoblasts transfected with siRNA for Pax3 and a control siRNA by Western blotting. (A and B) β-Actin was monitored as a loading control. (C and D) Relative Pax3, Bcl-2, and Bcl-xL protein expression levels shown in B normalized by β-actin expression were compared by Western blotting. (E) Luc activity was assessed after transfection with Bcl-2– or Bcl-xL–Luc reporter genes into wild-type or MyoD −/− myoblasts. Luc activity was also assessed after transfection with Bcl-2– or Bcl-xL–Luc reporter genes and the Pax3 expression vector or control empty vector into wild-type myoblasts. (F) Under differentiation conditions from day 0 to 3 or after UV exposure or treatment with thapsigargin (Thap) for 1 d, cell death was compared between wild-type myoblasts infected with a retrovirus vector expressing Pax3 and a control empty vector. (G) After UV exposure or treatment with thapsigargin for 1 d, caspase-3 activity was compared between wild-type and MyoD −/− myoblasts infected with a retrovirus vector expressing Pax3 and a control empty vector or transfected with siRNA for Pax3 and a control siRNA. *, P

    Journal: The Journal of Cell Biology

    Article Title: MyoD regulates apoptosis of myoblasts through microRNA-mediated down-regulation of Pax3

    doi: 10.1083/jcb.201006025

    Figure Lengend Snippet: Pax3 positively regulates Bcl-2 and Bcl-xL expression. (A) Pax3, Bcl-2, and Bcl-xL gene expression were compared between wild-type myoblasts infected with a retrovirus vector expressing Pax3 (Ect-Pax3) and a control empty vector or MyoD −/− myoblasts transfected with siRNA for Pax3 and a control siRNA (Cont) by RT-PCR. (B) Pax3, Bcl-2, and Bcl-xL protein expression were compared between wild-type myoblasts infected with a retrovirus vector expressing Pax3 and a control empty vector or MyoD −/− myoblasts transfected with siRNA for Pax3 and a control siRNA by Western blotting. (A and B) β-Actin was monitored as a loading control. (C and D) Relative Pax3, Bcl-2, and Bcl-xL protein expression levels shown in B normalized by β-actin expression were compared by Western blotting. (E) Luc activity was assessed after transfection with Bcl-2– or Bcl-xL–Luc reporter genes into wild-type or MyoD −/− myoblasts. Luc activity was also assessed after transfection with Bcl-2– or Bcl-xL–Luc reporter genes and the Pax3 expression vector or control empty vector into wild-type myoblasts. (F) Under differentiation conditions from day 0 to 3 or after UV exposure or treatment with thapsigargin (Thap) for 1 d, cell death was compared between wild-type myoblasts infected with a retrovirus vector expressing Pax3 and a control empty vector. (G) After UV exposure or treatment with thapsigargin for 1 d, caspase-3 activity was compared between wild-type and MyoD −/− myoblasts infected with a retrovirus vector expressing Pax3 and a control empty vector or transfected with siRNA for Pax3 and a control siRNA. *, P

    Article Snippet: Immunological staining for cell culture Immunostaining was performed with an anti-sarcomeric MHC (MF20; Developmental Studies Hybridoma Bank), anti-MyoD antibody (Ab-1; Thermo Fisher Scientific), anti-Pax3 antibodies (Developmental Studies Hybridoma Bank and EMD), anti-Pax7 antibody, and anti–c-met antibody (Santa Cruz Biotechnology, Inc.) followed by Alexa Fluor 488–conjugated anti–mouse IgG antibody (Invitrogen).

    Techniques: Expressing, Infection, Plasmid Preparation, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay

    Pax3/Pax7 and MRF expression identify distinct cell populations in the somite. ( A ) Pax3 and Pax7 are coexpressed in almost all dermomyotomal (DM), but not myotomal (My) cells at E9.75; transverse section of interlimb somites. Pax3 also marks dorsal root

    Journal: Genes & Development

    Article Title: Pax3/Pax7 mark a novel population of primitive myogenic cells during development

    doi: 10.1101/gad.345505

    Figure Lengend Snippet: Pax3/Pax7 and MRF expression identify distinct cell populations in the somite. ( A ) Pax3 and Pax7 are coexpressed in almost all dermomyotomal (DM), but not myotomal (My) cells at E9.75; transverse section of interlimb somites. Pax3 also marks dorsal root

    Article Snippet: For immunofluorescence , the antibodies used were Myf5 (C-terminal, Santa Cruz, polyclonal, 1/800), Mrf4 (Myf6 C-terminal, Santa Cruz, polyclonal, 1/200), Myogenin (F5D, monoclonal, 1/20), Pax7 (monoclonal, 1/20), and Pax3 (monoclonal, 1/200) from Developmental Studies Hybridoma Bank (NICHD), Pax3 (polyclonal, 1/200; kindly provided by J. Epstein, University of Pennsylvania, Philadelphia, PA), Myod (Dako, monoclonal, 1/20), phospho-met-Histone H3 (PM polyclonal, 1/5000; kindly provided by B. Mateescu, Institut Pasteur, Paris, France) ( ); β-gal (polyclonal, 1/200, kindly provided by O. Puijalon, Institut Pasteur, Paris, France), Ki67 (monoclonal, 1/100, BD Pharmingen), Myosin heavy chain (polyclonal, 1/400; kindly provided by G. Cossu, Stem Cell Research Institute, Milan, Italy), Desmin (monoclonal, 1/100, Dako), laminin (polyclonal, 1/200, Sigma), GFP (polyclonal, 1/200, AbCam), and activated Caspase-3 (polyclonal, 1/200; Cell Signaling).

    Techniques: Expressing

    Resident Pax7 + cells mark the emergence of satellite cells and do not require Pax3. ( A ) Immunohistochemistry of cryostat sections shows Pax7 + (anti-Pax7; arrowhead) cells associated with the muscle fiber by anti-laminin (green) staining in trunk muscles

    Journal: Genes & Development

    Article Title: Pax3/Pax7 mark a novel population of primitive myogenic cells during development

    doi: 10.1101/gad.345505

    Figure Lengend Snippet: Resident Pax7 + cells mark the emergence of satellite cells and do not require Pax3. ( A ) Immunohistochemistry of cryostat sections shows Pax7 + (anti-Pax7; arrowhead) cells associated with the muscle fiber by anti-laminin (green) staining in trunk muscles

    Article Snippet: For immunofluorescence , the antibodies used were Myf5 (C-terminal, Santa Cruz, polyclonal, 1/800), Mrf4 (Myf6 C-terminal, Santa Cruz, polyclonal, 1/200), Myogenin (F5D, monoclonal, 1/20), Pax7 (monoclonal, 1/20), and Pax3 (monoclonal, 1/200) from Developmental Studies Hybridoma Bank (NICHD), Pax3 (polyclonal, 1/200; kindly provided by J. Epstein, University of Pennsylvania, Philadelphia, PA), Myod (Dako, monoclonal, 1/20), phospho-met-Histone H3 (PM polyclonal, 1/5000; kindly provided by B. Mateescu, Institut Pasteur, Paris, France) ( ); β-gal (polyclonal, 1/200, kindly provided by O. Puijalon, Institut Pasteur, Paris, France), Ki67 (monoclonal, 1/100, BD Pharmingen), Myosin heavy chain (polyclonal, 1/400; kindly provided by G. Cossu, Stem Cell Research Institute, Milan, Italy), Desmin (monoclonal, 1/100, Dako), laminin (polyclonal, 1/200, Sigma), GFP (polyclonal, 1/200, AbCam), and activated Caspase-3 (polyclonal, 1/200; Cell Signaling).

    Techniques: Immunohistochemistry, Staining

    PAX3-FKHR substantially down-regulates PAI-1 and MHC class I protein expression in mouse and human RMS cells. (A) FACS analysis of MHC class I surface expression. The dotted line represents the secondary antibody alone, and the continuous line represents staining with anti–MHC class I. The staining is representative of four separate experiments. (B) Western blot analysis examining PAI-1 protein expression in RD-CMV and RD-P3F clones and in several human RMS cell lines. In a longer exposure, very faint bands can be detected in the lanes corresponding to the RD-P3F clones 6, 2, and 18. The RMS type and the expression of endogenous PAX3 and PAX3-FKHR RNA (determined by Northern blot; not depicted) for each cell line is indicated. A, ARMS; E, ERMS. (C) FACS analysis of MHC class I surface expression on cells cultured in the presence of 20 ng/ml IFN-γ for 48 h. The dotted line represents the secondary antibody alone, the continuous line represents MHC class I expression in the absence of IFN-γ, and the shaded histogram represents MHC class I in the presence of 20 ng/ml IFN-γ. FACS staining is representative of three separate experiments. Mean fluorescence intensities from the FACS plots are also indicated. Error bars = SEM. (D) Western blot of PAI-1 expression after treatment with 20 ng/ml IFN-γ for 48 h is representative of two independent experiments. PAI-1 was detected at a molecular mass of ∼47 kD, and α-tubulin expression was used as a loading control. (E) PAI-1 expression determined by quantitative RT-PCR. Expression is normalized to GAPDH and expressed relative to RH30 expression. Error bars = SEM of triplicate estimations. (F) FACS histograms of CXCR4 surface expression. The dotted line represents isotype control antibody, and the continuous line represents anti-CXCR4 antibody.

    Journal: The Journal of Experimental Medicine

    Article Title: Coordinated oncogenic transformation and inhibition of host immune responses by the PAX3-FKHR fusion oncoprotein

    doi: 10.1084/jem.20050730

    Figure Lengend Snippet: PAX3-FKHR substantially down-regulates PAI-1 and MHC class I protein expression in mouse and human RMS cells. (A) FACS analysis of MHC class I surface expression. The dotted line represents the secondary antibody alone, and the continuous line represents staining with anti–MHC class I. The staining is representative of four separate experiments. (B) Western blot analysis examining PAI-1 protein expression in RD-CMV and RD-P3F clones and in several human RMS cell lines. In a longer exposure, very faint bands can be detected in the lanes corresponding to the RD-P3F clones 6, 2, and 18. The RMS type and the expression of endogenous PAX3 and PAX3-FKHR RNA (determined by Northern blot; not depicted) for each cell line is indicated. A, ARMS; E, ERMS. (C) FACS analysis of MHC class I surface expression on cells cultured in the presence of 20 ng/ml IFN-γ for 48 h. The dotted line represents the secondary antibody alone, the continuous line represents MHC class I expression in the absence of IFN-γ, and the shaded histogram represents MHC class I in the presence of 20 ng/ml IFN-γ. FACS staining is representative of three separate experiments. Mean fluorescence intensities from the FACS plots are also indicated. Error bars = SEM. (D) Western blot of PAI-1 expression after treatment with 20 ng/ml IFN-γ for 48 h is representative of two independent experiments. PAI-1 was detected at a molecular mass of ∼47 kD, and α-tubulin expression was used as a loading control. (E) PAI-1 expression determined by quantitative RT-PCR. Expression is normalized to GAPDH and expressed relative to RH30 expression. Error bars = SEM of triplicate estimations. (F) FACS histograms of CXCR4 surface expression. The dotted line represents isotype control antibody, and the continuous line represents anti-CXCR4 antibody.

    Article Snippet: All HRP-labeled secondary antibodies were from Santa Cruz Biotechnology, Inc. For immunoprecipitation, 200 μl of lysate from 5 × 106 cells was precipitated with 2 μl of anti-STAT3 (rabbit polyclonal antibody; Upstate Biotechnology), anti STATs 2, 4, 5, and 6 (Santa Cruz Biotechnology, Inc.), anti-PAX3 (N19; Santa Cruz Biotechnology, Inc.), or antiestrogen receptor rabbit polyclonal (MC-20; Santa Cruz Biotechnology, Inc.) antibodies using protein A sepharose (GE Healthcare) in accordance with the manufacturer's instructions.

    Techniques: Expressing, FACS, Staining, Western Blot, Clone Assay, Northern Blot, Cell Culture, Fluorescence, Quantitative RT-PCR

    PAX3-FKHR suppresses local inflammatory and immunological responses and causes more rapid in vivo growth rate in immunodeficient mice. Tumor growth rates and tumor infiltrating cells of PAX3-FKHR expressing (C23) or control (CMV) cells grown in immunodeficient (A) or syngeneic C57BL/6 (B) mice. Each line represents the tumor growth curve of an individual animal. Tumor-infiltrating cells were determined by FACS analysis. Histograms indicate the percentage of total cells that were gated as viable cells for the analysis of tumor-infiltrating cells. Closed bars, tumors from vector-transfected 76-9 cells; open bars, tumors derived from 76-9–P3F-C23 cells. For DC activation analysis, CD11c cells were gated, and respective activation markers were enumerated. Error bars = SEM of four tumors per group.

    Journal: The Journal of Experimental Medicine

    Article Title: Coordinated oncogenic transformation and inhibition of host immune responses by the PAX3-FKHR fusion oncoprotein

    doi: 10.1084/jem.20050730

    Figure Lengend Snippet: PAX3-FKHR suppresses local inflammatory and immunological responses and causes more rapid in vivo growth rate in immunodeficient mice. Tumor growth rates and tumor infiltrating cells of PAX3-FKHR expressing (C23) or control (CMV) cells grown in immunodeficient (A) or syngeneic C57BL/6 (B) mice. Each line represents the tumor growth curve of an individual animal. Tumor-infiltrating cells were determined by FACS analysis. Histograms indicate the percentage of total cells that were gated as viable cells for the analysis of tumor-infiltrating cells. Closed bars, tumors from vector-transfected 76-9 cells; open bars, tumors derived from 76-9–P3F-C23 cells. For DC activation analysis, CD11c cells were gated, and respective activation markers were enumerated. Error bars = SEM of four tumors per group.

    Article Snippet: All HRP-labeled secondary antibodies were from Santa Cruz Biotechnology, Inc. For immunoprecipitation, 200 μl of lysate from 5 × 106 cells was precipitated with 2 μl of anti-STAT3 (rabbit polyclonal antibody; Upstate Biotechnology), anti STATs 2, 4, 5, and 6 (Santa Cruz Biotechnology, Inc.), anti-PAX3 (N19; Santa Cruz Biotechnology, Inc.), or antiestrogen receptor rabbit polyclonal (MC-20; Santa Cruz Biotechnology, Inc.) antibodies using protein A sepharose (GE Healthcare) in accordance with the manufacturer's instructions.

    Techniques: In Vivo, Mouse Assay, Expressing, FACS, Plasmid Preparation, Transfection, Derivative Assay, Activation Assay

    PAX3-FKHR interacts with STAT3. (A) Transient transfection assay using PRS9-CAT reporter showing specific inducible activity of PAX3-FKHR in clone 15 on addition of 4HT. Error bars = SEM of triplicate transfections. (B) Coimmunoprecipitation of STAT3 with estrogen receptor–tagged PAX3-FKHR is seen only in presence of 4HT. The IP is representative of three separate experiments. The high molecular mass band of ∼200 kD shown here was not seen in all replicate experiments. (bottom) Replica blot probed with STAT5. (C) Immunoprecipitations from 293T cells transfected with PAX3-FKHR, truncated PAX3-FKHR lacking FKHR regions, or empty vector. (left) Lysates are detected with an anti-PAX3 antibody. Immunoprecipitates are detected with an anti-STAT3 antibody. The IP data are representative of three separate experiments. (right) Replica blots from the same transfections probed with antibodies against STATs 2 (113 kD), 4 (81 kD), 5 (95 kD), and 6 (100 kD). No staining with STATs 2, 4, or 6 was observed when the membrane was subject to longer exposures. (D) Coimmunoprecipitation experiment to show that an anti–NH 2 terminus PAX3 antibody and anti-STAT3 antibody are both able to precipitate STAT3 from RH30 cells. The blot is representative of two independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Coordinated oncogenic transformation and inhibition of host immune responses by the PAX3-FKHR fusion oncoprotein

    doi: 10.1084/jem.20050730

    Figure Lengend Snippet: PAX3-FKHR interacts with STAT3. (A) Transient transfection assay using PRS9-CAT reporter showing specific inducible activity of PAX3-FKHR in clone 15 on addition of 4HT. Error bars = SEM of triplicate transfections. (B) Coimmunoprecipitation of STAT3 with estrogen receptor–tagged PAX3-FKHR is seen only in presence of 4HT. The IP is representative of three separate experiments. The high molecular mass band of ∼200 kD shown here was not seen in all replicate experiments. (bottom) Replica blot probed with STAT5. (C) Immunoprecipitations from 293T cells transfected with PAX3-FKHR, truncated PAX3-FKHR lacking FKHR regions, or empty vector. (left) Lysates are detected with an anti-PAX3 antibody. Immunoprecipitates are detected with an anti-STAT3 antibody. The IP data are representative of three separate experiments. (right) Replica blots from the same transfections probed with antibodies against STATs 2 (113 kD), 4 (81 kD), 5 (95 kD), and 6 (100 kD). No staining with STATs 2, 4, or 6 was observed when the membrane was subject to longer exposures. (D) Coimmunoprecipitation experiment to show that an anti–NH 2 terminus PAX3 antibody and anti-STAT3 antibody are both able to precipitate STAT3 from RH30 cells. The blot is representative of two independent experiments.

    Article Snippet: All HRP-labeled secondary antibodies were from Santa Cruz Biotechnology, Inc. For immunoprecipitation, 200 μl of lysate from 5 × 106 cells was precipitated with 2 μl of anti-STAT3 (rabbit polyclonal antibody; Upstate Biotechnology), anti STATs 2, 4, 5, and 6 (Santa Cruz Biotechnology, Inc.), anti-PAX3 (N19; Santa Cruz Biotechnology, Inc.), or antiestrogen receptor rabbit polyclonal (MC-20; Santa Cruz Biotechnology, Inc.) antibodies using protein A sepharose (GE Healthcare) in accordance with the manufacturer's instructions.

    Techniques: Transient Transfection Assay, Activity Assay, Transfection, Plasmid Preparation, Staining

    The presence of PAX3-FKHR causes the STAT3-dependent generation of immunoinhibitory secreted factors. (A–E) Flow cytometric analysis of HLA-DR and CD86 expression on viable CD11c + -gated DCs generated in CM from various cell lines (RD Vect +/− 4HT and RD P3F-ER +/− 4HT; 293T transfected with combinations of empty vector, truncated PAX3, PAX3-FKHR, PAX3-FKHR mutants, dominant negative STAT3, or siRNA against STAT3; and RH18 or RH30) 24 h after KLH activation. As indicated, conditioned media were obtained after growth of cell lines in the presence of a specific STAT3 inhibitory phosphopeptide (PY*) or control nonphosphorylated peptide (PY). G48S, paired domain mutant PAX3-FKHR; N269, homeodomain mutant of PAX3-FKHR; STAT3-β, dominant-negative form of STAT3. Data are the representative result of between two and three independent experiments. Conditioned media were collected from confluent cell lines 48 h after passaging. All FACS plots have logarithmic scales. Percentages refer to the percentage of CD11c-positive cells in the top right quadrant or indicated gates. (F) Stat-3 expression in tumor cells increases IL-10 cytokine production. Detection of human IL-10 secretion in tumor cells expressing inducible P3F-ER. Results are presented as the SEM of triplicate samples and are representative of two independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Coordinated oncogenic transformation and inhibition of host immune responses by the PAX3-FKHR fusion oncoprotein

    doi: 10.1084/jem.20050730

    Figure Lengend Snippet: The presence of PAX3-FKHR causes the STAT3-dependent generation of immunoinhibitory secreted factors. (A–E) Flow cytometric analysis of HLA-DR and CD86 expression on viable CD11c + -gated DCs generated in CM from various cell lines (RD Vect +/− 4HT and RD P3F-ER +/− 4HT; 293T transfected with combinations of empty vector, truncated PAX3, PAX3-FKHR, PAX3-FKHR mutants, dominant negative STAT3, or siRNA against STAT3; and RH18 or RH30) 24 h after KLH activation. As indicated, conditioned media were obtained after growth of cell lines in the presence of a specific STAT3 inhibitory phosphopeptide (PY*) or control nonphosphorylated peptide (PY). G48S, paired domain mutant PAX3-FKHR; N269, homeodomain mutant of PAX3-FKHR; STAT3-β, dominant-negative form of STAT3. Data are the representative result of between two and three independent experiments. Conditioned media were collected from confluent cell lines 48 h after passaging. All FACS plots have logarithmic scales. Percentages refer to the percentage of CD11c-positive cells in the top right quadrant or indicated gates. (F) Stat-3 expression in tumor cells increases IL-10 cytokine production. Detection of human IL-10 secretion in tumor cells expressing inducible P3F-ER. Results are presented as the SEM of triplicate samples and are representative of two independent experiments.

    Article Snippet: All HRP-labeled secondary antibodies were from Santa Cruz Biotechnology, Inc. For immunoprecipitation, 200 μl of lysate from 5 × 106 cells was precipitated with 2 μl of anti-STAT3 (rabbit polyclonal antibody; Upstate Biotechnology), anti STATs 2, 4, 5, and 6 (Santa Cruz Biotechnology, Inc.), anti-PAX3 (N19; Santa Cruz Biotechnology, Inc.), or antiestrogen receptor rabbit polyclonal (MC-20; Santa Cruz Biotechnology, Inc.) antibodies using protein A sepharose (GE Healthcare) in accordance with the manufacturer's instructions.

