anti-sox10 Search Results


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  • 91
    Thermo Fisher anti sox10
    Oligodendrocyte dynamics during motor skill learning. ( a ) Experimental design: all mice (approximately equal numbers of male and female) were given tamoxifen by gavage on 4 successive days (P60 to P63 inclusive), then EdU was administered in the drinking water for 10 days (P75 to P84) before transferring the mice to cages equipped with a complex wheel for up to 8 days. ( b ) Subcortical white matter of wild type mice housed with a wheel for 8 days (“8-day runners”). The great majority (~97%) of EdU + cells were also <t>Sox10</t> + oligodendrocyte lineage cells. At this time point there is a mixture of CC1-negative presumptive OPs (arrowhead) and CC1 + newly-formed oligodendrocytes (arrows). Images are representative of > 3 similar experiments. ( c , d ) Numbers of newly-generated (EdU + ) oligodendrocyte lineage cells at different developmental stages in 2-day runners versus control littermates, housed without a wheel (“non-runners”). The number of EdU + CC1 + newly-formed oligodendrocytes is the same in 2-day runners and non-runners, both in motor cortex ( c ) and underlying white matter (WM) ( d ). The number of recently generated OPs (EdU + Pdgfra + ) is decreased in 2-day runners compared to non-runners, with a reciprocal increase in the number of newly-differentiating oligodendrocytes (EdU + Pdgfra – CC1 – ). ( e , f ) Production of (EdU + CC1 + ) new myelinating oligodendrocytes is accelerated in both motor cortex ( e ) and subcortical white matter ( f ) of runners versus non-runners. The new oligodendrocytes accumulate between 4 and 8 days running. The number of new oligodendrocytes is strongly reduced in P-Myrf –/– mice, both runners and non-runners, compared to wild type mice (non-runner 8 days, Motor cortex: p =0.00091, t= –4.71, df=9; Sub-cortical white matter: p
    Anti Sox10, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti sox10 antibodies
    Hes5 regulates the expression of Mash1 and <t>Sox10.</t> RT–PCR of RNA isolated from Hes5−/−mice during the first 11 days of postnatal development revealed higher levels of Mash1 and Sox10 (arrows) compared to Hes5+/+ siblings ( A ). The increased levels of Mash1 and Sox10 transcripts were confirmed by quantitative PCR. The levels were normalized to GAPDH and expressed as percentage of the values detected in Hes5+/+ ( B ). The bar graphs represent the average and standard deviation ( * P ⩽0.05, *** P ⩽0.0005). Immunohistochemistry confirmed the higher level of Sox10 expression in the corpus callosum of p11 Hes5−/− mice (scale bar=25 μm) ( C ). RT–PCR of samples isolated from transfected progenitors supported the inhibitory effect of Hes5 levels on Sox10 and Mash1 expression ( D ). Note that the levels of other transcription factors (i.e. Olig1, Olig2, Id2) was similar in Hes5 and pcDNA-transected controls (D). The deletion of the DNA-binding domain of Hes5 (tHes5) abolished the ability of this molecule to inhibit Sox10 and Mash1 ( E ).
    Anti Sox10 Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti sox10
    Expression of <t>Sox10</t> was comparable in transgenic mice and controls. (A) Tissue lysates ( n =3) from 3-day-old sciatic nerves of transgenic (Tg) and control (Ctrl) mice were used for immunoblotting with an anti-Sox10 or actin antibody. (B) The scanned bands were densitometrically analyzed for quantification.
    Anti Sox10, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boster Bio anti sox10
    Pterygium stem cells induced spheres retain characteristic of stem cells. The spheres expressed multi-lineage stem cells markers including SOX2, SOX9, <t>SOX10,</t> NESTIN, VIMENTIN and CD44. IgG-Cy3 (red) was used as the secondary antibody. The nuclei were counterstained with Hoechst 33342 (blue). The scale bars are 50 μm.
    Anti Sox10, supplied by Boster Bio, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam anti sox10 antibody
    ILA molecular characterization in rhesus macaque ( Macaca mulatta ) cerebral cortex. ILA express Vimentin, APC, S100β, AQP4, and do not express Olig2, <t>Sox10,</t> NeuN, MAP2, Iba1. (a) GFAP (green)–Vimentin (red), (b) GFAP (green)–APC
    Anti Sox10 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Marque sox10
    Histological comparison of the embryo injection melanoma model and TEAZ melanoma model. (A) The left images show a melanoma created by injection of an mitfa :BRAF V600E -tdTomato (fusion) transgene into a tp53 −/− background ( n =1). Right images show a TEAZ-based melanoma created by electroporation of miniCoopR:GFP plus ubb :Cas9 plus zfU6 :sgRNA against Rb1 ( n =1) (example shown is fish at 16 weeks also shown in Fig. 2 A). (B) H E staining of both tumors shows similar histology, although with increased melanin pigmentation in the TEAZ tumor (also shown in Fig. 3 A). (C,D) Antibody staining against BRAF V600E shows that both tumors are widely BRAF V600E positive, which correlates with high levels of phospho-ERK staining. (E) Reflecting the neural crest origin of melanocytes, both tumors show strong nuclear expression of <t>SOX10.</t> Images are visualized at 4× and 40×. Scale bars: 500 μm (4×) and 50 μm (40×). Dashed line boxes indicate the area enlarged at 40×.
    Sox10, supplied by Cell Marque, used in various techniques. Bioz Stars score: 92/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology anti sox10
    <t>Sox10</t> is necessary and sufficient for the activity of ERBB3 _MCS6 in vitro . (A) Luciferase activities of wild-type ERBB3_ MCS6 and TFBS mutations in ERBB3_ MCS6 in melan-A cell line. The position of each TFBS in ERBB3_ MCS6 is shown on top and the corresponding mutation in each TFBS is shown next to the luciferase value for that construct. * indicates statistical significance. (B) Luciferase activity of WT ERBB3_ MCS6 in melan-A cells in mock-transfected cells and in cells with transient Sox10 and Ap2 knockdown. (C) Western blot to confirm knockdown of Sox10 and Ap2 protein upon siRNA treatment. Tubulin antibody was used as a loading control. (D) Western blot showing Erbb3 protein levels in melan-a cells upon transient transfection with Sox10 and Ap2 siRNA or mock-transfected cells. Tubulin antibody was used as a loading control. (E) Luciferase activity of ERBB3 _MCS6 upon knockdown of Sox10 in S16 cells using a dominant negative SOX10 mutant (E189X) under a CMV prmoter. (F) Luciferase assay of WT and SOXE-2m ERBB3_ MCS6 in Neuro2A cells when transiently co-transfected with an empty expression vector (pcDNA.31) and Sox10 cDNA. Cell lysates were collected 24 hours post transfection. (G) Luciferase assay of WT ERBB3_ MCS6 in Neuro2A cells when transiently co-transfected with equal amounts of WT and Sox10-ΔSTP cDNA either individually or in combination. Cell lysates were collected 24 hours post transfection. All luciferase values are normalized to renilla internal control and shown as fold-change compared to promoter only construct (pe1B) with standard deviation. (H) Real-time PCR of Erbb3 transcript levels upon expression of WT and Sox10-ΔSTP cDNA individually and in combination. Values are normalized to 18s internal control and are shown as fold-change compared to promoter only construct (pcDNA3.1) with standard error. (I) Real-time PCR of ChIP against Sox10 in untreated S16 cells and in S16 cells treated with Sox10 morpholino. Black bar indicates enrichment upon Sox10 ChIP whereas grey bar indicates enrichment with non-specific IgG. Error bars indicate standard deviation of technical replicates in real-time PCR.
    Anti Sox10, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 270 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biocare Medical sox10
    <t>Sox10</t> is necessary and sufficient for the activity of ERBB3 _MCS6 in vitro . (A) Luciferase activities of wild-type ERBB3_ MCS6 and TFBS mutations in ERBB3_ MCS6 in melan-A cell line. The position of each TFBS in ERBB3_ MCS6 is shown on top and the corresponding mutation in each TFBS is shown next to the luciferase value for that construct. * indicates statistical significance. (B) Luciferase activity of WT ERBB3_ MCS6 in melan-A cells in mock-transfected cells and in cells with transient Sox10 and Ap2 knockdown. (C) Western blot to confirm knockdown of Sox10 and Ap2 protein upon siRNA treatment. Tubulin antibody was used as a loading control. (D) Western blot showing Erbb3 protein levels in melan-a cells upon transient transfection with Sox10 and Ap2 siRNA or mock-transfected cells. Tubulin antibody was used as a loading control. (E) Luciferase activity of ERBB3 _MCS6 upon knockdown of Sox10 in S16 cells using a dominant negative SOX10 mutant (E189X) under a CMV prmoter. (F) Luciferase assay of WT and SOXE-2m ERBB3_ MCS6 in Neuro2A cells when transiently co-transfected with an empty expression vector (pcDNA.31) and Sox10 cDNA. Cell lysates were collected 24 hours post transfection. (G) Luciferase assay of WT ERBB3_ MCS6 in Neuro2A cells when transiently co-transfected with equal amounts of WT and Sox10-ΔSTP cDNA either individually or in combination. Cell lysates were collected 24 hours post transfection. All luciferase values are normalized to renilla internal control and shown as fold-change compared to promoter only construct (pe1B) with standard deviation. (H) Real-time PCR of Erbb3 transcript levels upon expression of WT and Sox10-ΔSTP cDNA individually and in combination. Values are normalized to 18s internal control and are shown as fold-change compared to promoter only construct (pcDNA3.1) with standard error. (I) Real-time PCR of ChIP against Sox10 in untreated S16 cells and in S16 cells treated with Sox10 morpholino. Black bar indicates enrichment upon Sox10 ChIP whereas grey bar indicates enrichment with non-specific IgG. Error bars indicate standard deviation of technical replicates in real-time PCR.
    Sox10, supplied by Biocare Medical, used in various techniques. Bioz Stars score: 91/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GeneTex anti sox10
    Systemic <t>Sox10</t> activation in SCs following amputation. (A) Scheme of the experiment. (B–H) Caudal fin of adult fish was amputated ( t = 0, blue arrow ), and the axon cytoskeleton ( red ) and immature SCs ( white ) were visualized over time through immunofluorescence staining for the axonal marker, acetylated tubulin ( red ), and the immature SC marker, Sox10 ( white ), respectively. Immunostaining in uncut fin (B) or after amputation (C–H) . (B–G) The upper panel shows the distal part of the amputated fin, and the lower panel shows a more proximal part. (H′) Shows a higher magnification of (H) . Dotted line : amputation plane. Dashed line : distal part of the fin. Scale bars = 50 μ M . R, ray; IR, inter-ray. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
    Anti Sox10, supplied by GeneTex, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Abcam anti sox10 antibody epr4007
    Systemic <t>Sox10</t> activation in SCs following amputation. (A) Scheme of the experiment. (B–H) Caudal fin of adult fish was amputated ( t = 0, blue arrow ), and the axon cytoskeleton ( red ) and immature SCs ( white ) were visualized over time through immunofluorescence staining for the axonal marker, acetylated tubulin ( red ), and the immature SC marker, Sox10 ( white ), respectively. Immunostaining in uncut fin (B) or after amputation (C–H) . (B–G) The upper panel shows the distal part of the amputated fin, and the lower panel shows a more proximal part. (H′) Shows a higher magnification of (H) . Dotted line : amputation plane. Dashed line : distal part of the fin. Scale bars = 50 μ M . R, ray; IR, inter-ray. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
    Anti Sox10 Antibody Epr4007, supplied by Abcam, used in various techniques. Bioz Stars score: 88/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc sox10
    Proliferative defects in TSC2-SCKO nerves. ( A ) Quantification of glial nuclei in sciatic nerve semithin cross-sections from control and TSC2-SCKO mice at the indicated postnatal ages reveals progressively increasing numbers of developing SCs in the mutant nerves. n = 3 mice per genotype at each age. ( B , Left ) Fluorescence microscopy analysis of immunostained teased fiber preparations from tibial nerves of P28 control and TSC2-SCKO mice using the indicated markers. Note increased numbers of <t>SOX10</t> + SCs (arrows) associated with axon segments in TSC2-SCKO nerves compared with control. (Scale bars, 10 µm.) ( Right ) Quantification of SOX10 + SCs along TUJ1 + axons ( n = 3 mice per genotype). ( C ) Quantitative immunofluorescence of longitudinal sciatic nerve sections (confocal z -series projections) from control and TSC2-SCKO mice at ages P14 (c-Casp3) and P21 (Ki67, p-H3) to demonstrate increased cell cycling ( Top ; arrows depict Ki67 + cells) and mitotic events ( Middle ; arrows depict p-H3 + cells), and induction of SC apoptosis ( Bottom ; arrows depcit c-Casp3 + cells) in the mutant ( n = 3 mice per genotype at each age). (Scale bars, 50 µm.) ( D , Upper ) Schematic illustrating cyclin expression during cell cycle, and checkpoint control by the restriction (R) point. ( Lower ) Western blots of sciatic nerve lysates from control and TSC2-SCKO mice at age P7 (three mice per group), probed with the indicated antibodies, demonstrating that TSC2-deficient SCs bear elevated levels of cyclin B1 (marker for M-phase) and D1 (marker for G 1 /S-phase). ( E ) Western blots of sciatic nerve lysates from control and TSC2-SCKO mice at age P7 (three mice per group) probed with the indicated antibodies, demonstrating hyperphosphorylation and thus inactivation of Rb in the mutant allowing uncontrolled passage through the G 1 restriction (R) point of the cell cycle.
    Sox10, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems sox10
    Morphological and immunophenotypical changes induced by BrdU glial differentiation treatment . A . Morphological changes in SK-N-ER cells (Phase contrast microscopy; 100×). B. GD2 expression levels after 15 days of treatment in I-type cell line SK-N-Be2C. C Calcyclin, ( D ) <t>Sox10</t> and ( E ) S100 expression after 21 days of BrdU treatment in N-type cell line LA1-55N. F . SA-β-GAL positive cells (arrow) after 21 days of BrdU treatment in the I-type SK-N-ER (Phase contrast; 100×). Control cell images are reported in the small squares. Scale bar: 50 μm.
    Sox10, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti sox10
    Mef2c -F1 is a bona fide Endothelin-responsive enhancer of Mef2c . (A) Schematics of the mouse Mef2c locus showing exons 4-6 (vertical black lines) and the Mef2c -F1 enhancer (green box). The Mef2c F1 Δ and Mef2c -null ( Mef2c Δ ) knockout strategies are indicated. (B) Number of live and dead offspring of each indicated genotype from Mef2c +/F1 Δ × Mef2c +/Δ intercrosses. Note that 23/23 wt and heterozygous (het) offspring survived, whereas only 2/7 Mef2c F1 Δ / Δ survived (Fisher's exact test, P =0.0001). (C-F″) The Mef2c -F1 enhancer is required for ET-1 to induce endogenous MEF2C expression in trunk neural crest (NC) cells. ET-1 induced endogenous MEF2C protein expression in trunk neural crest cells (marked by <t>Sox10</t> immunofluorescence) in ET-1-treated (D-D″) but not in PBS-treated (C-C″) explants. Note MEF2C expression in skeletal muscle (SkM) in both PBS- and ET-1-treated explants (C′,D′). ET-1 treatment failed to induce endogenous MEF2C protein expression in trunk neural crest cells in Mef2c F1 Δ /F1 Δ explants (F). Note the absence of co-expression of MEF2C and Sox10 in Mef2c F1 Δ /F1 Δ explants treated with either PBS (E-E″) or ET-1 (F-F″). NT, neural tube. Scale bars: 100 µm.
    Anti Sox10, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore sox10
    PEDF induces <t>DCX+/Sox10+</t> cells in the adult SVZ Saline or PEDF (300ng/ml) was infused into the lateral ventricle of adult wild type mice for 7 days before sacrifice. (A–B) Confocal images of saline-infused SVZ double-immunostained for DCX and Sox10. No DCX+ cells expressed Sox10 in the SVZ. (C–D) Orthogonal (C) and Z-series stack (D) confocal images of PEDF-infused SVZ. PEDF infusion induced DCX+/Sox10+ cells in the SVZ. The Insets in D show magnified images for the area indicated by a rectangle. Scale bar = 20μm.
    Sox10, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Oligodendrocyte dynamics during motor skill learning. ( a ) Experimental design: all mice (approximately equal numbers of male and female) were given tamoxifen by gavage on 4 successive days (P60 to P63 inclusive), then EdU was administered in the drinking water for 10 days (P75 to P84) before transferring the mice to cages equipped with a complex wheel for up to 8 days. ( b ) Subcortical white matter of wild type mice housed with a wheel for 8 days (“8-day runners”). The great majority (~97%) of EdU + cells were also Sox10 + oligodendrocyte lineage cells. At this time point there is a mixture of CC1-negative presumptive OPs (arrowhead) and CC1 + newly-formed oligodendrocytes (arrows). Images are representative of > 3 similar experiments. ( c , d ) Numbers of newly-generated (EdU + ) oligodendrocyte lineage cells at different developmental stages in 2-day runners versus control littermates, housed without a wheel (“non-runners”). The number of EdU + CC1 + newly-formed oligodendrocytes is the same in 2-day runners and non-runners, both in motor cortex ( c ) and underlying white matter (WM) ( d ). The number of recently generated OPs (EdU + Pdgfra + ) is decreased in 2-day runners compared to non-runners, with a reciprocal increase in the number of newly-differentiating oligodendrocytes (EdU + Pdgfra – CC1 – ). ( e , f ) Production of (EdU + CC1 + ) new myelinating oligodendrocytes is accelerated in both motor cortex ( e ) and subcortical white matter ( f ) of runners versus non-runners. The new oligodendrocytes accumulate between 4 and 8 days running. The number of new oligodendrocytes is strongly reduced in P-Myrf –/– mice, both runners and non-runners, compared to wild type mice (non-runner 8 days, Motor cortex: p =0.00091, t= –4.71, df=9; Sub-cortical white matter: p