    Techniques: Flow Cytometry, Expressing, Generated, Transfection, Plasmid Preparation, Dominant Negative Mutation, Activation Assay, Mutagenesis, Passaging, FACS

    PAX3-FKHR causes both up- and down-regulation of target genes. (A) PAX3-FKHR protein function in 76-9 and RD cells stably transfected with pBK-CMV-P3F as determined by transient transfection assays using the specific PAX3 reporter plasmid PRS-9 linked to CAT. This contains six direct repeats of the PAX3 paired domain and homeodomain consensus sequences upstream of a CAT reporter. CAT activity is plotted in arbitrary units ± SEM of triplicate samples. This assay is representative of four separate experiments. (B) Matrigel invasion assay to show that PAX3-FKHR increases the invasive ability of 76-9 cells. Percent invasion was calculated as: (number of invasive cells / total number of cells) × 100. Mean values ± SEM of quadruplicate samples are plotted. PAX3-FKHR–expressing 76-9–P3F-C24 cells (and 76-9–P3F-C23 cells; not depicted) were significantly more invasive than 76-9–CMV cells. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Coordinated oncogenic transformation and inhibition of host immune responses by the PAX3-FKHR fusion oncoprotein

    doi: 10.1084/jem.20050730

    Figure Lengend Snippet: PAX3-FKHR causes both up- and down-regulation of target genes. (A) PAX3-FKHR protein function in 76-9 and RD cells stably transfected with pBK-CMV-P3F as determined by transient transfection assays using the specific PAX3 reporter plasmid PRS-9 linked to CAT. This contains six direct repeats of the PAX3 paired domain and homeodomain consensus sequences upstream of a CAT reporter. CAT activity is plotted in arbitrary units ± SEM of triplicate samples. This assay is representative of four separate experiments. (B) Matrigel invasion assay to show that PAX3-FKHR increases the invasive ability of 76-9 cells. Percent invasion was calculated as: (number of invasive cells / total number of cells) × 100. Mean values ± SEM of quadruplicate samples are plotted. PAX3-FKHR–expressing 76-9–P3F-C24 cells (and 76-9–P3F-C23 cells; not depicted) were significantly more invasive than 76-9–CMV cells. *, P

    Article Snippet: All HRP-labeled secondary antibodies were from Santa Cruz Biotechnology, Inc. For immunoprecipitation, 200 μl of lysate from 5 × 106 cells was precipitated with 2 μl of anti-STAT3 (rabbit polyclonal antibody; Upstate Biotechnology), anti STATs 2, 4, 5, and 6 (Santa Cruz Biotechnology, Inc.), anti-PAX3 (N19; Santa Cruz Biotechnology, Inc.), or antiestrogen receptor rabbit polyclonal (MC-20; Santa Cruz Biotechnology, Inc.) antibodies using protein A sepharose (GE Healthcare) in accordance with the manufacturer's instructions.

    Techniques: Stable Transfection, Transfection, Plasmid Preparation, Activity Assay, Invasion Assay, Expressing

    PAX3-FKHR–expressing ARMS cells have an immunoinhibitory phenotype. (A) Numbers of neutrophils infiltrating PAX3-FKHR– expressing human ARMS versus ERMS tumors. The box whisker plot shows the first and third quartiles, median, and range. (B) Representative image of STAT3 immunohistochemistry of prechemotherapy tumor biopsies or surgical specimens. Three representative ARMS tumors are shown with one ERMS case for comparison.

    Journal: The Journal of Experimental Medicine

    Article Title: Coordinated oncogenic transformation and inhibition of host immune responses by the PAX3-FKHR fusion oncoprotein

    doi: 10.1084/jem.20050730

    Figure Lengend Snippet: PAX3-FKHR–expressing ARMS cells have an immunoinhibitory phenotype. (A) Numbers of neutrophils infiltrating PAX3-FKHR– expressing human ARMS versus ERMS tumors. The box whisker plot shows the first and third quartiles, median, and range. (B) Representative image of STAT3 immunohistochemistry of prechemotherapy tumor biopsies or surgical specimens. Three representative ARMS tumors are shown with one ERMS case for comparison.

    Article Snippet: All HRP-labeled secondary antibodies were from Santa Cruz Biotechnology, Inc. For immunoprecipitation, 200 μl of lysate from 5 × 106 cells was precipitated with 2 μl of anti-STAT3 (rabbit polyclonal antibody; Upstate Biotechnology), anti STATs 2, 4, 5, and 6 (Santa Cruz Biotechnology, Inc.), anti-PAX3 (N19; Santa Cruz Biotechnology, Inc.), or antiestrogen receptor rabbit polyclonal (MC-20; Santa Cruz Biotechnology, Inc.) antibodies using protein A sepharose (GE Healthcare) in accordance with the manufacturer's instructions.

    Techniques: Expressing, Whisker Assay, Immunohistochemistry

    The arrangement of HBMs pathways and pharyngeal arches in shark embryos. Pax3 , Dlx5 and Tbx1 expressions in sharks at stage 27 ( a ), 28 ( b ) and 29 ( c ). ( a , b ) Lateral and ( c ) ventrolateral views. Section in situ hybridization of Pax3 , Dlx5 and Tbx1 at stage 27 ( d – f ), 28 ( g – i ) and 29 ( k – m ). Transverse section levels are at the fourth branchial arch ( d – f ), the second branchial arch ( g – i ) and the hyoid arch ( j – m ), as indicated in ( a ), ( b ) and ( c ). Arrowheads indicate HBM precursors. Pharyngeal arches and the pericardium were in the same transverse plane. Note that HBM precursors passed through the lateral side of pericardium, and then extended medially at the hyoid arch. Adp, anterodorsal lateral line placode; AER, apical ectodermal ridge; ba, branchial arch mesoderm; bbm, basibranchial mesenchyme; dm, dermomyotome; drg, dorsal root ganglion; eb, ectomesenchyme of branchial arches; ed., endolymphatic duct; eh, ectomesenchyme of hyoid arch; em, ectomesenchyme of mandibular arch; gb, gill bud; ha, hyoid arch mesoderm; he, heart; hg, hatching gland; ih interhyoideus; ma, mandibular arch mesoderm; ne, nasal epithelium; np, nasal prominence; nt, neural tube; ot, otic vesicle; pcc, pericardial cavity; pcm, pericardial mesoderm; pe, pharyngeal endoderm; pfm, pectoral fin muscle; pohm, postotic paraxial head mesoderm; scl, sclerotome; thy thyroid gland. Scale bars on whole embryos, 200 μm. Scale bars on sections, 50 μm

    Journal: Zoological Letters

    Article Title: Development of hypobranchial muscles with special reference to the evolution of the vertebrate neck

    doi: 10.1186/s40851-018-0087-x

    Figure Lengend Snippet: The arrangement of HBMs pathways and pharyngeal arches in shark embryos. Pax3 , Dlx5 and Tbx1 expressions in sharks at stage 27 ( a ), 28 ( b ) and 29 ( c ). ( a , b ) Lateral and ( c ) ventrolateral views. Section in situ hybridization of Pax3 , Dlx5 and Tbx1 at stage 27 ( d – f ), 28 ( g – i ) and 29 ( k – m ). Transverse section levels are at the fourth branchial arch ( d – f ), the second branchial arch ( g – i ) and the hyoid arch ( j – m ), as indicated in ( a ), ( b ) and ( c ). Arrowheads indicate HBM precursors. Pharyngeal arches and the pericardium were in the same transverse plane. Note that HBM precursors passed through the lateral side of pericardium, and then extended medially at the hyoid arch. Adp, anterodorsal lateral line placode; AER, apical ectodermal ridge; ba, branchial arch mesoderm; bbm, basibranchial mesenchyme; dm, dermomyotome; drg, dorsal root ganglion; eb, ectomesenchyme of branchial arches; ed., endolymphatic duct; eh, ectomesenchyme of hyoid arch; em, ectomesenchyme of mandibular arch; gb, gill bud; ha, hyoid arch mesoderm; he, heart; hg, hatching gland; ih interhyoideus; ma, mandibular arch mesoderm; ne, nasal epithelium; np, nasal prominence; nt, neural tube; ot, otic vesicle; pcc, pericardial cavity; pcm, pericardial mesoderm; pe, pharyngeal endoderm; pfm, pectoral fin muscle; pohm, postotic paraxial head mesoderm; scl, sclerotome; thy thyroid gland. Scale bars on whole embryos, 200 μm. Scale bars on sections, 50 μm

    Article Snippet: Anti-PAX3 mouse monoclonal antibody (1/100 dilution, ab69856, Abcam) and anti-GFP rabbit polyclonal antibody (1/500 dilution, ab290, Abcam) were used for the primary antibody.

    Techniques: In Situ Hybridization, Periodic Counter-current Chromatography

    The arrangement of HBMs pathways and pharyngeal arches in lamprey embryos. Pax3/7 , DlxB and Tbx1/10A expressions in lampreys at stage 27 ( a ) and 28 ( b ) from the lateral view. Transverse sections of stage 27 embryos at the fifth branchial arch ( c – e ) and heart ( f – h ) levels, and stage 28 embryos at the second ( i - k ) and fourth branchial arch ( ln ) levels. Section levels are indicated in ( a ) and ( b ). Arrowheads indicate HBM precursors. Pharyngeal arches and the pericardium were not in the same transverse plane, and HBM precursors expanded lateral to the pharyngeal arches. ba, branchial arch mesoderm; dm, dermomyotome; eb, ectomesenchyme of branchial arches; he, heart; iom, infraoptic muscle; nt, neural tube; ot, otic vesicle; pcc, pericardial cavity; pe, pharyngeal endoderm; se, surface ectoderm; som, supraoptic muscle. Scale bars on whole embryos, 200 μm. Scale bars on sections, 50 μm

    Journal: Zoological Letters

    Article Title: Development of hypobranchial muscles with special reference to the evolution of the vertebrate neck

    doi: 10.1186/s40851-018-0087-x

    Figure Lengend Snippet: The arrangement of HBMs pathways and pharyngeal arches in lamprey embryos. Pax3/7 , DlxB and Tbx1/10A expressions in lampreys at stage 27 ( a ) and 28 ( b ) from the lateral view. Transverse sections of stage 27 embryos at the fifth branchial arch ( c – e ) and heart ( f – h ) levels, and stage 28 embryos at the second ( i - k ) and fourth branchial arch ( ln ) levels. Section levels are indicated in ( a ) and ( b ). Arrowheads indicate HBM precursors. Pharyngeal arches and the pericardium were not in the same transverse plane, and HBM precursors expanded lateral to the pharyngeal arches. ba, branchial arch mesoderm; dm, dermomyotome; eb, ectomesenchyme of branchial arches; he, heart; iom, infraoptic muscle; nt, neural tube; ot, otic vesicle; pcc, pericardial cavity; pe, pharyngeal endoderm; se, surface ectoderm; som, supraoptic muscle. Scale bars on whole embryos, 200 μm. Scale bars on sections, 50 μm

    Article Snippet: Anti-PAX3 mouse monoclonal antibody (1/100 dilution, ab69856, Abcam) and anti-GFP rabbit polyclonal antibody (1/500 dilution, ab290, Abcam) were used for the primary antibody.

    Techniques: Periodic Counter-current Chromatography

    Gene expression analysis of HBM precursors in chicken and mouse embryos. Pax3 , Dlx5 and Myf5 expression patterns in chicken HH20 ( a ) and HH23 ( b ), and mouse E10.25 ( c ) and E10.5 ( d ). Two-color in situ hybridization with Pax3 (red) and Dlx5 (blue) probes in chicken HH23 ( e ) and mouse E10.5 ( f ). Lateral views. Arrowheads indicate HBM precursors. Note that HBM precursors, marked by Pax3 and Myf5 expressions, developed medially at the level of hyoid arch at stage HH23 and E10.5 embryos. AER, apical ectodermal ridge; ba1, first branchial arch mesoderm; dm, dermomyotome; do, dorsal oblique; eb1, ectomesenchyme of first branchial arch; ed., endolymphatic duct; eh, ectomesenchyme of hyoid arch; ep, ectomesenchyme of pharyngeal arches; fm, forelimb muscles; ha, hyoid arch mesoderm; hc, hypoglossal cord; lr, lateral rectus; mr, medial rectus; my, myotome; ne, nasal epithelium; np, nasal prominence; nt, neural tube; ot, otic vesicle; pcm, pericardial mesoderm; sp., subpallium; V, trigeminal ganglion; vo, ventral oblique. Scale bars, 200 μm

    Journal: Zoological Letters

    Article Title: Development of hypobranchial muscles with special reference to the evolution of the vertebrate neck

    doi: 10.1186/s40851-018-0087-x

    Figure Lengend Snippet: Gene expression analysis of HBM precursors in chicken and mouse embryos. Pax3 , Dlx5 and Myf5 expression patterns in chicken HH20 ( a ) and HH23 ( b ), and mouse E10.25 ( c ) and E10.5 ( d ). Two-color in situ hybridization with Pax3 (red) and Dlx5 (blue) probes in chicken HH23 ( e ) and mouse E10.5 ( f ). Lateral views. Arrowheads indicate HBM precursors. Note that HBM precursors, marked by Pax3 and Myf5 expressions, developed medially at the level of hyoid arch at stage HH23 and E10.5 embryos. AER, apical ectodermal ridge; ba1, first branchial arch mesoderm; dm, dermomyotome; do, dorsal oblique; eb1, ectomesenchyme of first branchial arch; ed., endolymphatic duct; eh, ectomesenchyme of hyoid arch; ep, ectomesenchyme of pharyngeal arches; fm, forelimb muscles; ha, hyoid arch mesoderm; hc, hypoglossal cord; lr, lateral rectus; mr, medial rectus; my, myotome; ne, nasal epithelium; np, nasal prominence; nt, neural tube; ot, otic vesicle; pcm, pericardial mesoderm; sp., subpallium; V, trigeminal ganglion; vo, ventral oblique. Scale bars, 200 μm

    Article Snippet: Anti-PAX3 mouse monoclonal antibody (1/100 dilution, ab69856, Abcam) and anti-GFP rabbit polyclonal antibody (1/500 dilution, ab290, Abcam) were used for the primary antibody.

    Techniques: Expressing, In Situ Hybridization

    Trajectory of HBM precursors in mouse embryos. Transverse sections of mandibular, hyoid and branchial arch levels in Wnt1-Cre/R26R-H2B-EGFP embryos at stage E10.5. Bottom panels show the magnification of pharyngeal floor. Arrowheads indicate HBM precursors. PAX3 protein was detected on both sides of the EGFP-negative basibranchial mesenchyme and laterally surrounded by EGFP-positive neural crest cells at the hyoid arch. Bbm, basibranchial mesenchyme; cncc, cardiac neural crest cells; eb1–4, ectomesenchyme of first to fourth branchial arch; eh, ectomesenchyme of hyoid arch; em, ectomesenchyme of mandibular arch; ha, hyoid arch mesoderm; oft, outflow tract; ot, otic vesicle; ra, right atrium; thy thyroid gland. Scale bars, 50 μm

    Journal: Zoological Letters

    Article Title: Development of hypobranchial muscles with special reference to the evolution of the vertebrate neck

    doi: 10.1186/s40851-018-0087-x

    Figure Lengend Snippet: Trajectory of HBM precursors in mouse embryos. Transverse sections of mandibular, hyoid and branchial arch levels in Wnt1-Cre/R26R-H2B-EGFP embryos at stage E10.5. Bottom panels show the magnification of pharyngeal floor. Arrowheads indicate HBM precursors. PAX3 protein was detected on both sides of the EGFP-negative basibranchial mesenchyme and laterally surrounded by EGFP-positive neural crest cells at the hyoid arch. Bbm, basibranchial mesenchyme; cncc, cardiac neural crest cells; eb1–4, ectomesenchyme of first to fourth branchial arch; eh, ectomesenchyme of hyoid arch; em, ectomesenchyme of mandibular arch; ha, hyoid arch mesoderm; oft, outflow tract; ot, otic vesicle; ra, right atrium; thy thyroid gland. Scale bars, 50 μm

    Article Snippet: Anti-PAX3 mouse monoclonal antibody (1/100 dilution, ab69856, Abcam) and anti-GFP rabbit polyclonal antibody (1/500 dilution, ab290, Abcam) were used for the primary antibody.

    Techniques:

    Effect of inhibiting PAX3-FOXO1 phosphorylation on ARMS tumor cell proliferation. ( a ) RH30 or ( b ) RH4 ARMS tumor were cells stably transduced with empty vector (vector), wild-type PAX3-FOXO1 (WT) or the indicated PAX3-FOXO1 phosphomutants, as described in the Materials and Methods. Cells were plated and allowed to grow for up to seven days. On each day the cell density was determined using the CCK-8 cell counting kit, densities were graphed as a function of time, and doubling times were determined using GraphPad Prism 6 software. Error bars represent the standard deviation from three independent determinations and P -values were computed using non-parametric two-way analyses of variance comparing each treatment condition to results seen with the empty vector transduced negative control. (* P =0.01, ** P =0.003).

    Journal: Oncogenesis

    Article Title: Inhibiting phosphorylation of the oncogenic PAX3-FOXO1 reduces alveolar rhabdomyosarcoma phenotypes identifying novel therapy options

    doi: 10.1038/oncsis.2015.2

    Figure Lengend Snippet: Effect of inhibiting PAX3-FOXO1 phosphorylation on ARMS tumor cell proliferation. ( a ) RH30 or ( b ) RH4 ARMS tumor were cells stably transduced with empty vector (vector), wild-type PAX3-FOXO1 (WT) or the indicated PAX3-FOXO1 phosphomutants, as described in the Materials and Methods. Cells were plated and allowed to grow for up to seven days. On each day the cell density was determined using the CCK-8 cell counting kit, densities were graphed as a function of time, and doubling times were determined using GraphPad Prism 6 software. Error bars represent the standard deviation from three independent determinations and P -values were computed using non-parametric two-way analyses of variance comparing each treatment condition to results seen with the empty vector transduced negative control. (* P =0.01, ** P =0.003).

    Article Snippet: Dr Hollenbach and Mr Miller are co-authors on US Patent #8,304,521, 'Phospho-specific anti-Pax3 antibodies' and are co-signees on licensing agreements with Abcam and Kerafast to market the phospho-specific antibodies described in this manuscript.

    Techniques: Stable Transfection, Transduction, Plasmid Preparation, CCK-8 Assay, Cell Counting, Software, Standard Deviation, Negative Control

    Effect of inhibiting PAX3-FOXO1 phosphorylation on anchorage-independent growth. ( a and b ) RH30 or ( c and d ) RH4 ARMS tumor cells were stably transduced with empty vector (vector), wild-type PAX3-FOXO1 (WT) or the indicated PAX3-FOXO1 phosphomutants. ( a ) RH30 cells were plated in soft agar, incubated for 1 week, and the extent of transformation was determined with a fluorescent assay, as described in the Materials and Methods. Results are presented as Relative Fluorescence, error bars represent the standard deviation from three independent determinations and P -values were computed using non-parametric two-way analyses of variance comparing each treatment condition to results seen with the empty vector-transduced negative control (** P =0.002). ( b ) Representative images of colony formation. ( c ) Cells were grown to confluency, after which they were allowed to grow an additional 5 days. Total number of colonies was counted, the results are presented as Number of Foci, error bars represent the standard deviation from four independent determinations, and P -values were computed using non-parametric two-way analyses of variance comparing each treatment condition to results seen with the empty vector transduced negative control (** P —0.0006, **** P

    Journal: Oncogenesis

    Article Title: Inhibiting phosphorylation of the oncogenic PAX3-FOXO1 reduces alveolar rhabdomyosarcoma phenotypes identifying novel therapy options

    doi: 10.1038/oncsis.2015.2

    Figure Lengend Snippet: Effect of inhibiting PAX3-FOXO1 phosphorylation on anchorage-independent growth. ( a and b ) RH30 or ( c and d ) RH4 ARMS tumor cells were stably transduced with empty vector (vector), wild-type PAX3-FOXO1 (WT) or the indicated PAX3-FOXO1 phosphomutants. ( a ) RH30 cells were plated in soft agar, incubated for 1 week, and the extent of transformation was determined with a fluorescent assay, as described in the Materials and Methods. Results are presented as Relative Fluorescence, error bars represent the standard deviation from three independent determinations and P -values were computed using non-parametric two-way analyses of variance comparing each treatment condition to results seen with the empty vector-transduced negative control (** P =0.002). ( b ) Representative images of colony formation. ( c ) Cells were grown to confluency, after which they were allowed to grow an additional 5 days. Total number of colonies was counted, the results are presented as Number of Foci, error bars represent the standard deviation from four independent determinations, and P -values were computed using non-parametric two-way analyses of variance comparing each treatment condition to results seen with the empty vector transduced negative control (** P —0.0006, **** P

    Article Snippet: Dr Hollenbach and Mr Miller are co-authors on US Patent #8,304,521, 'Phospho-specific anti-Pax3 antibodies' and are co-signees on licensing agreements with Abcam and Kerafast to market the phospho-specific antibodies described in this manuscript.

    Techniques: Stable Transfection, Transduction, Plasmid Preparation, Incubation, Transformation Assay, Fluorescence, Standard Deviation, Negative Control

    Effect of inhibiting PAX3-FOXO1 phosphorylation on ARMS tumor cell migration. ( a ) ARMS tumor cells RH30 or RH4 were treated with increasing concentrations of LiCl or AR-A014418 or ( b ) stably transduced with empty vector (vector), wild-type PAX3-FOXO1 (WT) or the indicated PAX3-FOXO1 phosphomutants, as described in the Materials and Methods. A scratch was introduced and migration into the wound was monitored by time-lapse microscopy. Individual cells were tracked and their velocities were determined using ImageJ software. For both panels, results are presented as velocity with error bars representing the standard deviation from 60 to 80 independent determinations. P -values were computed using non-parametric two-way analyses of variance comparing each treatment condition to results with non-treated cells ( a ) or to the empty vector transduced negative control ( b ). (* P =0.07, ** P =0.003, *** P =0.0001 **** P

    Journal: Oncogenesis

    Article Title: Inhibiting phosphorylation of the oncogenic PAX3-FOXO1 reduces alveolar rhabdomyosarcoma phenotypes identifying novel therapy options

    doi: 10.1038/oncsis.2015.2

    Figure Lengend Snippet: Effect of inhibiting PAX3-FOXO1 phosphorylation on ARMS tumor cell migration. ( a ) ARMS tumor cells RH30 or RH4 were treated with increasing concentrations of LiCl or AR-A014418 or ( b ) stably transduced with empty vector (vector), wild-type PAX3-FOXO1 (WT) or the indicated PAX3-FOXO1 phosphomutants, as described in the Materials and Methods. A scratch was introduced and migration into the wound was monitored by time-lapse microscopy. Individual cells were tracked and their velocities were determined using ImageJ software. For both panels, results are presented as velocity with error bars representing the standard deviation from 60 to 80 independent determinations. P -values were computed using non-parametric two-way analyses of variance comparing each treatment condition to results with non-treated cells ( a ) or to the empty vector transduced negative control ( b ). (* P =0.07, ** P =0.003, *** P =0.0001 **** P

    Article Snippet: Dr Hollenbach and Mr Miller are co-authors on US Patent #8,304,521, 'Phospho-specific anti-Pax3 antibodies' and are co-signees on licensing agreements with Abcam and Kerafast to market the phospho-specific antibodies described in this manuscript.