    Journal: Nature neuroscience

    Article Title: Rapid production of new oligodendrocytes is required in the earliest stages of motor skill learning

    doi: 10.1038/nn.4351

    Figure Lengend Snippet: Oligodendrocyte dynamics during motor skill learning. ( a ) Experimental design: all mice (approximately equal numbers of male and female) were given tamoxifen by gavage on 4 successive days (P60 to P63 inclusive), then EdU was administered in the drinking water for 10 days (P75 to P84) before transferring the mice to cages equipped with a complex wheel for up to 8 days. ( b ) Subcortical white matter of wild type mice housed with a wheel for 8 days (“8-day runners”). The great majority (~97%) of EdU + cells were also Sox10 + oligodendrocyte lineage cells. At this time point there is a mixture of CC1-negative presumptive OPs (arrowhead) and CC1 + newly-formed oligodendrocytes (arrows). Images are representative of > 3 similar experiments. ( c , d ) Numbers of newly-generated (EdU + ) oligodendrocyte lineage cells at different developmental stages in 2-day runners versus control littermates, housed without a wheel (“non-runners”). The number of EdU + CC1 + newly-formed oligodendrocytes is the same in 2-day runners and non-runners, both in motor cortex ( c ) and underlying white matter (WM) ( d ). The number of recently generated OPs (EdU + Pdgfra + ) is decreased in 2-day runners compared to non-runners, with a reciprocal increase in the number of newly-differentiating oligodendrocytes (EdU + Pdgfra – CC1 – ). ( e , f ) Production of (EdU + CC1 + ) new myelinating oligodendrocytes is accelerated in both motor cortex ( e ) and subcortical white matter ( f ) of runners versus non-runners. The new oligodendrocytes accumulate between 4 and 8 days running. The number of new oligodendrocytes is strongly reduced in P-Myrf –/– mice, both runners and non-runners, compared to wild type mice (non-runner 8 days, Motor cortex: p =0.00091, t= –4.71, df=9; Sub-cortical white matter: p

    Article Snippet: The animals were caged with a complex wheel for different times and then were perfusion fixed and analyzed by immunolabeling floating cryosections (20 μm) with monoclonal CC1, anti-Sox10, and anti-Pdgfra followed by detection of EdU using the AlexaFluor-555 Click-iT detection kit (Invitrogen).

    Techniques: Mouse Assay, Transferring, Generated

    OPCs ectopically exit the spinal cord in erbb3b −/− larvae. A–D , Lateral views of the spinal cord with dorsal to the top and anterior to the left. A , At 55 hpf in nkx2.2a:megfp;sox10:mrfp larvae, sox10 + peripheral glial cells (arrowheads) associate with spinal motor axons in the periphery. B , In contrast, sox10 + glial cells fail to associate with spinal motor axons in nkx2.2a:megfp;sox10:mrfp;erbb3b −/− larvae at 53 hpf. C , Frames captured from a 17 h time-lapse movie of a nkx2.2a:megfp;olig2:dsred;erbb3b −/− larva beginning at 57 hpf. Because the olig2:dsred transgene labels both motor axons and OPCs, an arrowhead is used to identify the OPC. An nkx2.2a + ;olig2 + OPC (arrowhead) in the ventral spinal cord migrates toward the MEP TZ and ectopically exits the CNS. Bottom left corner, Numbers indicate the time that has elapsed from the first frame of the figure. D , Lateral view of 2 dpf nkx2.2a;olig2:dsred wildtype and erbb3b −/− larvae labeled with a Sox10 antibody (blue, arrowheads). E , Quantification of Sox10 + glial cells along peripheral motor axons in wildtype and erbb3b −/− larvae at 2, 3, 6, and 8 dpf. Sox10 + glial cells were significantly reduced in erbb3b −/− larvae at all time points evaluated ( p

    Journal: The Journal of Neuroscience

    Article Title: Perineurial Glial Plasticity and the Role of TGF-β in the Development of the Blood–Nerve Barrier

    doi: 10.1523/JNEUROSCI.2875-16.2017

    Figure Lengend Snippet: OPCs ectopically exit the spinal cord in erbb3b −/− larvae. A–D , Lateral views of the spinal cord with dorsal to the top and anterior to the left. A , At 55 hpf in nkx2.2a:megfp;sox10:mrfp larvae, sox10 + peripheral glial cells (arrowheads) associate with spinal motor axons in the periphery. B , In contrast, sox10 + glial cells fail to associate with spinal motor axons in nkx2.2a:megfp;sox10:mrfp;erbb3b −/− larvae at 53 hpf. C , Frames captured from a 17 h time-lapse movie of a nkx2.2a:megfp;olig2:dsred;erbb3b −/− larva beginning at 57 hpf. Because the olig2:dsred transgene labels both motor axons and OPCs, an arrowhead is used to identify the OPC. An nkx2.2a + ;olig2 + OPC (arrowhead) in the ventral spinal cord migrates toward the MEP TZ and ectopically exits the CNS. Bottom left corner, Numbers indicate the time that has elapsed from the first frame of the figure. D , Lateral view of 2 dpf nkx2.2a;olig2:dsred wildtype and erbb3b −/− larvae labeled with a Sox10 antibody (blue, arrowheads). E , Quantification of Sox10 + glial cells along peripheral motor axons in wildtype and erbb3b −/− larvae at 2, 3, 6, and 8 dpf. Sox10 + glial cells were significantly reduced in erbb3b −/− larvae at all time points evaluated ( p

    Article Snippet: The primary antibodies used in this study include the following: mouse anti-acetylated tubulin (1:5000, Sigma catalog #T7451, RRID:AB_609894), a rabbit antibody to Sox10 (1:5000, Thermo Fisher Scientific catalog #Kucenaslab_001, RRID:AB_2637056) , a mouse antibody to zonula occludens-1 (ZO-1; 1:200, Invitrogen catalog #33–9100, RRID:AB_2533147), and a rabbit antibody to anti-phospho-Smad3 (1:175, Abcam catalog #ab52903, RRID:AB_882596) ( ).

    Techniques: Labeling

    Perineurial glial proliferation is normal in erbb3b −/− larvae. All images are lateral views of the spinal cord, with dorsal to the top and anterior to the left. A , Frames captured from a 16 h time-lapse movie of a 55 hpf nkx2.2a:megfp;sox10:mrfp;erbb3b −/− larva reveals that perineurial glial proliferation (dots) occurs despite the presence of an ectopically located OPC (arrowhead). B , Frames captured from a 13 h time-lapse movie of a 59 hpf nkx2.2a:megfp;sox10:mrfp;erbb3b −/− larva reveals that perineurial glial proliferation (dots) also occurs in the absence of ectopically located OPCs. Bottom left corner, Numbers indicate the time that has elapsed from the first frame of the figure. Scale bars, 25 μm.

    Journal: The Journal of Neuroscience

    Article Title: Perineurial Glial Plasticity and the Role of TGF-β in the Development of the Blood–Nerve Barrier

    doi: 10.1523/JNEUROSCI.2875-16.2017

    Figure Lengend Snippet: Perineurial glial proliferation is normal in erbb3b −/− larvae. All images are lateral views of the spinal cord, with dorsal to the top and anterior to the left. A , Frames captured from a 16 h time-lapse movie of a 55 hpf nkx2.2a:megfp;sox10:mrfp;erbb3b −/− larva reveals that perineurial glial proliferation (dots) occurs despite the presence of an ectopically located OPC (arrowhead). B , Frames captured from a 13 h time-lapse movie of a 59 hpf nkx2.2a:megfp;sox10:mrfp;erbb3b −/− larva reveals that perineurial glial proliferation (dots) also occurs in the absence of ectopically located OPCs. Bottom left corner, Numbers indicate the time that has elapsed from the first frame of the figure. Scale bars, 25 μm.

    Article Snippet: The primary antibodies used in this study include the following: mouse anti-acetylated tubulin (1:5000, Sigma catalog #T7451, RRID:AB_609894), a rabbit antibody to Sox10 (1:5000, Thermo Fisher Scientific catalog #Kucenaslab_001, RRID:AB_2637056) , a mouse antibody to zonula occludens-1 (ZO-1; 1:200, Invitrogen catalog #33–9100, RRID:AB_2533147), and a rabbit antibody to anti-phospho-Smad3 (1:175, Abcam catalog #ab52903, RRID:AB_882596) ( ).

    Techniques:

    Perineurial glial development in zebrafish. A , Diagram of a zebrafish embryo identifying the CNS, which consists of the brain and spinal cord (red), and the peripheral spinal motor nerves (green). B , Diagram representing a cross sectional view of an adult peripheral motor nerve. Axons are wrapped by peripheral myelinating glial cells (myelin sheath, red) and are surrounded by the endoneurium (purple). Several myelinated axons are bundled by the perineurium (green) to form a fascicle. The epineurium (yellow) then encases several fascicles to form a nerve. C–E , All images are lateral views of the spinal cord, with dorsal to the top and anterior to the left. In wildtype nkx2.2a:megfp;sox10:mrfp zebrafish larvae, nkx2.2a + perineurial glia (arrowheads) exit the CNS by ∼52 hpf ( C ), proliferate ( D ), and extend ( E ) until full ensheathment of the motor nerve is achieved. Scale bar, 25 μm.

    Journal: The Journal of Neuroscience

    Article Title: Perineurial Glial Plasticity and the Role of TGF-β in the Development of the Blood–Nerve Barrier

    doi: 10.1523/JNEUROSCI.2875-16.2017

    Figure Lengend Snippet: Perineurial glial development in zebrafish. A , Diagram of a zebrafish embryo identifying the CNS, which consists of the brain and spinal cord (red), and the peripheral spinal motor nerves (green). B , Diagram representing a cross sectional view of an adult peripheral motor nerve. Axons are wrapped by peripheral myelinating glial cells (myelin sheath, red) and are surrounded by the endoneurium (purple). Several myelinated axons are bundled by the perineurium (green) to form a fascicle. The epineurium (yellow) then encases several fascicles to form a nerve. C–E , All images are lateral views of the spinal cord, with dorsal to the top and anterior to the left. In wildtype nkx2.2a:megfp;sox10:mrfp zebrafish larvae, nkx2.2a + perineurial glia (arrowheads) exit the CNS by ∼52 hpf ( C ), proliferate ( D ), and extend ( E ) until full ensheathment of the motor nerve is achieved. Scale bar, 25 μm.

    Article Snippet: The primary antibodies used in this study include the following: mouse anti-acetylated tubulin (1:5000, Sigma catalog #T7451, RRID:AB_609894), a rabbit antibody to Sox10 (1:5000, Thermo Fisher Scientific catalog #Kucenaslab_001, RRID:AB_2637056) , a mouse antibody to zonula occludens-1 (ZO-1; 1:200, Invitrogen catalog #33–9100, RRID:AB_2533147), and a rabbit antibody to anti-phospho-Smad3 (1:175, Abcam catalog #ab52903, RRID:AB_882596) ( ).