    Techniques: Migration, Stable Transfection, Transduction, Plasmid Preparation, Time-lapse Microscopy, Software, Standard Deviation, Negative Control

    Effect of inhibiting PAX3-FOXO1 phosphorylation on ARMS tumor cell invasion. ( a ) ARMS tumor cells RH30 were treated with increasing concentrations of LiCl or AR-A014418 or ( b ) stably transduced with empty vector (vector), wild-type PAX3-FOXO1 (WT) or the indicated PAX3-FOXO1 phosphomutants, as described in the Materials and Methods. Invasion capacity was determined using the BD Biocoat Tumor Invasion system, a Matrigel-based invasion assay, as described in the Methods. Results are presented as Relative Invasion with non-treated cells ( a ) or empty vector-transduced cells ( b ) being given a relative value of 100. Error bars represent the standard deviation from three independent determinations and P -values were computed using non-parametric two-way analyses of variance comparing each treatment condition to results with non-treated cells ( a ) or to the empty vector-transduced negative control ( b ). (* P =0.02, *** P =0.0003).

    Journal: Oncogenesis

    Article Title: Inhibiting phosphorylation of the oncogenic PAX3-FOXO1 reduces alveolar rhabdomyosarcoma phenotypes identifying novel therapy options

    doi: 10.1038/oncsis.2015.2

    Figure Lengend Snippet: Effect of inhibiting PAX3-FOXO1 phosphorylation on ARMS tumor cell invasion. ( a ) ARMS tumor cells RH30 were treated with increasing concentrations of LiCl or AR-A014418 or ( b ) stably transduced with empty vector (vector), wild-type PAX3-FOXO1 (WT) or the indicated PAX3-FOXO1 phosphomutants, as described in the Materials and Methods. Invasion capacity was determined using the BD Biocoat Tumor Invasion system, a Matrigel-based invasion assay, as described in the Methods. Results are presented as Relative Invasion with non-treated cells ( a ) or empty vector-transduced cells ( b ) being given a relative value of 100. Error bars represent the standard deviation from three independent determinations and P -values were computed using non-parametric two-way analyses of variance comparing each treatment condition to results with non-treated cells ( a ) or to the empty vector-transduced negative control ( b ). (* P =0.02, *** P =0.0003).

    Article Snippet: Dr Hollenbach and Mr Miller are co-authors on US Patent #8,304,521, 'Phospho-specific anti-Pax3 antibodies' and are co-signees on licensing agreements with Abcam and Kerafast to market the phospho-specific antibodies described in this manuscript.

    Techniques: Stable Transfection, Transduction, Plasmid Preparation, Invasion Assay, Standard Deviation, Negative Control

    Expression and phosphorylation of endogenous PAX3-FOXO1 or PAX3-FOXO1 phosphomutants. ( a ) Total extracts were made from RH30 (left panel) or RH4 (right panel) ARMS tumor cells treated with increasing concentrations of LiCl or AR-A014418. The presence of endogenous PAX3-FOXO1 (bottom panel) or endogenous PAX3-FOXO1 phosphorylated at Ser201 (top panel) was determined by western blot analysis on 25 μg of total cell extract using an antibody specific for PAX3 or the Ser201 phospho-specific antibody. ( b ) Phospho-PAX3-FOXO1 and total PAX3-FOXO1 were quantified by densitometry after which phospho-PAX3-FOXO1 was normalized for total PAX3-FOXO1. Results are plotted as relative phosphorylation with non-treated cells being given a value of 100. Error bars represent the standard deviation from three independent determinations and P -values were computed using non-parametric two-way analyses of variance comparing each treatment condition to results with non-treated cells. (* P =0.03, ** P =0.004). ( c ) Total cell extracts were made from RH30 (top panels) or RH4 (bottom panels) cells stably transduced with vector only (vector), wild-type PAX3-FOXO1 (WT) or the indicated PAX3-FOXO1 phospho-mutant. The presence of ectopically expressed PAX3-FOXO1 was determined by immunoprecipitation-western blot analysis, as described in the Materials and Methods.

    Journal: Oncogenesis

    Article Title: Inhibiting phosphorylation of the oncogenic PAX3-FOXO1 reduces alveolar rhabdomyosarcoma phenotypes identifying novel therapy options

    doi: 10.1038/oncsis.2015.2

    Figure Lengend Snippet: Expression and phosphorylation of endogenous PAX3-FOXO1 or PAX3-FOXO1 phosphomutants. ( a ) Total extracts were made from RH30 (left panel) or RH4 (right panel) ARMS tumor cells treated with increasing concentrations of LiCl or AR-A014418. The presence of endogenous PAX3-FOXO1 (bottom panel) or endogenous PAX3-FOXO1 phosphorylated at Ser201 (top panel) was determined by western blot analysis on 25 μg of total cell extract using an antibody specific for PAX3 or the Ser201 phospho-specific antibody. ( b ) Phospho-PAX3-FOXO1 and total PAX3-FOXO1 were quantified by densitometry after which phospho-PAX3-FOXO1 was normalized for total PAX3-FOXO1. Results are plotted as relative phosphorylation with non-treated cells being given a value of 100. Error bars represent the standard deviation from three independent determinations and P -values were computed using non-parametric two-way analyses of variance comparing each treatment condition to results with non-treated cells. (* P =0.03, ** P =0.004). ( c ) Total cell extracts were made from RH30 (top panels) or RH4 (bottom panels) cells stably transduced with vector only (vector), wild-type PAX3-FOXO1 (WT) or the indicated PAX3-FOXO1 phospho-mutant. The presence of ectopically expressed PAX3-FOXO1 was determined by immunoprecipitation-western blot analysis, as described in the Materials and Methods.

    Article Snippet: Dr Hollenbach and Mr Miller are co-authors on US Patent #8,304,521, 'Phospho-specific anti-Pax3 antibodies' and are co-signees on licensing agreements with Abcam and Kerafast to market the phospho-specific antibodies described in this manuscript.

    Techniques: Expressing, Western Blot, Standard Deviation, Stable Transfection, Transduction, Plasmid Preparation, Mutagenesis, Immunoprecipitation

    Expression of phosphorylated Pax3-FOXO1 in human primary tumor. Tumor sections were derived from a right lung transbronchial biopsy from a 17-year-old patient with a primary ARMS tumor testing positive for the t(2:13)(q35;q14) chromosomal translocation by FISH analysis. Sections were stained with hematoxylin and eosin ( a , d and f ), or antibodies that recognize Pax3 ( b and g ), Pax3 phosphorylated at Ser201 ( c and h ) or Pax3 phosphorylated at Ser205 ( e and i ) and magnifications are indicated in each panel.

    Journal: Oncogenesis

    Article Title: Inhibiting phosphorylation of the oncogenic PAX3-FOXO1 reduces alveolar rhabdomyosarcoma phenotypes identifying novel therapy options

    doi: 10.1038/oncsis.2015.2

    Figure Lengend Snippet: Expression of phosphorylated Pax3-FOXO1 in human primary tumor. Tumor sections were derived from a right lung transbronchial biopsy from a 17-year-old patient with a primary ARMS tumor testing positive for the t(2:13)(q35;q14) chromosomal translocation by FISH analysis. Sections were stained with hematoxylin and eosin ( a , d and f ), or antibodies that recognize Pax3 ( b and g ), Pax3 phosphorylated at Ser201 ( c and h ) or Pax3 phosphorylated at Ser205 ( e and i ) and magnifications are indicated in each panel.

    Article Snippet: Dr Hollenbach and Mr Miller are co-authors on US Patent #8,304,521, 'Phospho-specific anti-Pax3 antibodies' and are co-signees on licensing agreements with Abcam and Kerafast to market the phospho-specific antibodies described in this manuscript.

    Techniques: Expressing, Derivative Assay, Translocation Assay, Fluorescence In Situ Hybridization, Staining

    Abnormal gene expression in the patches. A–R , In situ hybridization of E12.5 and E16.5 Otx1 cre/+ ; Otx2 +/− and conditional mutants with Gli1 ( A , F ), Gli2 ( B , G ), Gli3 ( C , H ), Ptch1 ( D , I ), Smo ( E , J ), Lim1 ( K , O ), Pax3 ( L , P ), Pax7 ( M , Q ), and Gbx2 ( N , R ) probes. Note that Gbx2 is lost in the patches in which the expression of Lim1 , Pax3 , and Pax7 is activated. For control embryos, the sections at E12.5 from A–D and from E–N are two different groups of adjacent sections; similarly, also for the conditional mutant, the sections at E12.5 from A–D and from E–N are two groups of adjacent sections belonging to two different embryos. Th, Thalamus.

    Journal: The Journal of Neuroscience

    Article Title: Otx2 Controls Identity and Fate of Glutamatergic Progenitors of the Thalamus by Repressing GABAergic Differentiation

    doi: 10.1523/JNEUROSCI.1097-06.2006

    Figure Lengend Snippet: Abnormal gene expression in the patches. A–R , In situ hybridization of E12.5 and E16.5 Otx1 cre/+ ; Otx2 +/− and conditional mutants with Gli1 ( A , F ), Gli2 ( B , G ), Gli3 ( C , H ), Ptch1 ( D , I ), Smo ( E , J ), Lim1 ( K , O ), Pax3 ( L , P ), Pax7 ( M , Q ), and Gbx2 ( N , R ) probes. Note that Gbx2 is lost in the patches in which the expression of Lim1 , Pax3 , and Pax7 is activated. For control embryos, the sections at E12.5 from A–D and from E–N are two different groups of adjacent sections; similarly, also for the conditional mutant, the sections at E12.5 from A–D and from E–N are two groups of adjacent sections belonging to two different embryos. Th, Thalamus.

    Article Snippet: The rabbit antibodies were directed against Otx2 (1:5000; G. Corte, National Institute for Cancer Research, Genova, Italy), Pax3 (1:200; Zymed, San Francisco, CA), GABA (1:500; Sigma, St. Louis, MO), Lim1 (1:200; Chemicon, Temecula, CA), VGLUT2 (1:300; SySy, Goettingen, Germany), and Ph-H3 (the phosphorylated form of histone H3) (1:200; Upstate, Charlottesville, VA).

    Techniques: Expressing, In Situ Hybridization, Mutagenesis

    Cell-type identity in the patches. A–H , Combinatorial immunodetection with Otx2 ( A , C , E , H ), Pax3/7 ( A , C , D , F ) and Lim1 ( B , D , E–G ) antibodies. The sections at E10.5 and E16.5 are counterstained with Hoechst. The red color for Pax3/7 ( D ) is a pseudocolor. V, Ventricle.

    Journal: The Journal of Neuroscience

    Article Title: Otx2 Controls Identity and Fate of Glutamatergic Progenitors of the Thalamus by Repressing GABAergic Differentiation

    doi: 10.1523/JNEUROSCI.1097-06.2006

    Figure Lengend Snippet: Cell-type identity in the patches. A–H , Combinatorial immunodetection with Otx2 ( A , C , E , H ), Pax3/7 ( A , C , D , F ) and Lim1 ( B , D , E–G ) antibodies. The sections at E10.5 and E16.5 are counterstained with Hoechst. The red color for Pax3/7 ( D ) is a pseudocolor. V, Ventricle.

    Article Snippet: The rabbit antibodies were directed against Otx2 (1:5000; G. Corte, National Institute for Cancer Research, Genova, Italy), Pax3 (1:200; Zymed, San Francisco, CA), GABA (1:500; Sigma, St. Louis, MO), Lim1 (1:200; Chemicon, Temecula, CA), VGLUT2 (1:300; SySy, Goettingen, Germany), and Ph-H3 (the phosphorylated form of histone H3) (1:200; Upstate, Charlottesville, VA).

    Techniques: Immunodetection

    Activation of Mash1 in Otx2 − -Pax3/7 + progenitors and Otx2/Ngn2 synergistic cooperation. A–I , Combinatorial immunodetection with Mash1 ( A–F ), Pax3/7 ( A–C , G–I ), and Ngn2 ( D–I ) shows that, at E10.5, Mash1 is activated in a relevant fraction of Pax3/7 + cells ( A ) and maintained up to late gestation ( B , C ), whereas Ngn2 expression is unaffected at E10.5 ( D , G ) and then gradually lost ( E , F , H , I ). The green color for Pax3/7 ( A–C ) is a pseudocolor.

    Journal: The Journal of Neuroscience

    Article Title: Otx2 Controls Identity and Fate of Glutamatergic Progenitors of the Thalamus by Repressing GABAergic Differentiation

    doi: 10.1523/JNEUROSCI.1097-06.2006

    Figure Lengend Snippet: Activation of Mash1 in Otx2 − -Pax3/7 + progenitors and Otx2/Ngn2 synergistic cooperation. A–I , Combinatorial immunodetection with Mash1 ( A–F ), Pax3/7 ( A–C , G–I ), and Ngn2 ( D–I ) shows that, at E10.5, Mash1 is activated in a relevant fraction of Pax3/7 + cells ( A ) and maintained up to late gestation ( B , C ), whereas Ngn2 expression is unaffected at E10.5 ( D , G ) and then gradually lost ( E , F , H , I ). The green color for Pax3/7 ( A–C ) is a pseudocolor.

    Article Snippet: The rabbit antibodies were directed against Otx2 (1:5000; G. Corte, National Institute for Cancer Research, Genova, Italy), Pax3 (1:200; Zymed, San Francisco, CA), GABA (1:500; Sigma, St. Louis, MO), Lim1 (1:200; Chemicon, Temecula, CA), VGLUT2 (1:300; SySy, Goettingen, Germany), and Ph-H3 (the phosphorylated form of histone H3) (1:200; Upstate, Charlottesville, VA).

    Techniques: Activation Assay, Immunodetection, Expressing

    Proliferating activity and cell fate analysis. A–T , Immunodetection of Pax3/7 and BrdU ( A–D , I–L ), BrdU alone ( M–P ), and Ph-H3 ( E–H , Q–T ) in embryos exposed to a short pulse of BrdU at E10.5, E12.5, E13.5, and E16.5. Note that, at E12.5 and particularly at E13.5, the density of BrdU + and Ph-H3 + cells within the patches ( J , K , N , O , R , S ) is remarkably increased when compared with the unaffected neuroepithelium of conditional mutants (yellow arrow in J , K , N , O , R , S ) or Otx1 cre/+ ; Otx2 +/− control embryos ( B , C , F , G ). The red arrows in D point to the sporadic BrdU + cells detected at E16.5 in control embryos, and the white arrows in Q–T point to the Ph-H3 + cells in the patches. U–W , BrdU long-pulse experiments show that BrdU + cells labeled between E13.3 and E13.6 accumulate at E16.5 in the patches without mixing with those labeled along the unaffected neuroepithelium ( U ), and most of them are Lim1 + ( V ). Note that BrdU + -Lim1 − cells (arrowheads in the magnifications) may correspond to additional cell type(s) of the patches ( V , W ).

    Journal: The Journal of Neuroscience

    Article Title: Otx2 Controls Identity and Fate of Glutamatergic Progenitors of the Thalamus by Repressing GABAergic Differentiation

    doi: 10.1523/JNEUROSCI.1097-06.2006

    Figure Lengend Snippet: Proliferating activity and cell fate analysis. A–T , Immunodetection of Pax3/7 and BrdU ( A–D , I–L ), BrdU alone ( M–P ), and Ph-H3 ( E–H , Q–T ) in embryos exposed to a short pulse of BrdU at E10.5, E12.5, E13.5, and E16.5. Note that, at E12.5 and particularly at E13.5, the density of BrdU + and Ph-H3 + cells within the patches ( J , K , N , O , R , S ) is remarkably increased when compared with the unaffected neuroepithelium of conditional mutants (yellow arrow in J , K , N , O , R , S ) or Otx1 cre/+ ; Otx2 +/− control embryos ( B , C , F , G ). The red arrows in D point to the sporadic BrdU + cells detected at E16.5 in control embryos, and the white arrows in Q–T point to the Ph-H3 + cells in the patches. U–W , BrdU long-pulse experiments show that BrdU + cells labeled between E13.3 and E13.6 accumulate at E16.5 in the patches without mixing with those labeled along the unaffected neuroepithelium ( U ), and most of them are Lim1 + ( V ). Note that BrdU + -Lim1 − cells (arrowheads in the magnifications) may correspond to additional cell type(s) of the patches ( V , W ).

    Article Snippet: The rabbit antibodies were directed against Otx2 (1:5000; G. Corte, National Institute for Cancer Research, Genova, Italy), Pax3 (1:200; Zymed, San Francisco, CA), GABA (1:500; Sigma, St. Louis, MO), Lim1 (1:200; Chemicon, Temecula, CA), VGLUT2 (1:300; SySy, Goettingen, Germany), and Ph-H3 (the phosphorylated form of histone H3) (1:200; Upstate, Charlottesville, VA).

    Techniques: Activity Assay, Immunodetection, Labeling

    GABAergic fate switch of glutamatergic progenitors. A–C , In situ hybridization ( A , B ) and immunohistochemistry ( C ) of E16.5 Otx1 cre/+ ; Otx2 +/− and Otx1 cre/+ ; Otx2 flox/− embryos with VGLUT2 ( A ), Gad1 ( B ) probes, and GABA ( C ) antibody show that, in the patches, the lack of VGLUT2 expression correlates with the induction of GABAergic markers. D–G , Combinatorial immunodetection with GABA ( D , E ), VGLUT2 ( D , F , G ), and BrdU ( E , F ) antibodies in E16.5 conditional mutants labeled at E13.3 with BrdU shows that many of the BrdU + neurons are GABA + , although none of them are VGLUT2 + . H–M , Combinatorial immunodetection with GABA ( H–K ), Lim1 ( H , I , L , M ), and Pax3/7 ( J , K ) antibodies shows that GABA + neurons are Lim1 + . I , K , and M correspond to the magnification of the area demarcated in H , J , and L . The green color for VGLUT2 ( D ) is a pseudocolor.

    Journal: The Journal of Neuroscience

    Article Title: Otx2 Controls Identity and Fate of Glutamatergic Progenitors of the Thalamus by Repressing GABAergic Differentiation

    doi: 10.1523/JNEUROSCI.1097-06.2006

    Figure Lengend Snippet: GABAergic fate switch of glutamatergic progenitors. A–C , In situ hybridization ( A , B ) and immunohistochemistry ( C ) of E16.5 Otx1 cre/+ ; Otx2 +/− and Otx1 cre/+ ; Otx2 flox/− embryos with VGLUT2 ( A ), Gad1 ( B ) probes, and GABA ( C ) antibody show that, in the patches, the lack of VGLUT2 expression correlates with the induction of GABAergic markers. D–G , Combinatorial immunodetection with GABA ( D , E ), VGLUT2 ( D , F , G ), and BrdU ( E , F ) antibodies in E16.5 conditional mutants labeled at E13.3 with BrdU shows that many of the BrdU + neurons are GABA + , although none of them are VGLUT2 + . H–M , Combinatorial immunodetection with GABA ( H–K ), Lim1 ( H , I , L , M ), and Pax3/7 ( J , K ) antibodies shows that GABA + neurons are Lim1 + . I , K , and M correspond to the magnification of the area demarcated in H , J , and L . The green color for VGLUT2 ( D ) is a pseudocolor.

    Article Snippet: The rabbit antibodies were directed against Otx2 (1:5000; G. Corte, National Institute for Cancer Research, Genova, Italy), Pax3 (1:200; Zymed, San Francisco, CA), GABA (1:500; Sigma, St. Louis, MO), Lim1 (1:200; Chemicon, Temecula, CA), VGLUT2 (1:300; SySy, Goettingen, Germany), and Ph-H3 (the phosphorylated form of histone H3) (1:200; Upstate, Charlottesville, VA).