    Techniques:

    Perineurial barrier integrity is compromised in adult erbb3b −/− zebrafish. All images are cross sections of 8-month-old sox10:mrfp wildtype and erbb3b −/− zebrafish injected with a Dextran-647 dye (blue) into the muscle. A , In wildtype, the Dextran-647 dye (blue, arrowheads) remains localized to the perimeter of the motor nerve. B , In erbb3b −/− , the Dextran-647 dye (blue, arrowheads) infiltrates into the motor nerve. Scale bar, 25 μm.

    Journal: The Journal of Neuroscience

    Article Title: Perineurial Glial Plasticity and the Role of TGF-β in the Development of the Blood–Nerve Barrier

    doi: 10.1523/JNEUROSCI.2875-16.2017

    Figure Lengend Snippet: Perineurial barrier integrity is compromised in adult erbb3b −/− zebrafish. All images are cross sections of 8-month-old sox10:mrfp wildtype and erbb3b −/− zebrafish injected with a Dextran-647 dye (blue) into the muscle. A , In wildtype, the Dextran-647 dye (blue, arrowheads) remains localized to the perimeter of the motor nerve. B , In erbb3b −/− , the Dextran-647 dye (blue, arrowheads) infiltrates into the motor nerve. Scale bar, 25 μm.

    Article Snippet: The primary antibodies used in this study include the following: mouse anti-acetylated tubulin (1:5000, Sigma catalog #T7451, RRID:AB_609894), a rabbit antibody to Sox10 (1:5000, Thermo Fisher Scientific catalog #Kucenaslab_001, RRID:AB_2637056) , a mouse antibody to zonula occludens-1 (ZO-1; 1:200, Invitrogen catalog #33–9100, RRID:AB_2533147), and a rabbit antibody to anti-phospho-Smad3 (1:175, Abcam catalog #ab52903, RRID:AB_882596) ( ).

    Techniques: Injection

    Hes5 regulates the expression of Mash1 and Sox10. RT–PCR of RNA isolated from Hes5−/−mice during the first 11 days of postnatal development revealed higher levels of Mash1 and Sox10 (arrows) compared to Hes5+/+ siblings ( A ). The increased levels of Mash1 and Sox10 transcripts were confirmed by quantitative PCR. The levels were normalized to GAPDH and expressed as percentage of the values detected in Hes5+/+ ( B ). The bar graphs represent the average and standard deviation ( * P ⩽0.05, *** P ⩽0.0005). Immunohistochemistry confirmed the higher level of Sox10 expression in the corpus callosum of p11 Hes5−/− mice (scale bar=25 μm) ( C ). RT–PCR of samples isolated from transfected progenitors supported the inhibitory effect of Hes5 levels on Sox10 and Mash1 expression ( D ). Note that the levels of other transcription factors (i.e. Olig1, Olig2, Id2) was similar in Hes5 and pcDNA-transected controls (D). The deletion of the DNA-binding domain of Hes5 (tHes5) abolished the ability of this molecule to inhibit Sox10 and Mash1 ( E ).

    Journal: The EMBO Journal

    Article Title: A molecular insight of Hes5-dependent inhibition of myelin gene expression: old partners and new players

    doi: 10.1038/sj.emboj.7601352

    Figure Lengend Snippet: Hes5 regulates the expression of Mash1 and Sox10. RT–PCR of RNA isolated from Hes5−/−mice during the first 11 days of postnatal development revealed higher levels of Mash1 and Sox10 (arrows) compared to Hes5+/+ siblings ( A ). The increased levels of Mash1 and Sox10 transcripts were confirmed by quantitative PCR. The levels were normalized to GAPDH and expressed as percentage of the values detected in Hes5+/+ ( B ). The bar graphs represent the average and standard deviation ( * P ⩽0.05, *** P ⩽0.0005). Immunohistochemistry confirmed the higher level of Sox10 expression in the corpus callosum of p11 Hes5−/− mice (scale bar=25 μm) ( C ). RT–PCR of samples isolated from transfected progenitors supported the inhibitory effect of Hes5 levels on Sox10 and Mash1 expression ( D ). Note that the levels of other transcription factors (i.e. Olig1, Olig2, Id2) was similar in Hes5 and pcDNA-transected controls (D). The deletion of the DNA-binding domain of Hes5 (tHes5) abolished the ability of this molecule to inhibit Sox10 and Mash1 ( E ).

    Article Snippet: Commercially available anti-Sox10 antibodies were purchased from Chemicon, anti-MBP from Sternberg Monoclonals Incorporated and anti-Myc Tag from Upstate.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Mouse Assay, Real-time Polymerase Chain Reaction, Standard Deviation, Immunohistochemistry, Transfection, Binding Assay

    Functional interaction between Hes5 and Sox10 in the regulation of the MBP promoter. ( A ) Whole-cell lysates (wcl) from oligodendrocyte progenitors transfected with myc-tag Hes5 with or without Sox10 were used for co-immunoprecipitation studies, using antibodies against myc-tag for immunoprecipitation. The identity of the interacting proteins was defined by Western blot analysis, using antibodies specific for Sox10 or for myc-tagged Hes5. ( B ) Also, tHes5 retained the ability to bind to Sox10 as demonstrated by immunoprecipitation. ( C ) Luciferase reporter assay of cells cotransfected with MBP promoter driving reporter together with constant amounts of myc-tagged Hes5 plasmid (0.5 μg) and increasing amounts of Sox10 DNA (0.5, 1 and 1.5 μg) indicated that the inhibitory effect of Hes5 on the MBP promoter could be reversed by increasing concentrations of Sox10 ( * P ⩽0.05, *** P ⩽0.0005). ( D ) To confirm that increasing amounts of transfected Sox10 DNA corresponded to increased protein levels, we performed a Western blot analysis of protein extracts from cultures transfected with 0.5 μg of myc-tagged Hes5 plasmid together with 0.5 μg (+), 1 μg (++) and 1.5 μg (+++) of Sox10 plasmid. ( E ) ChIP was performed on samples isolated from untransfected (control), Hes5 transfected or Hes5 and Sox10 cotransfected progenitors, using antibodies against myc-tag. The immunoprecipitated products were amplified using the indicated primers. Note that in the absence of Sox10, Hes5 was recruited to the regions of the MBP promoter containing N- and E-boxes. However, in the presence of high levels of Sox10, Hes5 was displaced from the promoter.

    Journal: The EMBO Journal

    Article Title: A molecular insight of Hes5-dependent inhibition of myelin gene expression: old partners and new players

    doi: 10.1038/sj.emboj.7601352

    Figure Lengend Snippet: Functional interaction between Hes5 and Sox10 in the regulation of the MBP promoter. ( A ) Whole-cell lysates (wcl) from oligodendrocyte progenitors transfected with myc-tag Hes5 with or without Sox10 were used for co-immunoprecipitation studies, using antibodies against myc-tag for immunoprecipitation. The identity of the interacting proteins was defined by Western blot analysis, using antibodies specific for Sox10 or for myc-tagged Hes5. ( B ) Also, tHes5 retained the ability to bind to Sox10 as demonstrated by immunoprecipitation. ( C ) Luciferase reporter assay of cells cotransfected with MBP promoter driving reporter together with constant amounts of myc-tagged Hes5 plasmid (0.5 μg) and increasing amounts of Sox10 DNA (0.5, 1 and 1.5 μg) indicated that the inhibitory effect of Hes5 on the MBP promoter could be reversed by increasing concentrations of Sox10 ( * P ⩽0.05, *** P ⩽0.0005). ( D ) To confirm that increasing amounts of transfected Sox10 DNA corresponded to increased protein levels, we performed a Western blot analysis of protein extracts from cultures transfected with 0.5 μg of myc-tagged Hes5 plasmid together with 0.5 μg (+), 1 μg (++) and 1.5 μg (+++) of Sox10 plasmid. ( E ) ChIP was performed on samples isolated from untransfected (control), Hes5 transfected or Hes5 and Sox10 cotransfected progenitors, using antibodies against myc-tag. The immunoprecipitated products were amplified using the indicated primers. Note that in the absence of Sox10, Hes5 was recruited to the regions of the MBP promoter containing N- and E-boxes. However, in the presence of high levels of Sox10, Hes5 was displaced from the promoter.

    Article Snippet: Commercially available anti-Sox10 antibodies were purchased from Chemicon, anti-MBP from Sternberg Monoclonals Incorporated and anti-Myc Tag from Upstate.

    Techniques: Functional Assay, Transfection, Immunoprecipitation, Western Blot, Luciferase, Reporter Assay, Plasmid Preparation, Chromatin Immunoprecipitation, Isolation, Amplification

    Expression of Sox10 was comparable in transgenic mice and controls. (A) Tissue lysates ( n =3) from 3-day-old sciatic nerves of transgenic (Tg) and control (Ctrl) mice were used for immunoblotting with an anti-Sox10 or actin antibody. (B) The scanned bands were densitometrically analyzed for quantification.

    Journal: Data in Brief

    Article Title: Data supporting the role of Fyn in initiating myelination in the peripheral nervous system

    doi: 10.1016/j.dib.2016.03.096

    Figure Lengend Snippet: Expression of Sox10 was comparable in transgenic mice and controls. (A) Tissue lysates ( n =3) from 3-day-old sciatic nerves of transgenic (Tg) and control (Ctrl) mice were used for immunoblotting with an anti-Sox10 or actin antibody. (B) The scanned bands were densitometrically analyzed for quantification.

    Article Snippet: The following antibodies were used: polyclonal anti-MPZ and monoclonal anti-actin from MBL (Aichi, Japan); polyclonal anti-CNPase, monoclonal anti-pan-Akt, and monoclonal phosphorylated pan-Akt (active, phosphorylated Ser-473) from Cell Signaling Technology (Danvers, MA, USA); anti-Krox20, anti-Oct6, and anti-Sox10 from Abcam (Cambridge, UK); and anti-V5 epitope from Nacalai Tesque.