    Techniques: In Situ Hybridization, Immunohistochemistry, Expressing, Immunodetection, Labeling

    UVR-induced melanoma development in melanocyte-specific DOT1L conditional knockout mice. a Experimental design for UVR-induced melanoma development in the DOT1L flox/flox /Tyr-CreERT2 mice. b Melanoma incidence in wild-type and DOT1L flox/flox /Tyr-CreERT2 mice with or without UVB irradiation. c Image of a representative DOT1L flox/flox /Tyr-CreERT2 cutaneous melanoma. d H E staining of a representative cutaneous melanoma. Scale bar, 500 μm (upper panel) and 50 μm (lower panel). e Immunohistochemistry staining of S100, tyrosinase, Pax3, and Mart-1 of the representative cutaneous melanoma. Scale bar, 100 μm. f The relative mRNA expression of Pax3 and tyrosinase in mouse melanomas comparing to those of normal skin tissues. *** p

    Journal: Nature Communications

    Article Title: The protective role of DOT1L in UV-induced melanomagenesis

    doi: 10.1038/s41467-017-02687-7

    Figure Lengend Snippet: UVR-induced melanoma development in melanocyte-specific DOT1L conditional knockout mice. a Experimental design for UVR-induced melanoma development in the DOT1L flox/flox /Tyr-CreERT2 mice. b Melanoma incidence in wild-type and DOT1L flox/flox /Tyr-CreERT2 mice with or without UVB irradiation. c Image of a representative DOT1L flox/flox /Tyr-CreERT2 cutaneous melanoma. d H E staining of a representative cutaneous melanoma. Scale bar, 500 μm (upper panel) and 50 μm (lower panel). e Immunohistochemistry staining of S100, tyrosinase, Pax3, and Mart-1 of the representative cutaneous melanoma. Scale bar, 100 μm. f The relative mRNA expression of Pax3 and tyrosinase in mouse melanomas comparing to those of normal skin tissues. *** p

    Article Snippet: Immunohistochemistry Tissue samples for immunohistochemistry analysis were sectioned at 4 µm thick and underwent antigen heat retrieval using EDTA 105 °C for 10 min. Antibodies include: anti-S-100 (1:100, Dako North America, Inc. Carpinteria, CA, USA), anti-Tyrosinase (1:100, T311, Dako North America), anti-Pax3 (1:100, 38-1801, Thermo Fisher Scientific Inc.), and Melan-A/MART-1 antibody (1:500, M2-9E3, Novus Biologicals, USA).

    Techniques: Knock-Out, Mouse Assay, Irradiation, Staining, Immunohistochemistry, Expressing

    Co-expression analysis of PAX3-positive epidermal melanocytes of normal skin. Epidermal melanocytes of normal skin show a variable differentiation status: less differentiated melanocytes co-expressing PAX3 (mouse monoclonal antibody, DSHB) and HES1 (A); more differentiated co-expressing PAX3 (rabbit polyclonal, Invitrogen) and MLANA (B); and mature melanocytes expressing only MLANA (B). Single HES1-labelled cells in the epidermis are keratinocytes (A). C) PAX3 (mouse monoclonal antibody, DSHB) and Ki67 co-expressing melanocytes are also observed in the epidermis of sun-exposed skin. Lines in (A), (B) and (C) demarcate the epidermal-dermal border (EDB) or epidermal surface (ES). D) Graph shows the distribution of differentiation marker expression in normal skin melanocytes with respect to melanocyte location (in epidermis, outer root sheath (ORS), or hair follicle bulb).

    Journal: PLoS ONE

    Article Title: PAX3 Expression in Normal Skin Melanocytes and Melanocytic Lesions (Naevi and Melanomas)

    doi: 10.1371/journal.pone.0009977

    Figure Lengend Snippet: Co-expression analysis of PAX3-positive epidermal melanocytes of normal skin. Epidermal melanocytes of normal skin show a variable differentiation status: less differentiated melanocytes co-expressing PAX3 (mouse monoclonal antibody, DSHB) and HES1 (A); more differentiated co-expressing PAX3 (rabbit polyclonal, Invitrogen) and MLANA (B); and mature melanocytes expressing only MLANA (B). Single HES1-labelled cells in the epidermis are keratinocytes (A). C) PAX3 (mouse monoclonal antibody, DSHB) and Ki67 co-expressing melanocytes are also observed in the epidermis of sun-exposed skin. Lines in (A), (B) and (C) demarcate the epidermal-dermal border (EDB) or epidermal surface (ES). D) Graph shows the distribution of differentiation marker expression in normal skin melanocytes with respect to melanocyte location (in epidermis, outer root sheath (ORS), or hair follicle bulb).

    Article Snippet: PAX3-specific staining was also confirmed using a different commercially available anti-PAX3 antibody (Invitrogen) ( ).

    Techniques: Expressing, Marker

    Co-expression of PAX3 with markers of cell survival and migration in melanocytic and melanoma cells. A) Double immunofluorescent staining showing PAX3 (mouse monoclonal antibody, DSHB) and BCL2L1 co-expression in representative samples of normal skin, naevus, primary melanoma and melanoma metastasis. B) PAX3 (mouse monoclonal antibody, DSHB) and MCAM co-expression in normal skin (epidermal melanocytes), naevus, primary melanoma and melanoma metastasis. Lines in (A) and (B) demarcate the epidermal-dermal border (EDB) or epidermal surface (ES). C) In contrast to the epidermal melanocytes, some PAX3-positive melanocytes in the outer root sheath (ORS) co-express MCAM. D) Graph showing the overall number of PAX3, BCL2L1 double-labelled cells in normal skins, naevi, primary melanomas and melanoma metastases. Each column represents a percentage of PAX3-positive cells that are also BCL2L1-positive, averaged across all samples. E) Graph showing the overall number of PAX3, MCAM double-labelled cells in normal skins, naevi, primary melanomas and melanoma metastases. Each column represents a percentage of PAX3-positive cells that are also MCAM-positive, averaged across all samples.

    Journal: PLoS ONE

    Article Title: PAX3 Expression in Normal Skin Melanocytes and Melanocytic Lesions (Naevi and Melanomas)

    doi: 10.1371/journal.pone.0009977

    Figure Lengend Snippet: Co-expression of PAX3 with markers of cell survival and migration in melanocytic and melanoma cells. A) Double immunofluorescent staining showing PAX3 (mouse monoclonal antibody, DSHB) and BCL2L1 co-expression in representative samples of normal skin, naevus, primary melanoma and melanoma metastasis. B) PAX3 (mouse monoclonal antibody, DSHB) and MCAM co-expression in normal skin (epidermal melanocytes), naevus, primary melanoma and melanoma metastasis. Lines in (A) and (B) demarcate the epidermal-dermal border (EDB) or epidermal surface (ES). C) In contrast to the epidermal melanocytes, some PAX3-positive melanocytes in the outer root sheath (ORS) co-express MCAM. D) Graph showing the overall number of PAX3, BCL2L1 double-labelled cells in normal skins, naevi, primary melanomas and melanoma metastases. Each column represents a percentage of PAX3-positive cells that are also BCL2L1-positive, averaged across all samples. E) Graph showing the overall number of PAX3, MCAM double-labelled cells in normal skins, naevi, primary melanomas and melanoma metastases. Each column represents a percentage of PAX3-positive cells that are also MCAM-positive, averaged across all samples.

    Article Snippet: PAX3-specific staining was also confirmed using a different commercially available anti-PAX3 antibody (Invitrogen) ( ).

    Techniques: Expressing, Migration, Staining

    Co-expression analysis of PAX3-positive follicular melanocytes. A) The transverse section of the hair follicle shows both PAX3 and HES1 co-expressing (enlarged in B), and single PAX3-expressing (enlarged in C), melanocytes in the outer root sheath (ORS). The line circumscribes the hair follicle. PAX3 was labelled with mouse monoclonal antibody (DSHB). D) The longitudinal section of the hair follicle shows PAX3 and MLANA co-expressing (enlarged in the insert on the left) and single PAX3-expressing (enlarged in the insert on the right) melanocytes in the outer root sheath (ORS). E) Single PAX3-expresing (arrows) and PAX3 and MLANA co-expressing (arrowheads) melanocytes in the matrix of the hair bulb. PAX3 was labelled with rabbit polyclonal antibody (Invitrogen).

    Journal: PLoS ONE

    Article Title: PAX3 Expression in Normal Skin Melanocytes and Melanocytic Lesions (Naevi and Melanomas)

    doi: 10.1371/journal.pone.0009977

    Figure Lengend Snippet: Co-expression analysis of PAX3-positive follicular melanocytes. A) The transverse section of the hair follicle shows both PAX3 and HES1 co-expressing (enlarged in B), and single PAX3-expressing (enlarged in C), melanocytes in the outer root sheath (ORS). The line circumscribes the hair follicle. PAX3 was labelled with mouse monoclonal antibody (DSHB). D) The longitudinal section of the hair follicle shows PAX3 and MLANA co-expressing (enlarged in the insert on the left) and single PAX3-expressing (enlarged in the insert on the right) melanocytes in the outer root sheath (ORS). E) Single PAX3-expresing (arrows) and PAX3 and MLANA co-expressing (arrowheads) melanocytes in the matrix of the hair bulb. PAX3 was labelled with rabbit polyclonal antibody (Invitrogen).

    Article Snippet: PAX3-specific staining was also confirmed using a different commercially available anti-PAX3 antibody (Invitrogen) ( ).

    Techniques: Expressing

    PAX3 expression in melanocytic and melanoma cells. A) Immunohistochemistry shows PAX3 expression (top panel) in representative samples of normal skin, naevus, primary melanoma and melanoma metastasis, compared to MITF expression in adjacent sections (bottom panel). B–D) Show the distribution of PAX3-positive melanocytes (arrowheads) in normal skin: along the hair follicle (early (B) and late (C) anagen); and in the epidermis (D). Arrows in (D) point to the cytoplasmic melanin deposit distinguishable from the nuclear PAX3 staining (arrowheads). Asterisk in (B) and (C) marks a dermal papilla. PAX3 was labelled with mouse monoclonal antibody (DSHB). E) PAX3 expression was analysed by RT-PCR and the graph shows the mean fold increase of PAX3 expression in naevi, primary melanomas and lymph node metastases normalised to the expression in normal skin.

    Journal: PLoS ONE

    Article Title: PAX3 Expression in Normal Skin Melanocytes and Melanocytic Lesions (Naevi and Melanomas)

    doi: 10.1371/journal.pone.0009977

    Figure Lengend Snippet: PAX3 expression in melanocytic and melanoma cells. A) Immunohistochemistry shows PAX3 expression (top panel) in representative samples of normal skin, naevus, primary melanoma and melanoma metastasis, compared to MITF expression in adjacent sections (bottom panel). B–D) Show the distribution of PAX3-positive melanocytes (arrowheads) in normal skin: along the hair follicle (early (B) and late (C) anagen); and in the epidermis (D). Arrows in (D) point to the cytoplasmic melanin deposit distinguishable from the nuclear PAX3 staining (arrowheads). Asterisk in (B) and (C) marks a dermal papilla. PAX3 was labelled with mouse monoclonal antibody (DSHB). E) PAX3 expression was analysed by RT-PCR and the graph shows the mean fold increase of PAX3 expression in naevi, primary melanomas and lymph node metastases normalised to the expression in normal skin.

    Article Snippet: PAX3-specific staining was also confirmed using a different commercially available anti-PAX3 antibody (Invitrogen) ( ).

    Techniques: Expressing, Immunohistochemistry, Staining, Reverse Transcription Polymerase Chain Reaction

    Co-expression of PAX3 and MITF in melanocytic and melanoma cells. A–C) Double immunofluorescent staining showing co-expression of PAX3 and MITF in: (A) the epidermal melanocytes of normal skin; (B) primary melanoma; and (C) in an A2058 metastatic melanoma cell line. Arrowheads in (A) show PAX3-positive normal epidermal melanocytes. Lines in (A) and (B) demarcate epidermal-dermal border (EDB). The variable PAX3 expression was clearly visible in the A2058 cell line (C). For all these experiments depicted in the figure, PAX3 was labelled with rabbit polyclonal antibody (Invitrogen). D) Graph showing the overall number of MITF and PAX3 double labelled cells in normal skins, naevi, primary melanomas and melanoma metastases. Each column represents a percentage of MITF-positive cells that are also PAX3-positive averaged across all samples. E) Double immunofluorescent staining shows PAX3 and MITF co-expressing melanocytes (yellow-orange) in the bulb of the hair follicle of normal skin. Note the single MITF-labelled melanocytes (green) at the base of the hair.

    Journal: PLoS ONE

    Article Title: PAX3 Expression in Normal Skin Melanocytes and Melanocytic Lesions (Naevi and Melanomas)

    doi: 10.1371/journal.pone.0009977

    Figure Lengend Snippet: Co-expression of PAX3 and MITF in melanocytic and melanoma cells. A–C) Double immunofluorescent staining showing co-expression of PAX3 and MITF in: (A) the epidermal melanocytes of normal skin; (B) primary melanoma; and (C) in an A2058 metastatic melanoma cell line. Arrowheads in (A) show PAX3-positive normal epidermal melanocytes. Lines in (A) and (B) demarcate epidermal-dermal border (EDB). The variable PAX3 expression was clearly visible in the A2058 cell line (C). For all these experiments depicted in the figure, PAX3 was labelled with rabbit polyclonal antibody (Invitrogen). D) Graph showing the overall number of MITF and PAX3 double labelled cells in normal skins, naevi, primary melanomas and melanoma metastases. Each column represents a percentage of MITF-positive cells that are also PAX3-positive averaged across all samples. E) Double immunofluorescent staining shows PAX3 and MITF co-expressing melanocytes (yellow-orange) in the bulb of the hair follicle of normal skin. Note the single MITF-labelled melanocytes (green) at the base of the hair.

    Article Snippet: PAX3-specific staining was also confirmed using a different commercially available anti-PAX3 antibody (Invitrogen) ( ).

    Techniques: Expressing, Staining

    Ex vivo correction of dystrophic iPS cells (a) Scheme of the ex vivo gene therapy approach, which involves (1) reprogramming of dystrophic donor-derived fibroblasts into iPS cells, (2) genetic repair of iPS cells with the μ-Utrn transgene using the Sleeping Beauty transposon system, (3) generation of myogenic progenitors from corrected iPS cells through Pax3 induction, and (4) transplantation of corrected myogenic precursors into dystrophic donor mice. (b) The Sleeping Beauty transposon system: the transposon contains the hEF1α-eIF4g promoter, the μUtrn gene, and an iresGFP. The whole transgene is flanked by the terminal inverted repeats (IR/DR, arrowheads) each containing two binding sites for the transposase (DR, yellow arrows). The transposase protein SB100X (red spheres) catalyzes integration of the transposon into the genome with high efficiency. (c) Left panel: Graphic bars represent percentage of GFP + cells obtained prior to each sorting. Green bars correspond to iPS cells nucleofected with pKT2/μUTRN-iresGFP/SB100X, while yellow bars represent controls, iPS cells transduced with pKT2/μUTRN-IresGFP only (no transposase). Data are mean ±S.E.M. of 3 independent samples. (d) FACS profile for GFP shows stable expression of μUTRN in corrected iPS cells (right panel), while control iPS cells are GFP − (left panel). (e) Flow cytometric analyses for Flk-1 and PDGFαR expression on day 5 EBs of Pax3-induced uncorrected- (upper panel) and corrected-iPS (lower panel) cells. The PDGFαR + Flk-1 − cell fraction was gated (red square on left panels) and analyzed for the expression of GFP, representing μUTRN + cells (middle panels), and mCherry, representing Pax3 + cells (right panels). (f) Phase contrast images of monolayers under proliferation (left) and differentiation (right) culture conditions. (g) Immunofluorescence staining for μUTRN (green) in proliferating iPS-derived myogenic progenitors (left) and their derivative myotubes (right). Cells are co-stained with DAPI (blue). Scale bar is 200μm. (h) qPCR analyses indicate relative expression of μ-utrophin in corrected iPS-derived myogenic cells under proliferation (P) and differentiation (D) conditions. Actin was used as house-keeping gene. Error bars represent S.E.M. from three replicates of three independent experiments. (i) Immunoblotting with anti-UTRN and anti-FLAG antibodies confirm the presence of 163-kDa μUTRN only in corrected cells.

    Journal: Nature communications

    Article Title: An ex vivo Gene Therapy Approach to Treat Muscular Dystrophy Using inducible Pluripotent Stem Cells

    doi: 10.1038/ncomms2550

    Figure Lengend Snippet: Ex vivo correction of dystrophic iPS cells (a) Scheme of the ex vivo gene therapy approach, which involves (1) reprogramming of dystrophic donor-derived fibroblasts into iPS cells, (2) genetic repair of iPS cells with the μ-Utrn transgene using the Sleeping Beauty transposon system, (3) generation of myogenic progenitors from corrected iPS cells through Pax3 induction, and (4) transplantation of corrected myogenic precursors into dystrophic donor mice. (b) The Sleeping Beauty transposon system: the transposon contains the hEF1α-eIF4g promoter, the μUtrn gene, and an iresGFP. The whole transgene is flanked by the terminal inverted repeats (IR/DR, arrowheads) each containing two binding sites for the transposase (DR, yellow arrows). The transposase protein SB100X (red spheres) catalyzes integration of the transposon into the genome with high efficiency. (c) Left panel: Graphic bars represent percentage of GFP + cells obtained prior to each sorting. Green bars correspond to iPS cells nucleofected with pKT2/μUTRN-iresGFP/SB100X, while yellow bars represent controls, iPS cells transduced with pKT2/μUTRN-IresGFP only (no transposase). Data are mean ±S.E.M. of 3 independent samples. (d) FACS profile for GFP shows stable expression of μUTRN in corrected iPS cells (right panel), while control iPS cells are GFP − (left panel). (e) Flow cytometric analyses for Flk-1 and PDGFαR expression on day 5 EBs of Pax3-induced uncorrected- (upper panel) and corrected-iPS (lower panel) cells. The PDGFαR + Flk-1 − cell fraction was gated (red square on left panels) and analyzed for the expression of GFP, representing μUTRN + cells (middle panels), and mCherry, representing Pax3 + cells (right panels). (f) Phase contrast images of monolayers under proliferation (left) and differentiation (right) culture conditions. (g) Immunofluorescence staining for μUTRN (green) in proliferating iPS-derived myogenic progenitors (left) and their derivative myotubes (right). Cells are co-stained with DAPI (blue). Scale bar is 200μm. (h) qPCR analyses indicate relative expression of μ-utrophin in corrected iPS-derived myogenic cells under proliferation (P) and differentiation (D) conditions. Actin was used as house-keeping gene. Error bars represent S.E.M. from three replicates of three independent experiments. (i) Immunoblotting with anti-UTRN and anti-FLAG antibodies confirm the presence of 163-kDa μUTRN only in corrected cells.

    Article Snippet: The following primary antibodies were used: anti-Pax3 (1:100; R & D Systems), anti-Myf5 (1:100; C-20 Santa Cruz), anti-MyoD (1:100; Pharmingen), and anti-MyHC (1:50; M20, Developmental Studies Hybridoma Bank).

    Techniques: Ex Vivo, Derivative Assay, Transplantation Assay, Mouse Assay, Binding Assay, Transduction, FACS, Expressing, Flow Cytometry, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction

    3F- and 4F-iPS cells can be differentiated into NCSC and MELSCs. (A) Gene expression analysis of a neural crest stem cell marker (Slug), a melanocyte stem cell marker (PAX3), a melanocytic marker (MITF), pluripotency marker (NANOG) in 3F-iPS cells, differentiated cells derived from EBs on fibronectin-coated plates in melanocyte differentiation medium at 7 days after differentiation (EB-DIFF), and 3F-iPS derived melanocytes (3F-iPS MEL). Transcript levels were normalized to GAPDH. The graphs show the average of two independent experiments. Error bar indicates mean±S.E.M. (B) Representative flow cytometry results for HNK-1 and p75 staining in 3F-iPS EB-derived cells cultured in melanocyte differentiation medium for one week. Dead cells were excluded using PI staining. (C) Immunocytochemistry revealing cells positive for NCSC markers (p75, HNK-1, and AP2α) in both 3F- and 4F-iPS cells derived EB-differentiated cells 7 days after differentiation. Scale bar, 50 µm. (D) Images show cells positive for neural crest cell marker SOX10, as well as possible melanocyte stem cell marker, PAX3 in both 3F- and 4F-iPS cell-derived EB-differentiated cells 7 days after differentiation. Scale bar, 50 µm and 10 µm (insert images).

    Journal: PLoS ONE

    Article Title: Generation of Human Melanocytes from Induced Pluripotent Stem Cells

    doi: 10.1371/journal.pone.0016182

    Figure Lengend Snippet: 3F- and 4F-iPS cells can be differentiated into NCSC and MELSCs. (A) Gene expression analysis of a neural crest stem cell marker (Slug), a melanocyte stem cell marker (PAX3), a melanocytic marker (MITF), pluripotency marker (NANOG) in 3F-iPS cells, differentiated cells derived from EBs on fibronectin-coated plates in melanocyte differentiation medium at 7 days after differentiation (EB-DIFF), and 3F-iPS derived melanocytes (3F-iPS MEL). Transcript levels were normalized to GAPDH. The graphs show the average of two independent experiments. Error bar indicates mean±S.E.M. (B) Representative flow cytometry results for HNK-1 and p75 staining in 3F-iPS EB-derived cells cultured in melanocyte differentiation medium for one week. Dead cells were excluded using PI staining. (C) Immunocytochemistry revealing cells positive for NCSC markers (p75, HNK-1, and AP2α) in both 3F- and 4F-iPS cells derived EB-differentiated cells 7 days after differentiation. Scale bar, 50 µm. (D) Images show cells positive for neural crest cell marker SOX10, as well as possible melanocyte stem cell marker, PAX3 in both 3F- and 4F-iPS cell-derived EB-differentiated cells 7 days after differentiation. Scale bar, 50 µm and 10 µm (insert images).

    Article Snippet: Then, cells were subjected to immunofluorescence staining using the following primary antibodies: α-fetoprotein (AFP) (1∶500, DAKO, Kyoto, Japan), α-smooth muscle actin (SMA) (1∶100, Sigma), AP2α (1∶50, Cell Signaling Technology, Beverly, MA), β-III-tubulin (1∶1000, Sigma), microphthalmia-associated transcription factor (MITF) (1∶50, Thermo Scientific, Rockford, IL), HNK-1 (1∶200, Millipore, Billerica, MA), NANOG (1∶1000, Reprocell, Tokyo, Japan), OCT3/4 (1∶200, BD Bioscience, San Diego, CA), p75 (1∶100, Advanced Targeting Systems), PAX3 (1∶100, R & D Systems), S100 (1∶500, DAKO), silver protein (SILV) (1∶50, DAKO), SOX10 (1∶100, Abcam), SSEA-4 (1∶200, Millipore), TRA-1-60 (1∶200, Millipore), TRA-1-81 (1∶200, Millipore), tyrosinase (TYR) (1∶100, Santa Cruz Biothechnology, Inc., Santa Cruz, CA), tyrosinase-related protein-1 (TYRP1) (1∶20, COVANCE, Princeton, NJ).

    Techniques: Expressing, Marker, Derivative Assay, Flow Cytometry, Cytometry, Staining, Cell Culture, Immunocytochemistry

    Neuroprogenitor migration in Ntn1 , Dcc , and Robo3 knockouts. a Schematic of the migration of the dorsal spinal cord progenitors and interneurons. VZ, ventricular zone. b Cross sections of the spinal cord electroporated with Actb-gfp ( βactin-gfp) . The closeup images are of the boxed area. The embryos were cultured for 20 h. GFP+ neurons from all three KOs migrate out of the VZ (demarcated by PAX3/7 staining) later than WT neurons. c Quantification of the ratio between GFP+ neurons within the VZ and the total GFP+ neurons. A higher percentage of neurons is seen within the VZ in all three KOs. Data are represented as the mean ± SEM (Student’s t -test; **, p

    Journal: Neural Development

    Article Title: Netrin1/DCC signaling promotes neuronal migration in the dorsal spinal cord

    doi: 10.1186/s13064-016-0074-x

    Figure Lengend Snippet: Neuroprogenitor migration in Ntn1 , Dcc , and Robo3 knockouts. a Schematic of the migration of the dorsal spinal cord progenitors and interneurons. VZ, ventricular zone. b Cross sections of the spinal cord electroporated with Actb-gfp ( βactin-gfp) . The closeup images are of the boxed area. The embryos were cultured for 20 h. GFP+ neurons from all three KOs migrate out of the VZ (demarcated by PAX3/7 staining) later than WT neurons. c Quantification of the ratio between GFP+ neurons within the VZ and the total GFP+ neurons. A higher percentage of neurons is seen within the VZ in all three KOs. Data are represented as the mean ± SEM (Student’s t -test; **, p

    Article Snippet: Antibodies used in the study include anti-PAX3/7 (PA1-107, Thermo Fisher, raised against PAX3 and cross reacts with PAX7), anti-BARHL2 (NBP2-32013, Novus Biologicals), anti-LHX5 (AF6290, R & D), anti-ISL1/2 (39.4D5, DSHB), anti-PAX6 (DSHB), anti-phospho-Histone H3 (9701, CST), anti-Ki67 (12202, CST), and anti-SOX2 (3728, CST).

    Techniques: Migration, Droplet Countercurrent Chromatography, Cell Culture, Staining

    Neuroprogenitors are generated normally in Ntn1 , Dcc , and Robo3 knockouts. a Immunohistochemistry of phospho-Histone H3, a mitotic marker, Ki67, a cell proliferation marker, SOX2, a neuroprogenitor marker, and PAX3/7, a dorsal progenitor marker in E10.5 spinal cord. b Quantification of phenotypes in (a). Data are normalized to WT and are represented as the mean ± SEM (Student’s t -test; ns, not significant). Scale bar, 50 μm

    Journal: Neural Development

    Article Title: Netrin1/DCC signaling promotes neuronal migration in the dorsal spinal cord

    doi: 10.1186/s13064-016-0074-x

    Figure Lengend Snippet: Neuroprogenitors are generated normally in Ntn1 , Dcc , and Robo3 knockouts. a Immunohistochemistry of phospho-Histone H3, a mitotic marker, Ki67, a cell proliferation marker, SOX2, a neuroprogenitor marker, and PAX3/7, a dorsal progenitor marker in E10.5 spinal cord. b Quantification of phenotypes in (a). Data are normalized to WT and are represented as the mean ± SEM (Student’s t -test; ns, not significant). Scale bar, 50 μm

    Article Snippet: Antibodies used in the study include anti-PAX3/7 (PA1-107, Thermo Fisher, raised against PAX3 and cross reacts with PAX7), anti-BARHL2 (NBP2-32013, Novus Biologicals), anti-LHX5 (AF6290, R & D), anti-ISL1/2 (39.4D5, DSHB), anti-PAX6 (DSHB), anti-phospho-Histone H3 (9701, CST), anti-Ki67 (12202, CST), and anti-SOX2 (3728, CST).

    Techniques: Generated, Droplet Countercurrent Chromatography, Immunohistochemistry, Marker

    Nkx3.2, but not Sox9, inhibits the expression of Pax3 and Pax7 in ovo A. Ectopic expression of Nkx3.2 in the dorsal somite cells. In situ hybridization (ISH) analysis of chicken embryos electroporated with pMES-Nkx3.2-GFP into the lumen of the newly formed somites two days after electroporation, using an Nkx3.2 RNA probe. 5 embryos were analyzed and all exhibited ectopic Nkx3.2 in the dorsal somitic cells. Left panel: whole mount ISH; right panel: a cross section of the whole mount ISH embryo. Arrows: exogenous Nkx3.2 expression. Arrowheads: endogenous Nkx3.2 expression. B. Ectopic expression of Sox9 in the dorsal somite cells. Immunocytochemistry analysis on sectioned embryos electroporated with pMES-Sox9-GFP two days after electroporation. 5 sections were analyzed and all cells that expressed GFP also expressed Sox9V5. GFP-positive Panel 1, V5 staining showing Sox9V5 expression in dorsal somite cells. A bright field view of the section was overlaid on the fluorescent image. Panel 2, magnified view of Sox9V5-positive cells. Panel 3, magnified view of GFP-positive cells. Panel 4, merged image (yellow) of Sox9V5 and GFP. C. Nkx3.2, but not Sox9, inhibited Pax3 and Pax7 expression. Embryos electroporated with either pMES-GFP (vector), pMES-Nkx3.2-GFP or pMES-Sox9V5-GFP into the lumen of the newly formed somites were whole mount fixed and serial sectioned. The sections were immunostained with antibodies against Pax3 and Pax7. Cells harboring the introduced DNA are GFP-positive, and thus are green. Only overlaid images are shown. Most of the cells that expressed the introduced plasmids were laterally located. However, the few medially-targeted cells exhibit the same phenotype as these laterally located cells. Panels 1-4, GFP electroporated embryos (Panels 1-2 are 10X views, and Panels 3-4 are 40X views). Panels 5-8, Nkx3.2 electroporated embryos (Panels 5-6 are 10X views, and Panels 7-8 are 40X views). Panels 9-12, Sox9V5 electroporated embryos (Panels 9-10 are 10X views, panels 11-12 are 40X views). Arrows: cells that express introduced DNA (GFP-positive), but are not Pax3 or Pax7 -positive (red). Arrowheads: cells that express introduced DNA (GFP-positive), and are also Pax3 or Pax7-positive (red), and are thus yellow. D. Quantification of the results of Fig.5C. Percentage of electroporated cells that express Pax3 and Pax7 was quantified. For each electroporated sample, a total number of 100-200 targeted cells from at least 3 embryos were analyzed under the confocal microscope. Only the target cells (GFP-positive) that were located in the normal Pax3 and Pax7 expression domains were used for counting. Pax3, blue. Pax7, orange. E. Nkx3.2 induced the expression of Sox9 in the dorsal somite cells. Embryos electroporated with pMES-Nkx3.2-GFP into the lumen of the newly formed somites were subjected to whole mount in situ hybridization and sectioning, using a Sox9 RNA probe. 5 embryos were analyzed. Panel 1, in situ hybridization of Sox9. Arrowhead: exogenous Sox9 expression. Arrow: endogenous Sox9 expression. A bright field view of the section was overlaid on the fluorescent image. Panel 2, Anti-GFP staining indicating the ectopic expression of the introduced DNA pMES-Nkx3.2-GFP (arrow). Panel 3, Overlay of the bright field ISH image with the GFP immunostaining image (arrow). Panel 4, merged bright field image of ISH and immunofluorescent staining of endogenous Pax3, showing ectopic Sox9 expression within the Pax3-expressing domain.

    Journal: Developmental biology

    Article Title: A gradient of Shh establishes mutually repressing somitic cell fates induced by Nkx3.2 and Pax3

    doi: 10.1016/j.ydbio.2008.08.024

    Figure Lengend Snippet: Nkx3.2, but not Sox9, inhibits the expression of Pax3 and Pax7 in ovo A. Ectopic expression of Nkx3.2 in the dorsal somite cells. In situ hybridization (ISH) analysis of chicken embryos electroporated with pMES-Nkx3.2-GFP into the lumen of the newly formed somites two days after electroporation, using an Nkx3.2 RNA probe. 5 embryos were analyzed and all exhibited ectopic Nkx3.2 in the dorsal somitic cells. Left panel: whole mount ISH; right panel: a cross section of the whole mount ISH embryo. Arrows: exogenous Nkx3.2 expression. Arrowheads: endogenous Nkx3.2 expression. B. Ectopic expression of Sox9 in the dorsal somite cells. Immunocytochemistry analysis on sectioned embryos electroporated with pMES-Sox9-GFP two days after electroporation. 5 sections were analyzed and all cells that expressed GFP also expressed Sox9V5. GFP-positive Panel 1, V5 staining showing Sox9V5 expression in dorsal somite cells. A bright field view of the section was overlaid on the fluorescent image. Panel 2, magnified view of Sox9V5-positive cells. Panel 3, magnified view of GFP-positive cells. Panel 4, merged image (yellow) of Sox9V5 and GFP. C. Nkx3.2, but not Sox9, inhibited Pax3 and Pax7 expression. Embryos electroporated with either pMES-GFP (vector), pMES-Nkx3.2-GFP or pMES-Sox9V5-GFP into the lumen of the newly formed somites were whole mount fixed and serial sectioned. The sections were immunostained with antibodies against Pax3 and Pax7. Cells harboring the introduced DNA are GFP-positive, and thus are green. Only overlaid images are shown. Most of the cells that expressed the introduced plasmids were laterally located. However, the few medially-targeted cells exhibit the same phenotype as these laterally located cells. Panels 1-4, GFP electroporated embryos (Panels 1-2 are 10X views, and Panels 3-4 are 40X views). Panels 5-8, Nkx3.2 electroporated embryos (Panels 5-6 are 10X views, and Panels 7-8 are 40X views). Panels 9-12, Sox9V5 electroporated embryos (Panels 9-10 are 10X views, panels 11-12 are 40X views). Arrows: cells that express introduced DNA (GFP-positive), but are not Pax3 or Pax7 -positive (red). Arrowheads: cells that express introduced DNA (GFP-positive), and are also Pax3 or Pax7-positive (red), and are thus yellow. D. Quantification of the results of Fig.5C. Percentage of electroporated cells that express Pax3 and Pax7 was quantified. For each electroporated sample, a total number of 100-200 targeted cells from at least 3 embryos were analyzed under the confocal microscope. Only the target cells (GFP-positive) that were located in the normal Pax3 and Pax7 expression domains were used for counting. Pax3, blue. Pax7, orange. E. Nkx3.2 induced the expression of Sox9 in the dorsal somite cells. Embryos electroporated with pMES-Nkx3.2-GFP into the lumen of the newly formed somites were subjected to whole mount in situ hybridization and sectioning, using a Sox9 RNA probe. 5 embryos were analyzed. Panel 1, in situ hybridization of Sox9. Arrowhead: exogenous Sox9 expression. Arrow: endogenous Sox9 expression. A bright field view of the section was overlaid on the fluorescent image. Panel 2, Anti-GFP staining indicating the ectopic expression of the introduced DNA pMES-Nkx3.2-GFP (arrow). Panel 3, Overlay of the bright field ISH image with the GFP immunostaining image (arrow). Panel 4, merged bright field image of ISH and immunofluorescent staining of endogenous Pax3, showing ectopic Sox9 expression within the Pax3-expressing domain.

    Article Snippet: Mouse anti-Pax3, anti-Pax7 and anti-Myosin antibodies were purchased from Developmental Studies Hybridoma Bank.

    Techniques: Expressing, In Ovo, In Situ Hybridization, Electroporation, Immunocytochemistry, Staining, Plasmid Preparation, Microscopy, Immunostaining

    A gradient of Shh patterns the somite into mutually repressing muscle and cartilage cell fates Low levels of Shh promote the maintenance of Pax3 expression induced by Wnt signals. Medium levels of Shh, along with Wnt signals, promote the expression of the myotomal factors Myf5 and MyoD. High levels of Shh promote the expression of the sclerotomal factors Nkx3.2, Sox9 and Pax1, and simultaneously repress expression of both dermomyotomal and myotomal markers. Mutual repression by Pax3 and Nkx3.2 ensures that cells adopt either a dermomyotomal or sclerotomal cell fate. While Pax3 induces the expression of MyoD/Myf5 and promotes somitic myogenesis, Nkx3.2 and Sox9 promote somitic chondrogenesis.

    Journal: Developmental biology

    Article Title: A gradient of Shh establishes mutually repressing somitic cell fates induced by Nkx3.2 and Pax3

    doi: 10.1016/j.ydbio.2008.08.024

    Figure Lengend Snippet: A gradient of Shh patterns the somite into mutually repressing muscle and cartilage cell fates Low levels of Shh promote the maintenance of Pax3 expression induced by Wnt signals. Medium levels of Shh, along with Wnt signals, promote the expression of the myotomal factors Myf5 and MyoD. High levels of Shh promote the expression of the sclerotomal factors Nkx3.2, Sox9 and Pax1, and simultaneously repress expression of both dermomyotomal and myotomal markers. Mutual repression by Pax3 and Nkx3.2 ensures that cells adopt either a dermomyotomal or sclerotomal cell fate. While Pax3 induces the expression of MyoD/Myf5 and promotes somitic myogenesis, Nkx3.2 and Sox9 promote somitic chondrogenesis.

    Article Snippet: Mouse anti-Pax3, anti-Pax7 and anti-Myosin antibodies were purchased from Developmental Studies Hybridoma Bank.

    Techniques: Expressing

    In situ hybridization analysis of Pax3, Nkx3.2 and Sox9 A. Whole mount in situ hybridization analysis of Pax3, Nkx3.2 and Sox9 on 12 somite (stage 10) chick embryos. Arrows mark the caudal to rostral levels of the somitic mesoderm. a, presomitic mesoderm (Psm). b, somite I. c, somite III. d, somite V. B. Sections of Pax3, Nkx3.2 and Sox9 ISH embryos at various axial levels. Panels 1-4, Pax3 expression. Panels 5-8, Nkx3.2 expression. Panels 9-12, Sox9 expression. Sections at the presomitic mesoderm level are shown in panels 4, 8 and 12. Sections at somite I level are shown in panels 3, 7 and 11. Sections at somite III level are shown in panels 2, 6 and 7. Sections at somite V level are shown in panels 1, 5 and 9.

    Journal: Developmental biology

    Article Title: A gradient of Shh establishes mutually repressing somitic cell fates induced by Nkx3.2 and Pax3

    doi: 10.1016/j.ydbio.2008.08.024

    Figure Lengend Snippet: In situ hybridization analysis of Pax3, Nkx3.2 and Sox9 A. Whole mount in situ hybridization analysis of Pax3, Nkx3.2 and Sox9 on 12 somite (stage 10) chick embryos. Arrows mark the caudal to rostral levels of the somitic mesoderm. a, presomitic mesoderm (Psm). b, somite I. c, somite III. d, somite V. B. Sections of Pax3, Nkx3.2 and Sox9 ISH embryos at various axial levels. Panels 1-4, Pax3 expression. Panels 5-8, Nkx3.2 expression. Panels 9-12, Sox9 expression. Sections at the presomitic mesoderm level are shown in panels 4, 8 and 12. Sections at somite I level are shown in panels 3, 7 and 11. Sections at somite III level are shown in panels 2, 6 and 7. Sections at somite V level are shown in panels 1, 5 and 9.

    Article Snippet: Mouse anti-Pax3, anti-Pax7 and anti-Myosin antibodies were purchased from Developmental Studies Hybridoma Bank.

    Techniques: In Situ Hybridization, Expressing

    Nkx3.2 and Sox9 inhibit Wnt3a-induced Pax3 expression in somite explants A. Somite explants immunostained for Pax3 expression following infection with retroviral encoded Nkx3.2HA, Sox9V5 or GFP. Somite IV-VI explants of stage 10 chicken embryos were cultured in Wnt3a conditioned medium for 5 days. Panels 1-4, RCAS-B-GFP infected explant, arrows indicating a cell that expressed GFP as well as Pax3. Panels 5-8, RCAS-B-Nkx3.2HA infected explant. Panels 9-12, RCAS-B-Sox9V5 infected explant, arrows indicating a cell that expressed Sox9V5 as well as Pax3. The inset within each panel shows a low powered view of each explant. Green: GFP, Nkx3.2HA and Sox9V5. Red, Pax3. Overlay, merged view of virus-expression (GFP, Nkx3.2 and Sox9) and Pax3. Dapi images are not overlaid with virus and Pax3 images so that yellow overlapping expression is more evident. No significant changes in cell numbers in these explants were observed. B. Quantification of the results of Fig. 3A. Percentage of virus-infected cells that express Pax3 was quantified. For each virus-infected sample, a total number of 500-1000 virus-infected cells from at least 5 different views were analyzed under the microscope. Standard deviations are shown. C. Somite explants immunostained for Collagen II expression following infection with retroviral encoded Nkx3.2HA, Sox9V5 or GFP. Somite explants were cultured under the same condition as described in Fig. 3A. Panels 1-4, RCAS-B-GFP infected explant. Panels 5-8, RCAS-B-Nkx3.2HA infected explant. Panels 9-12, RCAS-B-Sox9V5 infected explant. Green: GFP, Nkx3.2HA and Sox9V5. Red, Collagen II. Overlay, merged view of virus-expression (GFP, Nkx3.2 and Sox9), Collagen II and Dapi. The inset within each panel shows a low powered view of each explant. D . Nkx3.2 virus-infected cells do not adopt a cartilage fate in the presence of Wnt3a, but do express collagen II in the presence of exogenous BMP4. Somites IV-VI of stage 10 chicken embryos were cultured in either Wnt3a-conditioned medium (panels 1-4), Wnt3a-conditioned medium plus 100ng/ml BMP4 protein (panels 5-8), or control L-cell conditioned medium plus 100ng/ml BMP4 protein (panels 9-12). The explants were cultured altogether for 6 days before immunostaining. Green: GFP, Nkx3.2HA and Sox9V5. Red, Collagen II. Overlay, merged view of virus-expression (GFP, Nkx3.2 and Sox9), Collagen II and Dapi. The inset within each panel shows a low powered view of each explant.

    Journal: Developmental biology

    Article Title: A gradient of Shh establishes mutually repressing somitic cell fates induced by Nkx3.2 and Pax3

    doi: 10.1016/j.ydbio.2008.08.024

    Figure Lengend Snippet: Nkx3.2 and Sox9 inhibit Wnt3a-induced Pax3 expression in somite explants A. Somite explants immunostained for Pax3 expression following infection with retroviral encoded Nkx3.2HA, Sox9V5 or GFP. Somite IV-VI explants of stage 10 chicken embryos were cultured in Wnt3a conditioned medium for 5 days. Panels 1-4, RCAS-B-GFP infected explant, arrows indicating a cell that expressed GFP as well as Pax3. Panels 5-8, RCAS-B-Nkx3.2HA infected explant. Panels 9-12, RCAS-B-Sox9V5 infected explant, arrows indicating a cell that expressed Sox9V5 as well as Pax3. The inset within each panel shows a low powered view of each explant. Green: GFP, Nkx3.2HA and Sox9V5. Red, Pax3. Overlay, merged view of virus-expression (GFP, Nkx3.2 and Sox9) and Pax3. Dapi images are not overlaid with virus and Pax3 images so that yellow overlapping expression is more evident. No significant changes in cell numbers in these explants were observed. B. Quantification of the results of Fig. 3A. Percentage of virus-infected cells that express Pax3 was quantified. For each virus-infected sample, a total number of 500-1000 virus-infected cells from at least 5 different views were analyzed under the microscope. Standard deviations are shown. C. Somite explants immunostained for Collagen II expression following infection with retroviral encoded Nkx3.2HA, Sox9V5 or GFP. Somite explants were cultured under the same condition as described in Fig. 3A. Panels 1-4, RCAS-B-GFP infected explant. Panels 5-8, RCAS-B-Nkx3.2HA infected explant. Panels 9-12, RCAS-B-Sox9V5 infected explant. Green: GFP, Nkx3.2HA and Sox9V5. Red, Collagen II. Overlay, merged view of virus-expression (GFP, Nkx3.2 and Sox9), Collagen II and Dapi. The inset within each panel shows a low powered view of each explant. D . Nkx3.2 virus-infected cells do not adopt a cartilage fate in the presence of Wnt3a, but do express collagen II in the presence of exogenous BMP4. Somites IV-VI of stage 10 chicken embryos were cultured in either Wnt3a-conditioned medium (panels 1-4), Wnt3a-conditioned medium plus 100ng/ml BMP4 protein (panels 5-8), or control L-cell conditioned medium plus 100ng/ml BMP4 protein (panels 9-12). The explants were cultured altogether for 6 days before immunostaining. Green: GFP, Nkx3.2HA and Sox9V5. Red, Collagen II. Overlay, merged view of virus-expression (GFP, Nkx3.2 and Sox9), Collagen II and Dapi. The inset within each panel shows a low powered view of each explant.

    Article Snippet: Mouse anti-Pax3, anti-Pax7 and anti-Myosin antibodies were purchased from Developmental Studies Hybridoma Bank.