    Techniques: Expressing, Transgenic Assay, Mouse Assay

    Immunocytochemical characterization of hE-SCs. ( A ) Immunofluorescent staining indicated that hE-SCs expressed S100, P75NTR, GFAP, Sox10 and Krox20 proteins. With the increased purity of the hE-SCs, the positive rates of S100, P75NTR, GFAP, Sox10 and Krox20 were also increased. hE-SCs were immuno-positive for S100, P75NTR, GFAP, Sox10 and Krox20 (white arrow), while hE-FLCs were negative for staining (white triangle). (Scale bars = 100 μm) ( B ) The percentage of hE-SCs expressing S100, P75NTR, GFAP, Sox10 and Krox20. Data are presented as the mean ± S.E.M, *P

    Journal: Scientific Reports

    Article Title: Human eyelid adipose tissue-derived Schwann cells promote regeneration of a transected sciatic nerve

    doi: 10.1038/srep43248

    Figure Lengend Snippet: Immunocytochemical characterization of hE-SCs. ( A ) Immunofluorescent staining indicated that hE-SCs expressed S100, P75NTR, GFAP, Sox10 and Krox20 proteins. With the increased purity of the hE-SCs, the positive rates of S100, P75NTR, GFAP, Sox10 and Krox20 were also increased. hE-SCs were immuno-positive for S100, P75NTR, GFAP, Sox10 and Krox20 (white arrow), while hE-FLCs were negative for staining (white triangle). (Scale bars = 100 μm) ( B ) The percentage of hE-SCs expressing S100, P75NTR, GFAP, Sox10 and Krox20. Data are presented as the mean ± S.E.M, *P

    Article Snippet: The cells were then incubated with rabbit anti-glial fibrillary acidic protein (GFAP; 1:500; Abcam, Cambridge, UK), rabbit anti-P75NTR (1:500 diluted in PBS; Abcam, Cambridge, UK), rabbit anti-S100 (1:500; Dako, Glostrup, Denmark), rabbit anti-Sox10 (1:500 diluted in PBS; Abcam, Cambridge, UK), and rabbit anti-Krox10 (1:500 diluted in PBS; Abcam, Cambridge, UK) overnight at 4 °C.

    Techniques: Staining, Expressing

    UGT8 positively correlates with Sox10 and is a direct transcriptional target of Sox10. (A) Analysis of GSE25066 and TCGA datasets for the expression of UGT8 and Sox10 . The relative level of UGT8 was plotted against that of Sox10. Correlations were analyzed using Pearson’s correlation method and Spearman’s rank correlation test. (B) Box plots indicated Sox10 mRNA expression in different subtypes of breast cancer from GSE25066 and TCGA datasets. Comparisons were analyzed by one-way ANOVA. (C) Expression of UGT8 and Sox10 was analyzed by quantitative RT-PCR in SUM159 and MDA-MB436 cells infected with empty vector or Sox10-expressing vector. Data are shown as mean ± SD based on three independent experiments. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Inhibition of UGT8 suppresses basal-like breast cancer progression by attenuating sulfatide–αVβ5 axis

    doi: 10.1084/jem.20172048

    Figure Lengend Snippet: UGT8 positively correlates with Sox10 and is a direct transcriptional target of Sox10. (A) Analysis of GSE25066 and TCGA datasets for the expression of UGT8 and Sox10 . The relative level of UGT8 was plotted against that of Sox10. Correlations were analyzed using Pearson’s correlation method and Spearman’s rank correlation test. (B) Box plots indicated Sox10 mRNA expression in different subtypes of breast cancer from GSE25066 and TCGA datasets. Comparisons were analyzed by one-way ANOVA. (C) Expression of UGT8 and Sox10 was analyzed by quantitative RT-PCR in SUM159 and MDA-MB436 cells infected with empty vector or Sox10-expressing vector. Data are shown as mean ± SD based on three independent experiments. *, P

    Article Snippet: Antibodies against Sox10, Smad4, Smad5, Integrin αV, and Integrin αVβ5 were from Abcam.

    Techniques: Expressing, Quantitative RT-PCR, Multiple Displacement Amplification, Infection, Plasmid Preparation

    Pterygium stem cells induced spheres retain characteristic of stem cells. The spheres expressed multi-lineage stem cells markers including SOX2, SOX9, SOX10, NESTIN, VIMENTIN and CD44. IgG-Cy3 (red) was used as the secondary antibody. The nuclei were counterstained with Hoechst 33342 (blue). The scale bars are 50 μm.

    Journal: American Journal of Translational Research

    Article Title: Identification and differentiation therapy strategy of pterygium in vitro

    doi:

    Figure Lengend Snippet: Pterygium stem cells induced spheres retain characteristic of stem cells. The spheres expressed multi-lineage stem cells markers including SOX2, SOX9, SOX10, NESTIN, VIMENTIN and CD44. IgG-Cy3 (red) was used as the secondary antibody. The nuclei were counterstained with Hoechst 33342 (blue). The scale bars are 50 μm.

    Article Snippet: These cells and spheres were incubated with primary antibodies anti-SOX2, anti-NESTIN, anti-VIMENTIN, anti-CD44, anti-Ki67 (rabbit polyclonal; dilution 1:100: boster), anti-CD133 (rabbit polyclona; dilution 1:100: boster), anti-CD90 (rabbit polyclona; dilution 1:100: boster), anti-CD105 (rabbit polyclonal; dilution 1:100: boster), anti-YAP1 (rabbit polyclonal; dilution 1:100: boster), anti-P63 (rabbit polyclona; dilution 1:100: boster), anti-E-Cadherin (rabbit polyclonal; dilution 1:100: boster), anti-N-cadherin (rabbit polyclona; dilution 1:100: boster), anti-stat3 (rabbit monoclonal; dilution 1:300: abcam), anti-snail (rabbit monoclonal; dilution 1:300: abcam), anti-SOX9 (mouse polyclonal; dilution 1:100: boster), anti-SOX10 (mouse polyclonal; dilution 1:100: boster), at 4°C overnight and incubated with cy3-labeled secondary antibody (dilution 1:300, Invitrogen) at 37°C for 60 mins.

    Techniques:

    ILA molecular characterization in rhesus macaque ( Macaca mulatta ) cerebral cortex. ILA express Vimentin, APC, S100β, AQP4, and do not express Olig2, Sox10, NeuN, MAP2, Iba1. (a) GFAP (green)–Vimentin (red), (b) GFAP (green)–APC

    Journal: The Journal of comparative neurology

    Article Title: Cortical interlaminar astrocytes across the therian mammal radiation

    doi: 10.1002/cne.24605

    Figure Lengend Snippet: ILA molecular characterization in rhesus macaque ( Macaca mulatta ) cerebral cortex. ILA express Vimentin, APC, S100β, AQP4, and do not express Olig2, Sox10, NeuN, MAP2, Iba1. (a) GFAP (green)–Vimentin (red), (b) GFAP (green)–APC

    Article Snippet: For immunofluorescence staining, Macaca mulatta sections were blocked in blocking solution (10% donkey serum, 0.1% Triton X-100 in PBS) for 1 hr at RT, incubated overnight at 4°C with the following primary antibodies: APC (mouse monoclonal 1:300, #0P80, Millipore Sigma, RRID:AB_2057371), Aqp4 (rabbit polyclonal 1:400, #, AB3594, Millipore Sigma, RRID:AB_91530), GFAP (rabbit polyclonal 1:400, Z0334, former DAKO, now Agilent, RRID:AB_10013382), GFAP (mouse monoclonal 1:400, G3893, Millipore Sigma, RRID:AB_477010), Iba1 (rabbit polyclonal 1:400, #019–19,741, FUJIFILM Wako Pure Chemical Corporation, Richmond, VA, RRID:AB_839504), Ki67 (rabbit polyclonal 1:400, ab15580, Abcam, Cambridge, MA, RRID: AB_443209), MAP2 (rabbit polyclonal 1: 400, ab32454, Abcam, RRID: AB_776174), NeuN (mouse monoclonal 1:100, MAB377, Millipore Sigma, RRID:AB_2298772), Olig2 (rabbit polyclonal 1:100, #AB9610, Millipore Sigma, RRID:AB_570666), S100b (rabbit polyclonal 1:300, ab52642, Abcam, RRID:AB_882426), Sox10 (rabbit polyclonal 1:300, ab27655, Abcam, RRID:AB_778021).

    Techniques:

    Reduced cell adhesion and disorganized cytoskeleton of neural crest cell (NCC)-specific integrin-linked kinase (ILK) knockout (NKO) mutant NCCs. (A-D) In control NCCs, paxillin and parvin are expressed in a punctate pattern, associated with the focal adhesions. However, in ILK mutant NCCs, paxillin expression is diffuse, while parvin expression appears to be normal. (E,F) Adhesion and spreading of ILK mutant NCCs (ILK f/f ;Cre + ) on fibronectin (Fn), laminin (Lam), collagen (Col) and vitronectin (Vtn) are impaired. (G-I) Reduced Akt phosphorylation in NCCs of NKO mutant OFT. (J,K) Immunostaining with antibodies to actin and Sox10 show well-aligned actin filaments in control NCCs. However, ILK f/f ;Cre + mutant NCCs exhibit excessive small protrusions and a large accumulation of cortical actin in the cell cortex.