    Techniques: Expressing, Infection, Cell Culture, Microscopy, Immunostaining

    Effects of Nkx3.2 and Sox9 on surface-ectoderm induced Pax3 expression A. Pax3 expression in somite/ectoderm explants infected with GFP, Nkx3.2HA and Sox9V5. Stage 10 chicken embryo explants of somite IV-VI with surface ectoderm attached were cultured for 5 days. Panels 1-4, RCAS-B-GFP infected explant. Panels 5-8, RCAS-B-Nkx3.2HA infected explant. Panels 9-12, RCAS-B-Sox9V5 infected explant. The inset within each panel shows a low powered view of each explant. Arrows indicating cells that expressed GFP or Sox9V5 as well as Pax3. The inset within each panel shows a low powered view of each explant. Green: GFP, Nkx3.2HA and Sox9V5. Red, Pax3. Overlay, merged view of virus-expression (GFP, Nkx3.2 and Sox9) and Pax3. Dapi images are not overlaid with virus and Pax3 images so that yellow overlapping expression is more evident. No significant changes in cell numbers in these explants were observed. B. Quantification of the results of Fig.4A. Percentage of virus infected cells that express Pax3 was quantified. For each virus-infected sample, a total number of 500-1000 virus-infected cells from 5 different views were analyzed under the microscope. C. Somite/ectoderm explants immunostained for Collagen II expression following infection with retroviral encoded Nkx3.2HA, Sox9V5 or GFP. Somite explants were cultured under the same condition as described in Fig. 4A. Panels 1-4, RCAS-B-GFP infected explant. Panels 5-8, RCAS-B-Nkx3.2HA infected explant. Arrow, an Nkx3.2HA-expressing cell that also expresses a low level of Collagen II. Panels 9-12, RCAS-B-Sox9V5 infected explant. Green: GFP, Nkx3.2HA and Sox9V5. Red, Collagen II. Overlay, merged view of virus-expression (GFP, Nkx3.2 and Sox9), Collagen II and Dapi. The inset within each panel shows a low powered view of each explant. D . Nkx3.2 virus-infected cells adopt a cartilage fate in the presence of exogenous BMP4. Stage 10 chicken embryo explants of somite IV-VI with surface ectoderm attached were cultured for 6 days in either control medium (panels 1-4) or in medium supplemented with 100ng/ml BMP4 protein (panels 5-8). The inset within each panel shows a low powered view of each explant. Nkx3.2HA, green. Collagen II, red. Dapi, blue. Overlay, merged image of Nkx3.2HA, Collagen II and Dapi.

    Journal: Developmental biology

    Article Title: A gradient of Shh establishes mutually repressing somitic cell fates induced by Nkx3.2 and Pax3

    doi: 10.1016/j.ydbio.2008.08.024

    Figure Lengend Snippet: Effects of Nkx3.2 and Sox9 on surface-ectoderm induced Pax3 expression A. Pax3 expression in somite/ectoderm explants infected with GFP, Nkx3.2HA and Sox9V5. Stage 10 chicken embryo explants of somite IV-VI with surface ectoderm attached were cultured for 5 days. Panels 1-4, RCAS-B-GFP infected explant. Panels 5-8, RCAS-B-Nkx3.2HA infected explant. Panels 9-12, RCAS-B-Sox9V5 infected explant. The inset within each panel shows a low powered view of each explant. Arrows indicating cells that expressed GFP or Sox9V5 as well as Pax3. The inset within each panel shows a low powered view of each explant. Green: GFP, Nkx3.2HA and Sox9V5. Red, Pax3. Overlay, merged view of virus-expression (GFP, Nkx3.2 and Sox9) and Pax3. Dapi images are not overlaid with virus and Pax3 images so that yellow overlapping expression is more evident. No significant changes in cell numbers in these explants were observed. B. Quantification of the results of Fig.4A. Percentage of virus infected cells that express Pax3 was quantified. For each virus-infected sample, a total number of 500-1000 virus-infected cells from 5 different views were analyzed under the microscope. C. Somite/ectoderm explants immunostained for Collagen II expression following infection with retroviral encoded Nkx3.2HA, Sox9V5 or GFP. Somite explants were cultured under the same condition as described in Fig. 4A. Panels 1-4, RCAS-B-GFP infected explant. Panels 5-8, RCAS-B-Nkx3.2HA infected explant. Arrow, an Nkx3.2HA-expressing cell that also expresses a low level of Collagen II. Panels 9-12, RCAS-B-Sox9V5 infected explant. Green: GFP, Nkx3.2HA and Sox9V5. Red, Collagen II. Overlay, merged view of virus-expression (GFP, Nkx3.2 and Sox9), Collagen II and Dapi. The inset within each panel shows a low powered view of each explant. D . Nkx3.2 virus-infected cells adopt a cartilage fate in the presence of exogenous BMP4. Stage 10 chicken embryo explants of somite IV-VI with surface ectoderm attached were cultured for 6 days in either control medium (panels 1-4) or in medium supplemented with 100ng/ml BMP4 protein (panels 5-8). The inset within each panel shows a low powered view of each explant. Nkx3.2HA, green. Collagen II, red. Dapi, blue. Overlay, merged image of Nkx3.2HA, Collagen II and Dapi.

    Article Snippet: Mouse anti-Pax3, anti-Pax7 and anti-Myosin antibodies were purchased from Developmental Studies Hybridoma Bank.

    Techniques: Expressing, Infection, Cell Culture, Microscopy

    High level of Wnt signaling and Pax3 expression inhibit Nkx3.2 expression A. High level of Wnt signaling inhibits Nkx3.2 expression and promotes dorsal somite cell fates. RatB1a cells secreting Wnt1 were co-cultured with presomitic mesoderm explants of stage 10 chicken embryos in the presence or absence of N-SHH. Explants were harvested after 5 days culture for RT-PCR analysis of indicated genes. Lane 1, control RatB1a cells co-cultured with presomitic mesoderm explants. Lane 2, Wnt1-secreting RatB1a cells co-cultured with presomitic mesoderm explants. Lane 3, control RatB1a cells co-cultured with presomitic mesoderm explants in the presence of 250ng/ml N-SHH. Lane 4, Wnt1-secreting RatB1a cells co-cultured with presomitic mesoderm explants in the presence of 250ng/ml N-SHH. B. Forced expression of Pax3 inhibits the expression of Nkx3.2 and chondrogenic markers induced by N-SHH. Presomitic mesoderm explants were infected with avian retroviruses encoding either Pax3 or Alkaline phosphatase (AP, control). 500ng/ml N-SHH was present in all cultures. Explants were harvested after 5 days culture for RT-PCR analysis of indicated genes. Lanes 1 and 3, RCAS-A-AP infected explants. Lanes 2 and 4, RCAS-A-Pax3 infected explants.

    Journal: Developmental biology

    Article Title: A gradient of Shh establishes mutually repressing somitic cell fates induced by Nkx3.2 and Pax3

    doi: 10.1016/j.ydbio.2008.08.024

    Figure Lengend Snippet: High level of Wnt signaling and Pax3 expression inhibit Nkx3.2 expression A. High level of Wnt signaling inhibits Nkx3.2 expression and promotes dorsal somite cell fates. RatB1a cells secreting Wnt1 were co-cultured with presomitic mesoderm explants of stage 10 chicken embryos in the presence or absence of N-SHH. Explants were harvested after 5 days culture for RT-PCR analysis of indicated genes. Lane 1, control RatB1a cells co-cultured with presomitic mesoderm explants. Lane 2, Wnt1-secreting RatB1a cells co-cultured with presomitic mesoderm explants. Lane 3, control RatB1a cells co-cultured with presomitic mesoderm explants in the presence of 250ng/ml N-SHH. Lane 4, Wnt1-secreting RatB1a cells co-cultured with presomitic mesoderm explants in the presence of 250ng/ml N-SHH. B. Forced expression of Pax3 inhibits the expression of Nkx3.2 and chondrogenic markers induced by N-SHH. Presomitic mesoderm explants were infected with avian retroviruses encoding either Pax3 or Alkaline phosphatase (AP, control). 500ng/ml N-SHH was present in all cultures. Explants were harvested after 5 days culture for RT-PCR analysis of indicated genes. Lanes 1 and 3, RCAS-A-AP infected explants. Lanes 2 and 4, RCAS-A-Pax3 infected explants.

    Article Snippet: Mouse anti-Pax3, anti-Pax7 and anti-Myosin antibodies were purchased from Developmental Studies Hybridoma Bank.

    Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Infection

    Shh concentration gradient patterns dorsal-ventral fates of the somite A. In the presence of constant levels of Wnt3a signals, differing levels of Shh are necessary to induce either dorsal or ventral somite markers. Presomitic mesoderm explants were cultured for five days in Wnt3a-conditioned medium and increasing amounts of N-SHH. Lane 1: no N-SHH. Lane 2: 34ng/ml N-SHH. Lane 3: 67ng/ml N-SHH. Lane 4: 125ng/ml N-SHH. Lane 5: 250ng/ml N-SHH. Lane 6: 500ng/ml N-SHH. Lane 7: 1μg/ml N-SHH. Lane 8: 2μg/ml N-SHH. Gene expression was assayed by RT-PCR. B. Shh is required to maintain but not initiate the expression of Pax3 in presomitic mesoderm exposed to Wnt signals. Presomitic mesoderm explants from stage 10 chicken embryos were cultured for either one day (panels 1-4) or five days (panels 5-8) in either control conditioned medium (panels 1 and 5), control conditioned medium containing 125ng/ml N-SHH (panels 2 and 6), Wnt3a-conditioned medium (panels 3 and 7), or the combination of Wnt3a-conditioned medium plus 125ng/ml N-SHH (panels 4 and 8). Pax3 expression was analyzed by immunocytochemistry and confocal microscopy. The inset in each panel represent a whole mount Dapi staining of the explant. The Dapi images were taken at the same magnification. Few somitic cells migrated out of the explants at day 1. By day 5, the explants were much larger due to cell proliferation and migration. It is evident that more cells were present in Shh-treated explants. C. Wnt3a alone is sufficient to induce Pax3 expression in somite IV-VI. Somites IV-VI explants from stage 10 chicken embryos were cultured for five days in either control conditioned medium (panels 1 and 3) or Wnt3a-conditioned medium (panels 2 and 4). Confocal images of Pax3 expression (panels 1 and 2) and DAPI staining (panels 3 and 4) are displayed. D. Model showing differing levels of Shh signal leads to differing cell fates in the somite.

    Journal: Developmental biology

    Article Title: A gradient of Shh establishes mutually repressing somitic cell fates induced by Nkx3.2 and Pax3

    doi: 10.1016/j.ydbio.2008.08.024

    Figure Lengend Snippet: Shh concentration gradient patterns dorsal-ventral fates of the somite A. In the presence of constant levels of Wnt3a signals, differing levels of Shh are necessary to induce either dorsal or ventral somite markers. Presomitic mesoderm explants were cultured for five days in Wnt3a-conditioned medium and increasing amounts of N-SHH. Lane 1: no N-SHH. Lane 2: 34ng/ml N-SHH. Lane 3: 67ng/ml N-SHH. Lane 4: 125ng/ml N-SHH. Lane 5: 250ng/ml N-SHH. Lane 6: 500ng/ml N-SHH. Lane 7: 1μg/ml N-SHH. Lane 8: 2μg/ml N-SHH. Gene expression was assayed by RT-PCR. B. Shh is required to maintain but not initiate the expression of Pax3 in presomitic mesoderm exposed to Wnt signals. Presomitic mesoderm explants from stage 10 chicken embryos were cultured for either one day (panels 1-4) or five days (panels 5-8) in either control conditioned medium (panels 1 and 5), control conditioned medium containing 125ng/ml N-SHH (panels 2 and 6), Wnt3a-conditioned medium (panels 3 and 7), or the combination of Wnt3a-conditioned medium plus 125ng/ml N-SHH (panels 4 and 8). Pax3 expression was analyzed by immunocytochemistry and confocal microscopy. The inset in each panel represent a whole mount Dapi staining of the explant. The Dapi images were taken at the same magnification. Few somitic cells migrated out of the explants at day 1. By day 5, the explants were much larger due to cell proliferation and migration. It is evident that more cells were present in Shh-treated explants. C. Wnt3a alone is sufficient to induce Pax3 expression in somite IV-VI. Somites IV-VI explants from stage 10 chicken embryos were cultured for five days in either control conditioned medium (panels 1 and 3) or Wnt3a-conditioned medium (panels 2 and 4). Confocal images of Pax3 expression (panels 1 and 2) and DAPI staining (panels 3 and 4) are displayed. D. Model showing differing levels of Shh signal leads to differing cell fates in the somite.

    Article Snippet: Mouse anti-Pax3, anti-Pax7 and anti-Myosin antibodies were purchased from Developmental Studies Hybridoma Bank.

    Techniques: Concentration Assay, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Immunocytochemistry, Confocal Microscopy, Staining, Migration

    Co-localization of Pax3 and Foxo1 in primary mouse myoblasts. A. Percentages of Pax3 and Foxo1 co-localization in interphase nuclei of primary mouse myoblasts and MEFs as determined by FISH analysis using Pax3 and Foxo1 BAC probes. Myo = myoblasts; MEFs = mouse embryo fibroblasts. P2 and P3 represent the second or third passage of the myoblasts in culture, B. Compilation of eight independent Pax3 and Foxo1 co-localization experiments in interphase nuclei of primary fore limb and hind limb mouse myoblasts as determined by FISH analysis using Pax3 and Foxo1 BAC probes. Experiments1, 3, 5, 6, 7 and 8 were done with normal myoblasts, while experiments 2 and 4 were performed with Foxo1-inv+/+ myoblasts. This compilation includes the two fore limb and hind limb co-localization experiments shown in A.C. Confocal FISH micrographs showing co-localization of Pax3 (shown in green) and Foxo1 (shown in red) genes in interphase nuclei of mouse primary myoblasts. D. Q-RT-PCR analysis of Pax3 , Foxo1 , Pax7 expression in primary mouse fore limb and hind limb myoblasts.

    Journal: PLoS Genetics

    Article Title: Modeling of the Human Alveolar Rhabdomyosarcoma Pax3-Foxo1 Chromosome Translocation in Mouse Myoblasts Using CRISPR-Cas9 Nuclease

    doi: 10.1371/journal.pgen.1004951

    Figure Lengend Snippet: Co-localization of Pax3 and Foxo1 in primary mouse myoblasts. A. Percentages of Pax3 and Foxo1 co-localization in interphase nuclei of primary mouse myoblasts and MEFs as determined by FISH analysis using Pax3 and Foxo1 BAC probes. Myo = myoblasts; MEFs = mouse embryo fibroblasts. P2 and P3 represent the second or third passage of the myoblasts in culture, B. Compilation of eight independent Pax3 and Foxo1 co-localization experiments in interphase nuclei of primary fore limb and hind limb mouse myoblasts as determined by FISH analysis using Pax3 and Foxo1 BAC probes. Experiments1, 3, 5, 6, 7 and 8 were done with normal myoblasts, while experiments 2 and 4 were performed with Foxo1-inv+/+ myoblasts. This compilation includes the two fore limb and hind limb co-localization experiments shown in A.C. Confocal FISH micrographs showing co-localization of Pax3 (shown in green) and Foxo1 (shown in red) genes in interphase nuclei of mouse primary myoblasts. D. Q-RT-PCR analysis of Pax3 , Foxo1 , Pax7 expression in primary mouse fore limb and hind limb myoblasts.

    Article Snippet: After adding 2 μg anti-Pax3 antibody [ ] or anti-Foxo1 (C29H4) Rabbit mAb (Cell Signaling) Pax3, Foxo1 and Pax3-Foxo1 were immunoprecipitated overnight at 4°C.

    Techniques: Fluorescence In Situ Hybridization, BAC Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

    Analysis of the CRISPR-Cas9 induced t(1;3) in Foxo1-inv +/+ myoblasts. A. Schematic representation of the position of the Pax3 forward (P-F) and reverse (P-R) PCR primers upstream and downstream of the Pax3 breakpoint in intron 7 (DSB) and of the Foxo1 forward (F-F) and reverse (F-R) PCR primers upstream and downstream of the Foxo1 breakpoint in intron 1 (DSB). The P-F and F-R primer pair amplify the Pax3-Foxo1 fusion . B. Agarose gel showing the PCR products obtained using the P-F/F-R primer pair and DNA from Foxo1-inv +/+ hind limb DNA without (lane 1) and with (lane 2) CRISPR-Cas9 treatment, form Foxo1-inv +/+ fore limb DNA without (lane 3) and with (lane 4) CRISPR-Cas9 treatment, from wild type fore limb DNA with CRISPR-Cas9 treatment (lane 5), and from Foxo1-inv +/+ MEF DNA with CRISPR-Cas9 treatment (lane 6). M is 100 bp ladder molecular weight marker. The position of the expected size of the cleanly ligated Pax3-Foxo1 fusion fragment is indicated on the right (251 bp). C. Agarose gel showing the Pax3 PCR fragments containing the RH30-like Pax3 breakpoint from Foxo1-inv +/+ fore limb myoblast DNA not treated with CRISPR-Cas9 without (lane1) and with (lane 2) MaeIII digest, treated with CRISPR-Cas9 without (lane 3) and with (lane 4) MaeIII digest and Foxo1-inv +/+ MEF DNA treated with CRISPR-Cas9 without (lane 5) and with (lane 6) MaeIII digest. Expected fragment sizes are indicated on the right. Fully modified CRISPR-Cas9/NHEJ DSBs do not cut with MaeIII . Beneath the gel is a table with the relative fragment intensities as measured with a BIO-RAD ChemiDoc imaging system. Underneath the table is a histogram giving a graphic representation of the relative band intensities. D. Same analysis as in C but for the Foxo1 fragment containing the RH30-like breakpoint digested with DdeI . The table underneath shows the relative fragment intensities as measured with a BIO-RAD ChemiDoc imaging system. Underneath the table is histogram giving a graphic representation of the relative band intensities. E. DNA sequences of 39 cloned Pax3-Foxo1 PCR fragments from CRISPR-Cas9 treated fore limb (6 different) and of 34 cloned Pax3-Foxo1 PCR fragments from CRISPR-Cas9 treated hind limb (3 different) myoblasts. F. Co-localization frequencies of Pax3 and Foxo1 in Foxo1-inv +/+ fore limb, hind limb, and MEFs used in B. G. Agarose gel and DNA sequence of a partial Pax3-Foxo1 fusion cDNA fragment showing the correctly spliced Pax3 exon7- Foxo1 exon2 fusion after RT-PCR of RNA from CRISPR-Cas9 treated Foxo1-inv +/+ fore limb myoblasts.

    Journal: PLoS Genetics

    Article Title: Modeling of the Human Alveolar Rhabdomyosarcoma Pax3-Foxo1 Chromosome Translocation in Mouse Myoblasts Using CRISPR-Cas9 Nuclease

    doi: 10.1371/journal.pgen.1004951

    Figure Lengend Snippet: Analysis of the CRISPR-Cas9 induced t(1;3) in Foxo1-inv +/+ myoblasts. A. Schematic representation of the position of the Pax3 forward (P-F) and reverse (P-R) PCR primers upstream and downstream of the Pax3 breakpoint in intron 7 (DSB) and of the Foxo1 forward (F-F) and reverse (F-R) PCR primers upstream and downstream of the Foxo1 breakpoint in intron 1 (DSB). The P-F and F-R primer pair amplify the Pax3-Foxo1 fusion . B. Agarose gel showing the PCR products obtained using the P-F/F-R primer pair and DNA from Foxo1-inv +/+ hind limb DNA without (lane 1) and with (lane 2) CRISPR-Cas9 treatment, form Foxo1-inv +/+ fore limb DNA without (lane 3) and with (lane 4) CRISPR-Cas9 treatment, from wild type fore limb DNA with CRISPR-Cas9 treatment (lane 5), and from Foxo1-inv +/+ MEF DNA with CRISPR-Cas9 treatment (lane 6). M is 100 bp ladder molecular weight marker. The position of the expected size of the cleanly ligated Pax3-Foxo1 fusion fragment is indicated on the right (251 bp). C. Agarose gel showing the Pax3 PCR fragments containing the RH30-like Pax3 breakpoint from Foxo1-inv +/+ fore limb myoblast DNA not treated with CRISPR-Cas9 without (lane1) and with (lane 2) MaeIII digest, treated with CRISPR-Cas9 without (lane 3) and with (lane 4) MaeIII digest and Foxo1-inv +/+ MEF DNA treated with CRISPR-Cas9 without (lane 5) and with (lane 6) MaeIII digest. Expected fragment sizes are indicated on the right. Fully modified CRISPR-Cas9/NHEJ DSBs do not cut with MaeIII . Beneath the gel is a table with the relative fragment intensities as measured with a BIO-RAD ChemiDoc imaging system. Underneath the table is a histogram giving a graphic representation of the relative band intensities. D. Same analysis as in C but for the Foxo1 fragment containing the RH30-like breakpoint digested with DdeI . The table underneath shows the relative fragment intensities as measured with a BIO-RAD ChemiDoc imaging system. Underneath the table is histogram giving a graphic representation of the relative band intensities. E. DNA sequences of 39 cloned Pax3-Foxo1 PCR fragments from CRISPR-Cas9 treated fore limb (6 different) and of 34 cloned Pax3-Foxo1 PCR fragments from CRISPR-Cas9 treated hind limb (3 different) myoblasts. F. Co-localization frequencies of Pax3 and Foxo1 in Foxo1-inv +/+ fore limb, hind limb, and MEFs used in B. G. Agarose gel and DNA sequence of a partial Pax3-Foxo1 fusion cDNA fragment showing the correctly spliced Pax3 exon7- Foxo1 exon2 fusion after RT-PCR of RNA from CRISPR-Cas9 treated Foxo1-inv +/+ fore limb myoblasts.

    Article Snippet: After adding 2 μg anti-Pax3 antibody [ ] or anti-Foxo1 (C29H4) Rabbit mAb (Cell Signaling) Pax3, Foxo1 and Pax3-Foxo1 were immunoprecipitated overnight at 4°C.

    Techniques: CRISPR, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight, Marker, Modification, Non-Homologous End Joining, Imaging, Clone Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction

    Sequences and vectors used for the generation of an RH30-like t(1;3) in Foxo1-inv +/+ mouse myoblasts. A. Top 2 lines show the sequence alignment of human PAX3 intron 7 (hPAX3) with that of mouse Pax3 intron 7 (mPax3) across the PAX3 breakpoint (black arrow) in the A-RMS cell line RH30. Bottom 2 lines show the sequence alignment of human FOXO1 intron 1 (hFOXO1) with that of mouse Foxo1 intron 1 (mFoxo1) across the FOXO1 breakpoint (black arrow) in RH30. The line in the middle shows the sequence of the t(2;13) breakpoint (black arrow) in RH30. B. Top 2 lines show the sequence of the mouse Pax3 intron 7 across the RH30 breakpoint (black arrow). The targeting sequence of the sgRNA (light blue, underlined) and PAM sequence (orange, underlined) are indicated. hCas9 will generate a DSB 3 bp upstream of the PAM sequence. The bottom 2 lines show the sequence of the mouse Foxo1 intron 1 across the RH30 breakpoint (black arrow). The targeting sequence of the sgRNA (pink, underlined) and PAM sequence (purple, underlined) are indicated. Cas9 will generate a DSB 3 bp upstream of the PAM sequence. The two lines in the middle show the sequence of the clean fusion between Pax3 intron 7 and Foxo1 intron 1 after Cas9 cleavage in Foxo1-inv +/+ myoblasts. Black arrows indicate the position of the RH30 PAX3 and FOXO1 breakpoints. The Pax3 breakpoint is 25 bp downstream of that of the RH30 PAX3 breakpoint, while the Foxo1 breakpoint is 17 bp upstream of that of the RH30 FOXO1 breakpoint. C. At the top is a schematic representation of the pCL20C-hCas9-IRES-YFP lentiviral vector. Below that is a schematic representation of the dual pCL20C-hU6-mU6-βact-puro sgRNA lentiviral vector containing the human (hU6) and mouse (mU6) U6 promoters driving the Pax3 and Foxo1 sgRNAs, respectively. Puro represents the SV40 early promoter driven puromycin resistance gene. Below the vector are magnifications of the borders of the hU6-sgRNA and mU6-sgRNA portions of the vector indicating the AgeI and EcoNI target RNA cloning sites and the DNA polymerase III sgRNA transcriptional start (start) and termination sites (PolIII term). LTR = long terminal repeat sequence, MSCV = MSCV-LTR, hCAS9 is human codon optimized Cas9, YFP = yellow fluorescent protein, D. Sequence of the RH30-like Pax3 and Foxo1 sgRNAs in the pCL20c-hU6-mU6-βact-puro vector.

    Journal: PLoS Genetics

    Article Title: Modeling of the Human Alveolar Rhabdomyosarcoma Pax3-Foxo1 Chromosome Translocation in Mouse Myoblasts Using CRISPR-Cas9 Nuclease

    doi: 10.1371/journal.pgen.1004951

    Figure Lengend Snippet: Sequences and vectors used for the generation of an RH30-like t(1;3) in Foxo1-inv +/+ mouse myoblasts. A. Top 2 lines show the sequence alignment of human PAX3 intron 7 (hPAX3) with that of mouse Pax3 intron 7 (mPax3) across the PAX3 breakpoint (black arrow) in the A-RMS cell line RH30. Bottom 2 lines show the sequence alignment of human FOXO1 intron 1 (hFOXO1) with that of mouse Foxo1 intron 1 (mFoxo1) across the FOXO1 breakpoint (black arrow) in RH30. The line in the middle shows the sequence of the t(2;13) breakpoint (black arrow) in RH30. B. Top 2 lines show the sequence of the mouse Pax3 intron 7 across the RH30 breakpoint (black arrow). The targeting sequence of the sgRNA (light blue, underlined) and PAM sequence (orange, underlined) are indicated. hCas9 will generate a DSB 3 bp upstream of the PAM sequence. The bottom 2 lines show the sequence of the mouse Foxo1 intron 1 across the RH30 breakpoint (black arrow). The targeting sequence of the sgRNA (pink, underlined) and PAM sequence (purple, underlined) are indicated. Cas9 will generate a DSB 3 bp upstream of the PAM sequence. The two lines in the middle show the sequence of the clean fusion between Pax3 intron 7 and Foxo1 intron 1 after Cas9 cleavage in Foxo1-inv +/+ myoblasts. Black arrows indicate the position of the RH30 PAX3 and FOXO1 breakpoints. The Pax3 breakpoint is 25 bp downstream of that of the RH30 PAX3 breakpoint, while the Foxo1 breakpoint is 17 bp upstream of that of the RH30 FOXO1 breakpoint. C. At the top is a schematic representation of the pCL20C-hCas9-IRES-YFP lentiviral vector. Below that is a schematic representation of the dual pCL20C-hU6-mU6-βact-puro sgRNA lentiviral vector containing the human (hU6) and mouse (mU6) U6 promoters driving the Pax3 and Foxo1 sgRNAs, respectively. Puro represents the SV40 early promoter driven puromycin resistance gene. Below the vector are magnifications of the borders of the hU6-sgRNA and mU6-sgRNA portions of the vector indicating the AgeI and EcoNI target RNA cloning sites and the DNA polymerase III sgRNA transcriptional start (start) and termination sites (PolIII term). LTR = long terminal repeat sequence, MSCV = MSCV-LTR, hCAS9 is human codon optimized Cas9, YFP = yellow fluorescent protein, D. Sequence of the RH30-like Pax3 and Foxo1 sgRNAs in the pCL20c-hU6-mU6-βact-puro vector.

    Article Snippet: After adding 2 μg anti-Pax3 antibody [ ] or anti-Foxo1 (C29H4) Rabbit mAb (Cell Signaling) Pax3, Foxo1 and Pax3-Foxo1 were immunoprecipitated overnight at 4°C.

    Techniques: Sequencing, Plasmid Preparation, Clone Assay

    The reciprocal t(1;3) in myoblasts expresses Pax3-Foxo1 protein, which upregulates target gene expression. A-C. FISH analysis of an t(1;3)-enriched pool 1G3 myoblasts showing the reciprocal translocation. B and C show the magnified der(1) and der(3) chromosomes, respectively. D. Lysate of 1H3 (0%) and 1G3 [64% t(1;3)] myoblasts was immunoprecipitated with an anti-Pax3 antibody and immunoblotted with anti-Pax3 antibody (Pax3/Pax3) or anti-Foxo1 antibody (Pax3/Foxo1). The IP with anti-Foxo1 antibody was immunoblotted with anti-Pax3 antibody (Foxo1/Pax3) and an anti-Foxo1 antibody (Foxo1/Foxo1). The positions of the Pax3, Pax3-Foxo1 and Foxo1 bands are indicated.

    Journal: PLoS Genetics

    Article Title: Modeling of the Human Alveolar Rhabdomyosarcoma Pax3-Foxo1 Chromosome Translocation in Mouse Myoblasts Using CRISPR-Cas9 Nuclease

    doi: 10.1371/journal.pgen.1004951

    Figure Lengend Snippet: The reciprocal t(1;3) in myoblasts expresses Pax3-Foxo1 protein, which upregulates target gene expression. A-C. FISH analysis of an t(1;3)-enriched pool 1G3 myoblasts showing the reciprocal translocation. B and C show the magnified der(1) and der(3) chromosomes, respectively. D. Lysate of 1H3 (0%) and 1G3 [64% t(1;3)] myoblasts was immunoprecipitated with an anti-Pax3 antibody and immunoblotted with anti-Pax3 antibody (Pax3/Pax3) or anti-Foxo1 antibody (Pax3/Foxo1). The IP with anti-Foxo1 antibody was immunoblotted with anti-Pax3 antibody (Foxo1/Pax3) and an anti-Foxo1 antibody (Foxo1/Foxo1). The positions of the Pax3, Pax3-Foxo1 and Foxo1 bands are indicated.

    Article Snippet: After adding 2 μg anti-Pax3 antibody [ ] or anti-Foxo1 (C29H4) Rabbit mAb (Cell Signaling) Pax3, Foxo1 and Pax3-Foxo1 were immunoprecipitated overnight at 4°C.

    Techniques: Expressing, Fluorescence In Situ Hybridization, Translocation Assay, Immunoprecipitation

    Pax3 flox/flox /P0-Cre conditional knockout mice do not exhibit congenital hydrocephalus, indicating that restricted loss of Pax3 within the migratory neural crest is insufficient to cause defects. ( A ) P0-Cre/R26r lineage mapping in E10.5 control embryos reveals that the neural crest-colonized tissues, such as cranial-facial (green arrow), pharyngeal arches (blue arrows), and dorsal root ganglia (red arrow) are all positively labeled (blue staining). However, as P0-Cre is only expressed in neural crest derivatives after emigration from the neural tube, P0-Cre/R26r lineage mapping verified that the neural tube (white arrow), hindbrain, and brain are not labeled; ( B ) lateral view of P12 Pax3 flox/flox /P0-Cre (upper) and control (lower) littermates. Their lateral cranial profile is indistinguishable (indicated by dotted lines and compared side-by-side in insert). Also note normal pigmentation of Pax3 flox/flox /P0-Cre neonate: ( C,D ) coronal views of neonates in B , verifying Pax3 flox/flox /P0-Cre neonate lateral ventricles (arrows in C) are unaffected.

    Journal: Journal of developmental biology

    Article Title: Restricted Pax3 Deletion within the Neural Tube Results in Congenital Hydrocephalus

    doi: 10.3390/jdb4010007

    Figure Lengend Snippet: Pax3 flox/flox /P0-Cre conditional knockout mice do not exhibit congenital hydrocephalus, indicating that restricted loss of Pax3 within the migratory neural crest is insufficient to cause defects. ( A ) P0-Cre/R26r lineage mapping in E10.5 control embryos reveals that the neural crest-colonized tissues, such as cranial-facial (green arrow), pharyngeal arches (blue arrows), and dorsal root ganglia (red arrow) are all positively labeled (blue staining). However, as P0-Cre is only expressed in neural crest derivatives after emigration from the neural tube, P0-Cre/R26r lineage mapping verified that the neural tube (white arrow), hindbrain, and brain are not labeled; ( B ) lateral view of P12 Pax3 flox/flox /P0-Cre (upper) and control (lower) littermates. Their lateral cranial profile is indistinguishable (indicated by dotted lines and compared side-by-side in insert). Also note normal pigmentation of Pax3 flox/flox /P0-Cre neonate: ( C,D ) coronal views of neonates in B , verifying Pax3 flox/flox /P0-Cre neonate lateral ventricles (arrows in C) are unaffected.

    Article Snippet: Dilution of primary antibodies was 1:200 for goat anti-Pax3 (Santa Cruz sc-7748, Dallas, TX, USA); 1:200 for mouse anti-Pax7 (Hybridoma Bank, Iowa City, IA, USA); 1:25 for monoclonal rat anti-Ki67 (DAKO, Carpinteria, CA, USA); 1:1000 for rabbit anti-β-galactosidase (Molecular Probes, Eugene, OR, USA); and 1:200 for rabbit anti-β-Catenin (Sigma, St. Louis, MO, USA).

    Techniques: Knock-Out, Mouse Assay, Labeling, Staining

    Pax3 and Pax7 play a combinatorial role in cerebral ventricular development. ( A–C ) Lateral views of Pax3 +/Δ5 / Pax7 +/Δ2 compound heterozygous mutant ( A ) and control ( B ) P20 littermates, with lateral profiles depicted by dotted lines and compared side-by-side to highlight the large domed cranium in Pax3 +/− / Pax7 +/− mutant ( C ); ( D,E ) Skeletal preps of heads from animals in ( A,B ); only the compound mutant ( D ) displays sagittal (narrow arrows) and transverse (thick arrows) suture fusion defects; ( F,G ) Hematoxylin and eosin sections reveal that the mutant lateral ventricle ( F ) is dilated and there is a loss of structure, when compared to control (double asterisks, G). The normal anterior segment of the third ventricle (plus sign), aqueduct (asterisk), and cerebellum (c,b) are indicated; ( H,I ) high power view of stenotic connection between mutant third ventricle and aqueduct (arrow, H), compared to normal third ventricle-aqueduct junction in control ( I ); ( J,K ) Results of representative Western blot analysis ( J ) and densitometric quantification ( K ) of Pax3 and Pax7 expression levels in individual E10.5 whole embryo lysates of designated genotypes ( n = 3–5 of each genotype were analyzed). Note the gene dosage-dependent reduction (~50%) of corresponding protein level in various heterozygous compound offspring, relative to wild-type ( P3 +/+ / P7 +/+ ). Data are represented as mean ± SEM, * p

    Journal: Journal of developmental biology

    Article Title: Restricted Pax3 Deletion within the Neural Tube Results in Congenital Hydrocephalus

    doi: 10.3390/jdb4010007

    Figure Lengend Snippet: Pax3 and Pax7 play a combinatorial role in cerebral ventricular development. ( A–C ) Lateral views of Pax3 +/Δ5 / Pax7 +/Δ2 compound heterozygous mutant ( A ) and control ( B ) P20 littermates, with lateral profiles depicted by dotted lines and compared side-by-side to highlight the large domed cranium in Pax3 +/− / Pax7 +/− mutant ( C ); ( D,E ) Skeletal preps of heads from animals in ( A,B ); only the compound mutant ( D ) displays sagittal (narrow arrows) and transverse (thick arrows) suture fusion defects; ( F,G ) Hematoxylin and eosin sections reveal that the mutant lateral ventricle ( F ) is dilated and there is a loss of structure, when compared to control (double asterisks, G). The normal anterior segment of the third ventricle (plus sign), aqueduct (asterisk), and cerebellum (c,b) are indicated; ( H,I ) high power view of stenotic connection between mutant third ventricle and aqueduct (arrow, H), compared to normal third ventricle-aqueduct junction in control ( I ); ( J,K ) Results of representative Western blot analysis ( J ) and densitometric quantification ( K ) of Pax3 and Pax7 expression levels in individual E10.5 whole embryo lysates of designated genotypes ( n = 3–5 of each genotype were analyzed). Note the gene dosage-dependent reduction (~50%) of corresponding protein level in various heterozygous compound offspring, relative to wild-type ( P3 +/+ / P7 +/+ ). Data are represented as mean ± SEM, * p

    Article Snippet: Dilution of primary antibodies was 1:200 for goat anti-Pax3 (Santa Cruz sc-7748, Dallas, TX, USA); 1:200 for mouse anti-Pax7 (Hybridoma Bank, Iowa City, IA, USA); 1:25 for monoclonal rat anti-Ki67 (DAKO, Carpinteria, CA, USA); 1:1000 for rabbit anti-β-galactosidase (Molecular Probes, Eugene, OR, USA); and 1:200 for rabbit anti-β-Catenin (Sigma, St. Louis, MO, USA).

    Techniques: Mutagenesis, Western Blot, Expressing

    Analysis of Pax3 and Pax7 spatiotemporal expression patterns, Cre expression limits and cell proliferation during cerebral ventricular development. ( A–C ) In situ hybridization detection of Pax3 mRNA in E15.5 wild-type heads show active expression in ventricular epithelium (arrow) and multiple craniofacial structures ( A ). Coronal E14.5 sections cut at the subcommissural organ (SCO, indicated by the red arrow in C ) level of Pax3 flox/flox /Wnt1-Cre mutant ( B ) and control ( C ) reveal Pax3 is still expressed in the ventricular epithelium (white arrow) comparable to control ( C ), despite collapse of the ventricular opening in mutants; ( D,E ) Phase contrast images of B,C , illustrating the lack of a distinct subcommissural organ in the mutant D ); ( F,H ) Immunohistochemical detection of Pax3 ( F ) and Pax7 ( H ) protein on adjacent telencephalic vesicle sections at E10, reveals co-localization in the majority of the neuroepithelium, with especially robust expression (brown) in the lamina terminalis. The black arrows point to intense artificial staining due to tissue folding; ( G,I ) Immunohistochemical detection beta-galactosidase expression following lineage mapping at E13.5 for Pax7 −Cre /R26r ( G ) and Wnt1-Cre/R26r ( I ) following Cre-mediated activation of R26r reporter. While Wnt1-Cre marks the ependymal layer from third ventricle (3v) through aqueduct ( I ), Pax7 −Cre only marks the ependymal layer of 3v (arrow points to the 3v-aqueduct junction and asterisk indicates aqueduct). Scale bars: B–E = 100 µm; F,H = 200 µm; G,I = 50 µm.

    Journal: Journal of developmental biology

    Article Title: Restricted Pax3 Deletion within the Neural Tube Results in Congenital Hydrocephalus

    doi: 10.3390/jdb4010007

    Figure Lengend Snippet: Analysis of Pax3 and Pax7 spatiotemporal expression patterns, Cre expression limits and cell proliferation during cerebral ventricular development. ( A–C ) In situ hybridization detection of Pax3 mRNA in E15.5 wild-type heads show active expression in ventricular epithelium (arrow) and multiple craniofacial structures ( A ). Coronal E14.5 sections cut at the subcommissural organ (SCO, indicated by the red arrow in C ) level of Pax3 flox/flox /Wnt1-Cre mutant ( B ) and control ( C ) reveal Pax3 is still expressed in the ventricular epithelium (white arrow) comparable to control ( C ), despite collapse of the ventricular opening in mutants; ( D,E ) Phase contrast images of B,C , illustrating the lack of a distinct subcommissural organ in the mutant D ); ( F,H ) Immunohistochemical detection of Pax3 ( F ) and Pax7 ( H ) protein on adjacent telencephalic vesicle sections at E10, reveals co-localization in the majority of the neuroepithelium, with especially robust expression (brown) in the lamina terminalis. The black arrows point to intense artificial staining due to tissue folding; ( G,I ) Immunohistochemical detection beta-galactosidase expression following lineage mapping at E13.5 for Pax7 −Cre /R26r ( G ) and Wnt1-Cre/R26r ( I ) following Cre-mediated activation of R26r reporter. While Wnt1-Cre marks the ependymal layer from third ventricle (3v) through aqueduct ( I ), Pax7 −Cre only marks the ependymal layer of 3v (arrow points to the 3v-aqueduct junction and asterisk indicates aqueduct). Scale bars: B–E = 100 µm; F,H = 200 µm; G,I = 50 µm.

    Article Snippet: Dilution of primary antibodies was 1:200 for goat anti-Pax3 (Santa Cruz sc-7748, Dallas, TX, USA); 1:200 for mouse anti-Pax7 (Hybridoma Bank, Iowa City, IA, USA); 1:25 for monoclonal rat anti-Ki67 (DAKO, Carpinteria, CA, USA); 1:1000 for rabbit anti-β-galactosidase (Molecular Probes, Eugene, OR, USA); and 1:200 for rabbit anti-β-Catenin (Sigma, St. Louis, MO, USA).

    Techniques: Expressing, In Situ Hybridization, Mutagenesis, Immunohistochemistry, Staining, Activation Assay

    Histological analysis of ventricular dilation and tissue loss in Pax3 flox/flox /Wnt1-Cre mutants. ( A,B ) Histology revealed a narrow third ventricle (arrow) along the E12.5 mutant ( A ) anterior-posterior axis compared to control ( B ); ( C–H ) E13.5 coronal sections from anterior to posterior and stained for lacZ reporter (blue) and eosin (pink) of Pax3 flox/flox /Wnt1-Cre;R26r mutants ( C,E,G ) and Wnt1-Cre/R26r controls ( D,F,H ); Opening of the dorsal part of mutant third ventricle (3v) is narrow (arrow, C ) or contracted (broken arrow, E ) compared with the control ( D,F ). However, at this stage there is a connection between both mutant and control third ventricle and aqueduct (asterisk); ( I,J ) hematoxylin and eosin horizontal sections from P20 mutant ( I ) and control ( J ). Note mutant lateral ventricles (LV) are enlarged and fused due to loss of normal interventricular tissue (double asterisk, J); ( K–R ) hematoxylin and eosin stained transverse sections of E16.5 Pax3 flox/flox /Wnt1-Cre ( K–N ) and control brains ( O–R ); L,P are magnified in areas indicated in K,O and clearly show diminished opening of dorsal part of the mutant third ventricle and an under-developed subcommissural organ ( K,L ); as a consequence of likely impaired fluid flow, the mutant medial region (arrow, K ) is remarkably enlarged compared to control (arrow, O ). Additionally, mutant lateral ventricle (arrow, M ) is significantly dilated. Although the superior sagittal sinus (SSS) is easily recognizable in control ( R ) it is obscure and undersized in the Pax3 flox/flox /Wnt1-Cre mutants (arrow, N ); ( S,T ) enlarged images of boxed areas in I,J illustrate the lack of connection between the mutant third ventricles and aqueducts (asterisks) in mutants ( S ) but not in controls ( T ). Scale bars: A–D,F–I = 1 mm; E,J = 2 mm; S,T = 0.5 mm.