    Journal: BMC Biology

    Article Title: Requirement for integrin-linked kinase in neural crest migration and differentiation and outflow tract morphogenesis

    doi: 10.1186/1741-7007-11-107

    Figure Lengend Snippet: Reduced cell adhesion and disorganized cytoskeleton of neural crest cell (NCC)-specific integrin-linked kinase (ILK) knockout (NKO) mutant NCCs. (A-D) In control NCCs, paxillin and parvin are expressed in a punctate pattern, associated with the focal adhesions. However, in ILK mutant NCCs, paxillin expression is diffuse, while parvin expression appears to be normal. (E,F) Adhesion and spreading of ILK mutant NCCs (ILK f/f ;Cre + ) on fibronectin (Fn), laminin (Lam), collagen (Col) and vitronectin (Vtn) are impaired. (G-I) Reduced Akt phosphorylation in NCCs of NKO mutant OFT. (J,K) Immunostaining with antibodies to actin and Sox10 show well-aligned actin filaments in control NCCs. However, ILK f/f ;Cre + mutant NCCs exhibit excessive small protrusions and a large accumulation of cortical actin in the cell cortex.

    Article Snippet: The following primary antibodies were used: α-SMA (Abcam, ab7817), phosphohistone H3 (Cell Signaling, #9701), BrdU (Abcam), β-gal (Cappel, #55976), ILK (Sigma, I0783), laminin (Abcam, ab11575), fibronectin (Abcam, ab2413), elastin (Sigma, E4013), NCAM (Millipore, AB5032), TGFβ2 (Abcam, ab15539), p-Akt (Cell signaling, #9271), p-Smad1 (Cell Signaling, #9516), p-Smad2 (Abcam, ab5478), Sox10 (Abcam, ab27655), BMP2K (Abcam, ab115469), neurofilament (Developmental Studies Hybridoma Bank, 2H3), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology), parvin (Abcam) and paxillin (Abcam).

    Techniques: Knock-Out, Mutagenesis, Expressing, Laser Capture Microdissection, Immunostaining

    EMT inhibition induced by REC8 overexpression is reversed by REC8 ablation or EGR1 overexpression. REC8-overexpressed BGC823 cells were transiently transfected with REC8 shRNA or EGR1 overexpression plasmid. (A) The mRNA expression levels of EMT markers (SOX10, E-cadherin and vimentin) were assessed using qRT-PCR. (B) The protein levels of REC8, EGR1 and EMT markers (SOX10, E-cadherin and vimentin) were assessed using western blotting. (C) The location of β-catenin in gastric cancer cells was assessed using immunofluorescence assay (magnification, ×400). All the results were expressed as the mean ± SD of three independent experiments with triplicate wells.

    Journal: Oncology Reports

    Article Title: REC8 inhibits EMT by downregulating EGR1 in gastric cancer cells

    doi: 10.3892/or.2018.6244

    Figure Lengend Snippet: EMT inhibition induced by REC8 overexpression is reversed by REC8 ablation or EGR1 overexpression. REC8-overexpressed BGC823 cells were transiently transfected with REC8 shRNA or EGR1 overexpression plasmid. (A) The mRNA expression levels of EMT markers (SOX10, E-cadherin and vimentin) were assessed using qRT-PCR. (B) The protein levels of REC8, EGR1 and EMT markers (SOX10, E-cadherin and vimentin) were assessed using western blotting. (C) The location of β-catenin in gastric cancer cells was assessed using immunofluorescence assay (magnification, ×400). All the results were expressed as the mean ± SD of three independent experiments with triplicate wells.

    Article Snippet: After incubation with the specific primary antibodies: REC8 antibody (1:1,000; rabbit monoclonal, cat. no. ab192241; Abcam, San Francisco, CA, USA), EGR1 antibody (1:1,000; rabbit polyclonal, cat. no. ab6054; Abcam), TGFβ1 antibody (1:1,000; rabbit polyclonal, cat. no. ab92486; Abcam), SOX10 antibody (1:1,000; rabbit polyclonal, cat. no. ab108408; Abcam), vimentin antibody (1:1,000; rabbit monoclonal, cat. no. ab92547; Abcam), E-cadherin antibody (1:1,000; rabbit polyclonal, cat. no. 3195; Cell Signaling Technology, Danvers, MA, USA), GAPDH antibody (1:4,000; rabbit polyclonal, cat. no. 2118; Cell Signaling Technology), ATG12 antibody (1:1,000; mouse monoclonal, cat. no. sc-271688; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight, the membranes were incubated with HRP-conjugated secondary antibodies (1:10,000; cat no. A4416 and A6154; Sigma-Aldrich).

    Techniques: Inhibition, Over Expression, Transfection, shRNA, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence

    EMT is inhibited by REC8 overexpression. Gastric cancer cells BGC823 were transiently transfected with REC8 overexpression plasmid. (A) The mRNA expression levels of EMT markers (SOX10, E-cadherin and vimentin) were assessed using qRT-PCR. (B) The protein levels of EMT markers (SOX10, E-cadherin and vimentin) were assessed using western blotting. (C) The location of β-catenin in gastric cancer cells was assessed using immunofluorescence assay (magnification, ×400).

    Journal: Oncology Reports

    Article Title: REC8 inhibits EMT by downregulating EGR1 in gastric cancer cells

    doi: 10.3892/or.2018.6244

    Figure Lengend Snippet: EMT is inhibited by REC8 overexpression. Gastric cancer cells BGC823 were transiently transfected with REC8 overexpression plasmid. (A) The mRNA expression levels of EMT markers (SOX10, E-cadherin and vimentin) were assessed using qRT-PCR. (B) The protein levels of EMT markers (SOX10, E-cadherin and vimentin) were assessed using western blotting. (C) The location of β-catenin in gastric cancer cells was assessed using immunofluorescence assay (magnification, ×400).

    Article Snippet: After incubation with the specific primary antibodies: REC8 antibody (1:1,000; rabbit monoclonal, cat. no. ab192241; Abcam, San Francisco, CA, USA), EGR1 antibody (1:1,000; rabbit polyclonal, cat. no. ab6054; Abcam), TGFβ1 antibody (1:1,000; rabbit polyclonal, cat. no. ab92486; Abcam), SOX10 antibody (1:1,000; rabbit polyclonal, cat. no. ab108408; Abcam), vimentin antibody (1:1,000; rabbit monoclonal, cat. no. ab92547; Abcam), E-cadherin antibody (1:1,000; rabbit polyclonal, cat. no. 3195; Cell Signaling Technology, Danvers, MA, USA), GAPDH antibody (1:4,000; rabbit polyclonal, cat. no. 2118; Cell Signaling Technology), ATG12 antibody (1:1,000; mouse monoclonal, cat. no. sc-271688; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight, the membranes were incubated with HRP-conjugated secondary antibodies (1:10,000; cat no. A4416 and A6154; Sigma-Aldrich).

    Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence

    SOX9 binds to BEST1 proximal promoter in vivo in RPE. A , ChIP for SOX9 and SOX10 with bovine RPE. ChIP was performed using fresh bovine RPE cells with anti-SOX9 antibody AB5535 or anti-SOX10 antibody sc-17342 (labeled sc ) or ab25978 (labeled ab ). The

    Journal: The Journal of Biological Chemistry

    Article Title: SOX9, through Interaction with Microphthalmia-associated Transcription Factor (MITF) and OTX2, Regulates BEST1 Expression in the Retinal Pigment Epithelium *

    doi: 10.1074/jbc.M110.130294

    Figure Lengend Snippet: SOX9 binds to BEST1 proximal promoter in vivo in RPE. A , ChIP for SOX9 and SOX10 with bovine RPE. ChIP was performed using fresh bovine RPE cells with anti-SOX9 antibody AB5535 or anti-SOX10 antibody sc-17342 (labeled sc ) or ab25978 (labeled ab ). The

    Article Snippet: Antibodies used were anti-SOX9 antibody AB5535 (Chemicon, Temecula, CA), anti-SOX10 antibody sc-17342 (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-SOX10 antibody ab25978 (Abcam, Cambridge, UK).

    Techniques: In Vivo, Chromatin Immunoprecipitation, Labeling

    Histological comparison of the embryo injection melanoma model and TEAZ melanoma model. (A) The left images show a melanoma created by injection of an mitfa :BRAF V600E -tdTomato (fusion) transgene into a tp53 −/− background ( n =1). Right images show a TEAZ-based melanoma created by electroporation of miniCoopR:GFP plus ubb :Cas9 plus zfU6 :sgRNA against Rb1 ( n =1) (example shown is fish at 16 weeks also shown in Fig. 2 A). (B) H E staining of both tumors shows similar histology, although with increased melanin pigmentation in the TEAZ tumor (also shown in Fig. 3 A). (C,D) Antibody staining against BRAF V600E shows that both tumors are widely BRAF V600E positive, which correlates with high levels of phospho-ERK staining. (E) Reflecting the neural crest origin of melanocytes, both tumors show strong nuclear expression of SOX10. Images are visualized at 4× and 40×. Scale bars: 500 μm (4×) and 50 μm (40×). Dashed line boxes indicate the area enlarged at 40×.

    Journal: Disease Models & Mechanisms

    Article Title: Cancer modeling by Transgene Electroporation in Adult Zebrafish (TEAZ)

    doi: 10.1242/dmm.034561

    Figure Lengend Snippet: Histological comparison of the embryo injection melanoma model and TEAZ melanoma model. (A) The left images show a melanoma created by injection of an mitfa :BRAF V600E -tdTomato (fusion) transgene into a tp53 −/− background ( n =1). Right images show a TEAZ-based melanoma created by electroporation of miniCoopR:GFP plus ubb :Cas9 plus zfU6 :sgRNA against Rb1 ( n =1) (example shown is fish at 16 weeks also shown in Fig. 2 A). (B) H E staining of both tumors shows similar histology, although with increased melanin pigmentation in the TEAZ tumor (also shown in Fig. 3 A). (C,D) Antibody staining against BRAF V600E shows that both tumors are widely BRAF V600E positive, which correlates with high levels of phospho-ERK staining. (E) Reflecting the neural crest origin of melanocytes, both tumors show strong nuclear expression of SOX10. Images are visualized at 4× and 40×. Scale bars: 500 μm (4×) and 50 μm (40×). Dashed line boxes indicate the area enlarged at 40×.