    Journal: Journal of developmental biology

    Article Title: Restricted Pax3 Deletion within the Neural Tube Results in Congenital Hydrocephalus

    doi: 10.3390/jdb4010007

    Figure Lengend Snippet: Histological analysis of ventricular dilation and tissue loss in Pax3 flox/flox /Wnt1-Cre mutants. ( A,B ) Histology revealed a narrow third ventricle (arrow) along the E12.5 mutant ( A ) anterior-posterior axis compared to control ( B ); ( C–H ) E13.5 coronal sections from anterior to posterior and stained for lacZ reporter (blue) and eosin (pink) of Pax3 flox/flox /Wnt1-Cre;R26r mutants ( C,E,G ) and Wnt1-Cre/R26r controls ( D,F,H ); Opening of the dorsal part of mutant third ventricle (3v) is narrow (arrow, C ) or contracted (broken arrow, E ) compared with the control ( D,F ). However, at this stage there is a connection between both mutant and control third ventricle and aqueduct (asterisk); ( I,J ) hematoxylin and eosin horizontal sections from P20 mutant ( I ) and control ( J ). Note mutant lateral ventricles (LV) are enlarged and fused due to loss of normal interventricular tissue (double asterisk, J); ( K–R ) hematoxylin and eosin stained transverse sections of E16.5 Pax3 flox/flox /Wnt1-Cre ( K–N ) and control brains ( O–R ); L,P are magnified in areas indicated in K,O and clearly show diminished opening of dorsal part of the mutant third ventricle and an under-developed subcommissural organ ( K,L ); as a consequence of likely impaired fluid flow, the mutant medial region (arrow, K ) is remarkably enlarged compared to control (arrow, O ). Additionally, mutant lateral ventricle (arrow, M ) is significantly dilated. Although the superior sagittal sinus (SSS) is easily recognizable in control ( R ) it is obscure and undersized in the Pax3 flox/flox /Wnt1-Cre mutants (arrow, N ); ( S,T ) enlarged images of boxed areas in I,J illustrate the lack of connection between the mutant third ventricles and aqueducts (asterisks) in mutants ( S ) but not in controls ( T ). Scale bars: A–D,F–I = 1 mm; E,J = 2 mm; S,T = 0.5 mm.

    Article Snippet: Dilution of primary antibodies was 1:200 for goat anti-Pax3 (Santa Cruz sc-7748, Dallas, TX, USA); 1:200 for mouse anti-Pax7 (Hybridoma Bank, Iowa City, IA, USA); 1:25 for monoclonal rat anti-Ki67 (DAKO, Carpinteria, CA, USA); 1:1000 for rabbit anti-β-galactosidase (Molecular Probes, Eugene, OR, USA); and 1:200 for rabbit anti-β-Catenin (Sigma, St. Louis, MO, USA).

    Techniques: Mutagenesis, Staining, Flow Cytometry

    Lineage mapping reveals restricted loss of Pax3 results in abnormal opening to the brain third ventricle. ( A–E ) Coronal sections of E14.5 of Pax3 flox/flox /Wnt1-Cre; R26r mutant ( A,D ), Wnt1-Cre/R26r control ( B,E ) and Pax3 flox/flox /R26r control ( C ) littermates following β-galactosidase (brown) immunohistochemistry to unequivocally detect lacZ expression. The Wnt1-Cre -negative sample ( C ) serves as a negative control. Significantly, the epithelium lining the control third ventricle and subcommissural organ is specifically labeled ( B ), whereas in the mutant ( A ), although a group of cells is positively labeled, they failed to form a normal tubular structure in the mutant. Enlarged views from A,B (indicated via large dotted line boxed areas labeled D,E ) reveal lacZ -expressing elongated columnar epithelial cells surrounding a clear opening in control ( E ) but lacZ -expressing mutant cells fail to establish the normal opening to the third ventricle (arrow in D ). Additionally, double-headed arrows indicate the space in the lateral ventricles in all three genotypes, revealing a mild dilation of lateral ventricles in only the mutant ( A ). Inserts in A,B (solid line boxes) show a similar pattern of typical Wnt1-Cre/R26r expressing cells in both mutant and control great arteries (arrows) and thymus (asterisk), from more posterior sections adjacent to the outflow tract of the heart. ( F,G ) High-power view from small dotted line boxed areas in A,B , demonstrating that the superior sagittal sinus (SSS) and surrounding tissue are positively labeled for Wnt1-Cre/R26r lineage. Note however that the circumferential labeling pattern seen in control ( G ) is discontinuous in the mutant (arrows, F ). Scale bars: A–C = 400 µm; D,E = 130 µm; F,G = 50 µm.

    Journal: Journal of developmental biology

    Article Title: Restricted Pax3 Deletion within the Neural Tube Results in Congenital Hydrocephalus

    doi: 10.3390/jdb4010007

    Figure Lengend Snippet: Lineage mapping reveals restricted loss of Pax3 results in abnormal opening to the brain third ventricle. ( A–E ) Coronal sections of E14.5 of Pax3 flox/flox /Wnt1-Cre; R26r mutant ( A,D ), Wnt1-Cre/R26r control ( B,E ) and Pax3 flox/flox /R26r control ( C ) littermates following β-galactosidase (brown) immunohistochemistry to unequivocally detect lacZ expression. The Wnt1-Cre -negative sample ( C ) serves as a negative control. Significantly, the epithelium lining the control third ventricle and subcommissural organ is specifically labeled ( B ), whereas in the mutant ( A ), although a group of cells is positively labeled, they failed to form a normal tubular structure in the mutant. Enlarged views from A,B (indicated via large dotted line boxed areas labeled D,E ) reveal lacZ -expressing elongated columnar epithelial cells surrounding a clear opening in control ( E ) but lacZ -expressing mutant cells fail to establish the normal opening to the third ventricle (arrow in D ). Additionally, double-headed arrows indicate the space in the lateral ventricles in all three genotypes, revealing a mild dilation of lateral ventricles in only the mutant ( A ). Inserts in A,B (solid line boxes) show a similar pattern of typical Wnt1-Cre/R26r expressing cells in both mutant and control great arteries (arrows) and thymus (asterisk), from more posterior sections adjacent to the outflow tract of the heart. ( F,G ) High-power view from small dotted line boxed areas in A,B , demonstrating that the superior sagittal sinus (SSS) and surrounding tissue are positively labeled for Wnt1-Cre/R26r lineage. Note however that the circumferential labeling pattern seen in control ( G ) is discontinuous in the mutant (arrows, F ). Scale bars: A–C = 400 µm; D,E = 130 µm; F,G = 50 µm.

    Article Snippet: Dilution of primary antibodies was 1:200 for goat anti-Pax3 (Santa Cruz sc-7748, Dallas, TX, USA); 1:200 for mouse anti-Pax7 (Hybridoma Bank, Iowa City, IA, USA); 1:25 for monoclonal rat anti-Ki67 (DAKO, Carpinteria, CA, USA); 1:1000 for rabbit anti-β-galactosidase (Molecular Probes, Eugene, OR, USA); and 1:200 for rabbit anti-β-Catenin (Sigma, St. Louis, MO, USA).

    Techniques: Mutagenesis, Immunohistochemistry, Expressing, Negative Control, Labeling

    Marker analysis of anterior third ventricle morphogenesis. ( A,B ) Immunofluorescence detection of β-catenin expression in Pax3 flox/flox /Wnt1-Cre mutants ( A ) compared to control ( B ) E12.5 embryos. Red arrows point to the apical pole of the anterior side of third ventricles, revealing robust β-catenin in mutants but not control. However, β-Catenin expression is equally present in control and mutant lateral walls (white arrows). Inserts are negative controls, illustrating lack of autofluorescence; ( C,D ) TUNEL assay shows ectopic apoptosis in anterior wall of third ventricle in Pax3 flox/flox /Wnt1-Cre mutants (arrow, C ) but none in control; ( E,F ) Ki67 immunohistochemical analysis of reduced cell proliferation in Pax3 flox/flox /Wnt1-Cre mutants ( E ) compared to control ( F ) E12.5 embryos. Black dashed lines indicate the ventricular lumen and the white lines indicate the pseudostratified epithelium. Inserts from E12.5 lateral ventricles, exhibiting similar Ki67 labeling index in Pax3 flox/flox /Wnt1-Cre mutants ( E ) and control ( F ). Scale bars: A,B = 50 µm; C–F = 100 µm.

    Journal: Journal of developmental biology

    Article Title: Restricted Pax3 Deletion within the Neural Tube Results in Congenital Hydrocephalus

    doi: 10.3390/jdb4010007

    Figure Lengend Snippet: Marker analysis of anterior third ventricle morphogenesis. ( A,B ) Immunofluorescence detection of β-catenin expression in Pax3 flox/flox /Wnt1-Cre mutants ( A ) compared to control ( B ) E12.5 embryos. Red arrows point to the apical pole of the anterior side of third ventricles, revealing robust β-catenin in mutants but not control. However, β-Catenin expression is equally present in control and mutant lateral walls (white arrows). Inserts are negative controls, illustrating lack of autofluorescence; ( C,D ) TUNEL assay shows ectopic apoptosis in anterior wall of third ventricle in Pax3 flox/flox /Wnt1-Cre mutants (arrow, C ) but none in control; ( E,F ) Ki67 immunohistochemical analysis of reduced cell proliferation in Pax3 flox/flox /Wnt1-Cre mutants ( E ) compared to control ( F ) E12.5 embryos. Black dashed lines indicate the ventricular lumen and the white lines indicate the pseudostratified epithelium. Inserts from E12.5 lateral ventricles, exhibiting similar Ki67 labeling index in Pax3 flox/flox /Wnt1-Cre mutants ( E ) and control ( F ). Scale bars: A,B = 50 µm; C–F = 100 µm.

    Article Snippet: Dilution of primary antibodies was 1:200 for goat anti-Pax3 (Santa Cruz sc-7748, Dallas, TX, USA); 1:200 for mouse anti-Pax7 (Hybridoma Bank, Iowa City, IA, USA); 1:25 for monoclonal rat anti-Ki67 (DAKO, Carpinteria, CA, USA); 1:1000 for rabbit anti-β-galactosidase (Molecular Probes, Eugene, OR, USA); and 1:200 for rabbit anti-β-Catenin (Sigma, St. Louis, MO, USA).

    Techniques: Marker, Immunofluorescence, Expressing, Mutagenesis, TUNEL Assay, Immunohistochemistry, Labeling

    Pax3 flox/flox /Pax7 −Cre conditional knockout mice exhibit congenital hydrocephalus, indicating that restricted absence of Pax3 from the Pax7 lineage is sufficient to cause defects. ( A ) Lateral views of Pax3 flox/flox /Pax7 −Cre (top) and control (bottom) P20 pups, with lateral profiles depicted by dotted lines and compared side by side to highlight the domed cranium in Pax3 flox/flox /Pax7 −Cre mutant. Insert depicts Pax7 −Cre /R26r lineage mapping in an E9.5 control embryo, with positive lacZ -labeling (blue) in the cranial neural tube (black arrow) with negligible labeling of neural crest structures (red arrow); ( B–I ) Hematoxylin and eosin sections of P6 Pax3 flox/flox /Pax7 −Cre ( B,D,F,H ) and control ( C,E,G,I ) littermates. Note that mutant lateral ventricle is dilated and loss of tissue is apparent ( B ). Enlarged views of boxed areas in ( B,C ) reveals the lack of connection between the mutant third ventricle and aqueduct (asterisk, D ) compared to control littermate ( E ); Arrow in E indicates the control posterior part of the third ventricle connecting to the aqueduct (asterisk). When coronal sections of the posterior part of the control ( G ) third ventricles are compared to Pax3 flox/flox /Pax7 −Cre mutants ( F ), the opening of the mutant ventricle is significantly narrowed; high-power views of the mutant ( H ) and control ( I ) superior sagittal sinus (arrows) confirm that the mutant lumen size is significantly reduced. Scale bars: B,C = 2 mm; D,E = 500 µm; F–I = 100 µm.

    Journal: Journal of developmental biology

    Article Title: Restricted Pax3 Deletion within the Neural Tube Results in Congenital Hydrocephalus

    doi: 10.3390/jdb4010007

    Figure Lengend Snippet: Pax3 flox/flox /Pax7 −Cre conditional knockout mice exhibit congenital hydrocephalus, indicating that restricted absence of Pax3 from the Pax7 lineage is sufficient to cause defects. ( A ) Lateral views of Pax3 flox/flox /Pax7 −Cre (top) and control (bottom) P20 pups, with lateral profiles depicted by dotted lines and compared side by side to highlight the domed cranium in Pax3 flox/flox /Pax7 −Cre mutant. Insert depicts Pax7 −Cre /R26r lineage mapping in an E9.5 control embryo, with positive lacZ -labeling (blue) in the cranial neural tube (black arrow) with negligible labeling of neural crest structures (red arrow); ( B–I ) Hematoxylin and eosin sections of P6 Pax3 flox/flox /Pax7 −Cre ( B,D,F,H ) and control ( C,E,G,I ) littermates. Note that mutant lateral ventricle is dilated and loss of tissue is apparent ( B ). Enlarged views of boxed areas in ( B,C ) reveals the lack of connection between the mutant third ventricle and aqueduct (asterisk, D ) compared to control littermate ( E ); Arrow in E indicates the control posterior part of the third ventricle connecting to the aqueduct (asterisk). When coronal sections of the posterior part of the control ( G ) third ventricles are compared to Pax3 flox/flox /Pax7 −Cre mutants ( F ), the opening of the mutant ventricle is significantly narrowed; high-power views of the mutant ( H ) and control ( I ) superior sagittal sinus (arrows) confirm that the mutant lumen size is significantly reduced. Scale bars: B,C = 2 mm; D,E = 500 µm; F–I = 100 µm.

    Article Snippet: Dilution of primary antibodies was 1:200 for goat anti-Pax3 (Santa Cruz sc-7748, Dallas, TX, USA); 1:200 for mouse anti-Pax7 (Hybridoma Bank, Iowa City, IA, USA); 1:25 for monoclonal rat anti-Ki67 (DAKO, Carpinteria, CA, USA); 1:1000 for rabbit anti-β-galactosidase (Molecular Probes, Eugene, OR, USA); and 1:200 for rabbit anti-β-Catenin (Sigma, St. Louis, MO, USA).

    Techniques: Knock-Out, Mouse Assay, Mutagenesis, Labeling

    Pax3 flox/flox /Wnt1-Cre conditional knockout mice exhibit congenital hydrocephalus, indicating that restricted loss of Pax3 within both the neural tube andmigratory neural crest is sufficient to cause defects. ( A,B ) Dorsal view of postnatal (P) day 1 heads in Pax3 flox/flox /Wnt1-Cre ( A ) and control ( B ) littermates. Lines drawn at similar anatomical planes measure the width of the heads, showing mild enlargement in mutants ( A ); ( C,D ) Lateral view of P6 ( C ) and P12 ( D ) Pax3 flox/flox /Wnt1-Cre (top) and control (bottom) littermates. Note the generalized pigmentation defect in mutants; ( E ) Dashed lines from C,D illustrate the domed (red) mutant cranium; ( F,G ) Dorsal view of skeletal preparation of P20 Pax3 flox/flox /Wnt1-Cre ( F ) and control ( G ) heads; Note sagittal (arrowhead) and transverse (arrow) suture fusion defects in only the mutants ( F ); ( H,I ) Transverse views of P1 heads from animals in ( A,B ); significant dilation of lateral ventricle (arrows) is already present in Pax3 flox/flox /Wnt1-Cre mutants ( H ); dashed lines in control ( I ) indicate normal lateral ventricles; ( J,K ) Transverse view of P6 heads. Further dilation of lateral ventricles (large arrows) and loss of interventricular tissue (asterisk) is present in Pax3 flox/flox /Wnt1-Cre mutants ( J ). As a consequence of expansion of mutant lateral ventricle, a thinned cortex (red line) also results.

    Journal: Journal of developmental biology

    Article Title: Restricted Pax3 Deletion within the Neural Tube Results in Congenital Hydrocephalus

    doi: 10.3390/jdb4010007

    Figure Lengend Snippet: Pax3 flox/flox /Wnt1-Cre conditional knockout mice exhibit congenital hydrocephalus, indicating that restricted loss of Pax3 within both the neural tube andmigratory neural crest is sufficient to cause defects. ( A,B ) Dorsal view of postnatal (P) day 1 heads in Pax3 flox/flox /Wnt1-Cre ( A ) and control ( B ) littermates. Lines drawn at similar anatomical planes measure the width of the heads, showing mild enlargement in mutants ( A ); ( C,D ) Lateral view of P6 ( C ) and P12 ( D ) Pax3 flox/flox /Wnt1-Cre (top) and control (bottom) littermates. Note the generalized pigmentation defect in mutants; ( E ) Dashed lines from C,D illustrate the domed (red) mutant cranium; ( F,G ) Dorsal view of skeletal preparation of P20 Pax3 flox/flox /Wnt1-Cre ( F ) and control ( G ) heads; Note sagittal (arrowhead) and transverse (arrow) suture fusion defects in only the mutants ( F ); ( H,I ) Transverse views of P1 heads from animals in ( A,B ); significant dilation of lateral ventricle (arrows) is already present in Pax3 flox/flox /Wnt1-Cre mutants ( H ); dashed lines in control ( I ) indicate normal lateral ventricles; ( J,K ) Transverse view of P6 heads. Further dilation of lateral ventricles (large arrows) and loss of interventricular tissue (asterisk) is present in Pax3 flox/flox /Wnt1-Cre mutants ( J ). As a consequence of expansion of mutant lateral ventricle, a thinned cortex (red line) also results.

    Article Snippet: Dilution of primary antibodies was 1:200 for goat anti-Pax3 (Santa Cruz sc-7748, Dallas, TX, USA); 1:200 for mouse anti-Pax7 (Hybridoma Bank, Iowa City, IA, USA); 1:25 for monoclonal rat anti-Ki67 (DAKO, Carpinteria, CA, USA); 1:1000 for rabbit anti-β-galactosidase (Molecular Probes, Eugene, OR, USA); and 1:200 for rabbit anti-β-Catenin (Sigma, St. Louis, MO, USA).

    Techniques: Knock-Out, Mouse Assay, Mutagenesis

    5f restored neural tube development and marker expression in chick embryos on EDD 5. Glucose concentration ( A ) and Pax3 protein expression ( B ) were determined in chick embryos. The plasma glucose concentration was measured using a glucose oxidase-coupled spectrophotometric assay kit. Proteins were detected using the monoclonal antibody anti-Pax3 diluted 1:1000 (DSHB, USA) and visualized using anti-mouse IgG conjugated with horseradish peroxidase (HRP) and Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, USA) as the substrate of HRP. The abbreviations “CON”, “GLU”, “CL”, “CM”, “CH”, “EPA”, “EDA” mean “controlled”, “glucose treated”, “low concentration of 5f treated”, “mild concentration of 5f treated”, “high concentration of 5f treated”, “epalrestat treated”, “edaravone treated” groups, respectively. Values were expressed as mean ± SD in each group (n = 10). * P

    Journal: Scientific Reports

    Article Title: Bioactivity Focus of α-Cyano-4-hydroxycinnamic acid (CHCA) Leads to Effective Multifunctional Aldose Reductase Inhibitors

    doi: 10.1038/srep24942

    Figure Lengend Snippet: 5f restored neural tube development and marker expression in chick embryos on EDD 5. Glucose concentration ( A ) and Pax3 protein expression ( B ) were determined in chick embryos. The plasma glucose concentration was measured using a glucose oxidase-coupled spectrophotometric assay kit. Proteins were detected using the monoclonal antibody anti-Pax3 diluted 1:1000 (DSHB, USA) and visualized using anti-mouse IgG conjugated with horseradish peroxidase (HRP) and Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, USA) as the substrate of HRP. The abbreviations “CON”, “GLU”, “CL”, “CM”, “CH”, “EPA”, “EDA” mean “controlled”, “glucose treated”, “low concentration of 5f treated”, “mild concentration of 5f treated”, “high concentration of 5f treated”, “epalrestat treated”, “edaravone treated” groups, respectively. Values were expressed as mean ± SD in each group (n = 10). * P

    Article Snippet: Proteins were detected using the monoclonal antibody anti-Pax3 diluted 1:1000 (DSHB, USA) and visualized using anti-mouse IgG conjugated with horseradish peroxidase (HRP) and Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, USA) as the substrate of HRP.

    Techniques: Marker, Expressing, Concentration Assay, Spectrophotometric Assay, Western Blot

    Analysis of relative hair color gene expression for evidence of a transcriptome profile, gp100 expression and MC4R protein expression in the skin of Cdk5 knockdown mice. ( A ) Western blot analysis of MITF, TYRP1, TYRP2, PAX3, MC1R, gp100 and MC4R protein expression in the skin of Cdk5 knockdown mice comp ompared to the wild-type mouse ared with wild-type mice. ( B ) The abundance of the MITF, TYRP1, TYRP2, PAX3 and MC1R proteins was quantified using Image-Pro Plus software and normalized relative to the abundance of β-actin. Bars represent the mean ± standard error (n = 3). ***P

    Journal: Scientific Reports

    Article Title: Functional Role of Cyclin-Dependent Kinase 5 in the Regulation of Melanogenesis and Epidermal Structure

    doi: 10.1038/s41598-017-12567-1

    Figure Lengend Snippet: Analysis of relative hair color gene expression for evidence of a transcriptome profile, gp100 expression and MC4R protein expression in the skin of Cdk5 knockdown mice. ( A ) Western blot analysis of MITF, TYRP1, TYRP2, PAX3, MC1R, gp100 and MC4R protein expression in the skin of Cdk5 knockdown mice comp ompared to the wild-type mouse ared with wild-type mice. ( B ) The abundance of the MITF, TYRP1, TYRP2, PAX3 and MC1R proteins was quantified using Image-Pro Plus software and normalized relative to the abundance of β-actin. Bars represent the mean ± standard error (n = 3). ***P

    Article Snippet: Western blot analysis Western blot analysis was performed as described with the following primary antibodies: polyclonal rabbit anti-TYR antibody (1:1000, Santa Cruz Biotechnology), polyclonal rabbit anti-β-actin antibody (1:800, Sigma, Germany), polyclonal rabbit anti-K10 antibody (1:1000, Abcam), rabbit anti-TYRP1 (1:1000, Abcam), mouse anti-MITF (1:1000, Thermo, USA), rabbit anti-PAX3 (1:200, Bioss), rabbit anti-MC1R (1:700, Abcam), polyclonal rabbit anti-Cdk5 antibody (1:200, Abcam), rabbit anti-gp100 (1:500, Abcam) and polyclonal rabbit anti-MC4R antibody (1:500, Abcam), rabbit anti-IL1R (1:500, Abcam), rabbit anti-NASM antibody (1:500, Abcam) and rabbit TGFβIIR (1:500, Abcam).

    Techniques: Expressing, Mouse Assay, Western Blot, Software