    Article Snippet: Fish were sectioned at 5 µM and placed on Apex Adhesive slides, baked at 60°C, and then stained with H & E or antibodies against GFP (Abcam, ab183734, 1:100), BRAFV600E (Abcam, ab228461, 1:400), phospho-Rb1 (Cell Signaling Technology, 8516s, 1:400), phospho-ERK (Cell Signaling Technology, 4370, 1:100) or SOX10 (Cell Marque, 383A-76, 1:50).

    Techniques: Injection, Electroporation, Fluorescence In Situ Hybridization, Staining, Expressing

    Sox10 is necessary and sufficient for the activity of ERBB3 _MCS6 in vitro . (A) Luciferase activities of wild-type ERBB3_ MCS6 and TFBS mutations in ERBB3_ MCS6 in melan-A cell line. The position of each TFBS in ERBB3_ MCS6 is shown on top and the corresponding mutation in each TFBS is shown next to the luciferase value for that construct. * indicates statistical significance. (B) Luciferase activity of WT ERBB3_ MCS6 in melan-A cells in mock-transfected cells and in cells with transient Sox10 and Ap2 knockdown. (C) Western blot to confirm knockdown of Sox10 and Ap2 protein upon siRNA treatment. Tubulin antibody was used as a loading control. (D) Western blot showing Erbb3 protein levels in melan-a cells upon transient transfection with Sox10 and Ap2 siRNA or mock-transfected cells. Tubulin antibody was used as a loading control. (E) Luciferase activity of ERBB3 _MCS6 upon knockdown of Sox10 in S16 cells using a dominant negative SOX10 mutant (E189X) under a CMV prmoter. (F) Luciferase assay of WT and SOXE-2m ERBB3_ MCS6 in Neuro2A cells when transiently co-transfected with an empty expression vector (pcDNA.31) and Sox10 cDNA. Cell lysates were collected 24 hours post transfection. (G) Luciferase assay of WT ERBB3_ MCS6 in Neuro2A cells when transiently co-transfected with equal amounts of WT and Sox10-ΔSTP cDNA either individually or in combination. Cell lysates were collected 24 hours post transfection. All luciferase values are normalized to renilla internal control and shown as fold-change compared to promoter only construct (pe1B) with standard deviation. (H) Real-time PCR of Erbb3 transcript levels upon expression of WT and Sox10-ΔSTP cDNA individually and in combination. Values are normalized to 18s internal control and are shown as fold-change compared to promoter only construct (pcDNA3.1) with standard error. (I) Real-time PCR of ChIP against Sox10 in untreated S16 cells and in S16 cells treated with Sox10 morpholino. Black bar indicates enrichment upon Sox10 ChIP whereas grey bar indicates enrichment with non-specific IgG. Error bars indicate standard deviation of technical replicates in real-time PCR.

    Journal: BMC Developmental Biology

    Article Title: SOX10 directly modulates ERBB3 transcription via an intronic neural crest enhancer

    doi: 10.1186/1471-213X-11-40

    Figure Lengend Snippet: Sox10 is necessary and sufficient for the activity of ERBB3 _MCS6 in vitro . (A) Luciferase activities of wild-type ERBB3_ MCS6 and TFBS mutations in ERBB3_ MCS6 in melan-A cell line. The position of each TFBS in ERBB3_ MCS6 is shown on top and the corresponding mutation in each TFBS is shown next to the luciferase value for that construct. * indicates statistical significance. (B) Luciferase activity of WT ERBB3_ MCS6 in melan-A cells in mock-transfected cells and in cells with transient Sox10 and Ap2 knockdown. (C) Western blot to confirm knockdown of Sox10 and Ap2 protein upon siRNA treatment. Tubulin antibody was used as a loading control. (D) Western blot showing Erbb3 protein levels in melan-a cells upon transient transfection with Sox10 and Ap2 siRNA or mock-transfected cells. Tubulin antibody was used as a loading control. (E) Luciferase activity of ERBB3 _MCS6 upon knockdown of Sox10 in S16 cells using a dominant negative SOX10 mutant (E189X) under a CMV prmoter. (F) Luciferase assay of WT and SOXE-2m ERBB3_ MCS6 in Neuro2A cells when transiently co-transfected with an empty expression vector (pcDNA.31) and Sox10 cDNA. Cell lysates were collected 24 hours post transfection. (G) Luciferase assay of WT ERBB3_ MCS6 in Neuro2A cells when transiently co-transfected with equal amounts of WT and Sox10-ΔSTP cDNA either individually or in combination. Cell lysates were collected 24 hours post transfection. All luciferase values are normalized to renilla internal control and shown as fold-change compared to promoter only construct (pe1B) with standard deviation. (H) Real-time PCR of Erbb3 transcript levels upon expression of WT and Sox10-ΔSTP cDNA individually and in combination. Values are normalized to 18s internal control and are shown as fold-change compared to promoter only construct (pcDNA3.1) with standard error. (I) Real-time PCR of ChIP against Sox10 in untreated S16 cells and in S16 cells treated with Sox10 morpholino. Black bar indicates enrichment upon Sox10 ChIP whereas grey bar indicates enrichment with non-specific IgG. Error bars indicate standard deviation of technical replicates in real-time PCR.

    Article Snippet: The antibodies used were anti-Sox10 (sc-17342X, Santa Cruz Biotechnology, Santa Cruz, CA) and control anti-goat IgG (sc-2808, Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Activity Assay, In Vitro, Luciferase, Mutagenesis, Construct, Transfection, Western Blot, Dominant Negative Mutation, Expressing, Plasmid Preparation, Standard Deviation, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

    ERBB3_ MCS6 drives reporter expression in vivo in a pattern similar to erbb3b and under the control of sox10 . (A-D) Expression pattern of ERBB3_MCS driving eGFP in G1 transgenic 24-72hpf zebrafish embryos. Arrows indicate tissues where expression was noted in multiple founders. (E-H) eGFP expression pattern seen in multiple founders of SOXE-2m ERBB3 _MCS6 transgenic fish. Although expression is similar to WT ERBB3 _MCS6 at 24hpf, expression differs at later stages of development. (I-P) Results of morpholino knockdown of sox10 in ERBB3 _MCS6 transgenic embryos. (I-L) Normal expression of ERBB3_MCS6 in uninjected transgenic embryos at 24-48hpf. (M-P) Loss of reporter expression in embryos injected with a Sox10 morpholino at 24hpf and appearance of disorganized GFP expressing NC cells at 48hpf. Abbreviations: cranial neural crest (CNC), migratory crest (MC), pre-migratory crest (PMC), dorsal root ganglia (DRG), posterior lateral line (PLL), Schwann cells (SC), oligodendrocytes (OD) and notochord (NC).

    Journal: BMC Developmental Biology

    Article Title: SOX10 directly modulates ERBB3 transcription via an intronic neural crest enhancer

    doi: 10.1186/1471-213X-11-40

    Figure Lengend Snippet: ERBB3_ MCS6 drives reporter expression in vivo in a pattern similar to erbb3b and under the control of sox10 . (A-D) Expression pattern of ERBB3_MCS driving eGFP in G1 transgenic 24-72hpf zebrafish embryos. Arrows indicate tissues where expression was noted in multiple founders. (E-H) eGFP expression pattern seen in multiple founders of SOXE-2m ERBB3 _MCS6 transgenic fish. Although expression is similar to WT ERBB3 _MCS6 at 24hpf, expression differs at later stages of development. (I-P) Results of morpholino knockdown of sox10 in ERBB3 _MCS6 transgenic embryos. (I-L) Normal expression of ERBB3_MCS6 in uninjected transgenic embryos at 24-48hpf. (M-P) Loss of reporter expression in embryos injected with a Sox10 morpholino at 24hpf and appearance of disorganized GFP expressing NC cells at 48hpf. Abbreviations: cranial neural crest (CNC), migratory crest (MC), pre-migratory crest (PMC), dorsal root ganglia (DRG), posterior lateral line (PLL), Schwann cells (SC), oligodendrocytes (OD) and notochord (NC).

    Article Snippet: The antibodies used were anti-Sox10 (sc-17342X, Santa Cruz Biotechnology, Santa Cruz, CA) and control anti-goat IgG (sc-2808, Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Expressing, In Vivo, Transgenic Assay, Fluorescence In Situ Hybridization, Injection

    Egr2 activation of the Mpz intron element depends on Sox10 binding sites A) The Mpz intron reporters (wild type and Sox10 mut) were cotransfected in B16/F10 cells with an expression plasmid for Egr2 (30 ng). The putative Sox10- and Egr2-binding sites are indicated by ovals and squares, respectively. The filled ovals in the diagram represent the intron element that contains mutant Sox10-binding sites. The sequence alignment shows conservation of the Sox10-binding sites in mouse, rat and human Mpz loci, with mutated bases indicated by lines over the sequence. Fold induction is calculated relative to the activity of each reporter alone. Means and standard deviations of six replicate assays are shown. The fold induction of the Mpz intron reporter is significantly more than the fold induction of the Mpz intron Sox10 mut reporter (P

    Journal: Neuron glia biology

    Article Title: Interactions of Sox10 and Egr2 in Myelin Gene Regulation

    doi: 10.1017/S1740925X08000173

    Figure Lengend Snippet: Egr2 activation of the Mpz intron element depends on Sox10 binding sites A) The Mpz intron reporters (wild type and Sox10 mut) were cotransfected in B16/F10 cells with an expression plasmid for Egr2 (30 ng). The putative Sox10- and Egr2-binding sites are indicated by ovals and squares, respectively. The filled ovals in the diagram represent the intron element that contains mutant Sox10-binding sites. The sequence alignment shows conservation of the Sox10-binding sites in mouse, rat and human Mpz loci, with mutated bases indicated by lines over the sequence. Fold induction is calculated relative to the activity of each reporter alone. Means and standard deviations of six replicate assays are shown. The fold induction of the Mpz intron reporter is significantly more than the fold induction of the Mpz intron Sox10 mut reporter (P

    Article Snippet: ChIP analysis of myelinating rat sciatic nerve at postnatal days 5 and 15 was performed as described ( ; ), by mincing pooled sciatic nerves from 8–13 rat pups in 1% formaldehyde for 25 min. Polyclonal antibodies directed against Egr2 (Covance) and Sox10 (Santa Cruz sc-17342) were employed in the immunoprecipitations.

    Techniques: Activation Assay, Binding Assay, Expressing, Plasmid Preparation, Mutagenesis, Sequencing, Activity Assay

    Model for activation of the Mpz gene by Egr2/Sox10 This model depicts a potential mechanism for the induction of Mpz ) indicates that Sox10 is responsible for the low basal level of Mpz expression in embryonic Schwann cells. B) Our work indicates that the large induction of Mpz expression that accompanies Egr2 induction in myelinating Schwann cells can be at least partially attributed to Egr2-dependent binding of Sox10 to an enhancer in the first intron of the Mpz gene. In turn, Sox10 is largely required for the large induction of Mpz expression by Egr2, perhaps by serving an architectural function because of its ability to bend DNA. It is proposed that similar configurations of Sox10/Egr2 sites are involved in induction of other myelin genes.

    Journal: Neuron glia biology

    Article Title: Interactions of Sox10 and Egr2 in Myelin Gene Regulation

    doi: 10.1017/S1740925X08000173

    Figure Lengend Snippet: Model for activation of the Mpz gene by Egr2/Sox10 This model depicts a potential mechanism for the induction of Mpz ) indicates that Sox10 is responsible for the low basal level of Mpz expression in embryonic Schwann cells. B) Our work indicates that the large induction of Mpz expression that accompanies Egr2 induction in myelinating Schwann cells can be at least partially attributed to Egr2-dependent binding of Sox10 to an enhancer in the first intron of the Mpz gene. In turn, Sox10 is largely required for the large induction of Mpz expression by Egr2, perhaps by serving an architectural function because of its ability to bend DNA. It is proposed that similar configurations of Sox10/Egr2 sites are involved in induction of other myelin genes.

    Article Snippet: ChIP analysis of myelinating rat sciatic nerve at postnatal days 5 and 15 was performed as described ( ; ), by mincing pooled sciatic nerves from 8–13 rat pups in 1% formaldehyde for 25 min. Polyclonal antibodies directed against Egr2 (Covance) and Sox10 (Santa Cruz sc-17342) were employed in the immunoprecipitations.

    Techniques: Activation Assay, Expressing, Binding Assay

    Conserved configuration of Egr2/Sox10-binding sites in the first intron of the Mpz gene The six exons of Mpz and the location of a conserved intron element containing Egr2 and Sox10 sites (gray squares and open ovals, respetively). DNase I footprinting analysis of a fragment (+984–+1500) of the mouse Mpz ). M=marker lane

    Journal: Neuron glia biology

    Article Title: Interactions of Sox10 and Egr2 in Myelin Gene Regulation

    doi: 10.1017/S1740925X08000173

    Figure Lengend Snippet: Conserved configuration of Egr2/Sox10-binding sites in the first intron of the Mpz gene The six exons of Mpz and the location of a conserved intron element containing Egr2 and Sox10 sites (gray squares and open ovals, respetively). DNase I footprinting analysis of a fragment (+984–+1500) of the mouse Mpz ). M=marker lane

    Article Snippet: ChIP analysis of myelinating rat sciatic nerve at postnatal days 5 and 15 was performed as described ( ; ), by mincing pooled sciatic nerves from 8–13 rat pups in 1% formaldehyde for 25 min. Polyclonal antibodies directed against Egr2 (Covance) and Sox10 (Santa Cruz sc-17342) were employed in the immunoprecipitations.

    Techniques: Binding Assay, Footprinting, Marker

    Binding of Egr2 to a conserved module in the periaxin gene A) Cross-linked chromatin was prepared from the S16 rat Schwann cell line. Sonicated chromatin was immunoprecipitated with antibodies directed against either Egr2 (filled bars) or purified rabbit IgG (open bars) as a negative control. Purified DNA was then analyzed by quantitative PCR using primers designed for the Mpz upstream region (−2.3 kb) as a negative control. Additional primer sets were used to detect binding of Egr2 at −1.7 kb upstream of the Mag gene and 4.5 kb downstream of the periaxin transcription start site. Percentage recovery relative to input was determined using quantitative PCR. These data are representative of two independent ChIP experiments. B) Similar ChIP assays were performed using pooled sciatic nerves obtained from a rat litter at postnatal day 15. An additional primer set in the Mpz intron element (IN1) was used as a positive control. These data are representative of assays performed with two independent litters of rat pups. C) A ChIP assay for Sox10 binding to the indicated sites in pooled rat sciatic nerve (postnatal day 15) was performed as described above.

    Journal: Neuron glia biology

    Article Title: Interactions of Sox10 and Egr2 in Myelin Gene Regulation

    doi: 10.1017/S1740925X08000173

    Figure Lengend Snippet: Binding of Egr2 to a conserved module in the periaxin gene A) Cross-linked chromatin was prepared from the S16 rat Schwann cell line. Sonicated chromatin was immunoprecipitated with antibodies directed against either Egr2 (filled bars) or purified rabbit IgG (open bars) as a negative control. Purified DNA was then analyzed by quantitative PCR using primers designed for the Mpz upstream region (−2.3 kb) as a negative control. Additional primer sets were used to detect binding of Egr2 at −1.7 kb upstream of the Mag gene and 4.5 kb downstream of the periaxin transcription start site. Percentage recovery relative to input was determined using quantitative PCR. These data are representative of two independent ChIP experiments. B) Similar ChIP assays were performed using pooled sciatic nerves obtained from a rat litter at postnatal day 15. An additional primer set in the Mpz intron element (IN1) was used as a positive control. These data are representative of assays performed with two independent litters of rat pups. C) A ChIP assay for Sox10 binding to the indicated sites in pooled rat sciatic nerve (postnatal day 15) was performed as described above.

    Article Snippet: ChIP analysis of myelinating rat sciatic nerve at postnatal days 5 and 15 was performed as described ( ; ), by mincing pooled sciatic nerves from 8–13 rat pups in 1% formaldehyde for 25 min. Polyclonal antibodies directed against Egr2 (Covance) and Sox10 (Santa Cruz sc-17342) were employed in the immunoprecipitations.

    Techniques: Binding Assay, Sonication, Immunoprecipitation, Purification, Negative Control, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Positive Control

    Several Egr family members interact with Sox10 A) The Mpz intron reporters (wild type and Sox10 mut) were cotransfected in B16/F10 cells with expression plasmids for Egr2, Egr3 (either 25ng or 100ng), and Egr1 (either 50ng or 200ng). The putative Sox10- and Egr2-binding sites are indicated by ovals and squares, respectively. The filled ovals in the diagram represent mutated Sox10 sites. Fold-induction is calculated relative to the activity of each reporter alone. Means and standard deviations of two replicate assays are shown. The fold inductions of the Mpz intron reporter by Egr1, Egr2, and Egr3 are statistically significant compared to that of the Mpz intron Sox10 mut reporter (P

    Journal: Neuron glia biology

    Article Title: Interactions of Sox10 and Egr2 in Myelin Gene Regulation

    doi: 10.1017/S1740925X08000173

    Figure Lengend Snippet: Several Egr family members interact with Sox10 A) The Mpz intron reporters (wild type and Sox10 mut) were cotransfected in B16/F10 cells with expression plasmids for Egr2, Egr3 (either 25ng or 100ng), and Egr1 (either 50ng or 200ng). The putative Sox10- and Egr2-binding sites are indicated by ovals and squares, respectively. The filled ovals in the diagram represent mutated Sox10 sites. Fold-induction is calculated relative to the activity of each reporter alone. Means and standard deviations of two replicate assays are shown. The fold inductions of the Mpz intron reporter by Egr1, Egr2, and Egr3 are statistically significant compared to that of the Mpz intron Sox10 mut reporter (P

    Article Snippet: ChIP analysis of myelinating rat sciatic nerve at postnatal days 5 and 15 was performed as described ( ; ), by mincing pooled sciatic nerves from 8–13 rat pups in 1% formaldehyde for 25 min. Polyclonal antibodies directed against Egr2 (Covance) and Sox10 (Santa Cruz sc-17342) were employed in the immunoprecipitations.

    Techniques: Expressing, Binding Assay, Activity Assay

    Subcellular localization of Sox10. A , endogenous Sox10 was detected in OBL21 cells by immunofluorescence. Nuclei were stained with 4′,6-diamidino-2-phenylindole ( DAPI ). The four sets of images show nuclear ( a ), cytoplasmic ( b ), and nucleocytoplasmic

    Journal: The Journal of Biological Chemistry

    Article Title: The Armadillo Repeat-containing Protein, ARMCX3, Physically and Functionally Interacts with the Developmental Regulatory Factor Sox10 *

    doi: 10.1074/jbc.M901177200

    Figure Lengend Snippet: Subcellular localization of Sox10. A , endogenous Sox10 was detected in OBL21 cells by immunofluorescence. Nuclei were stained with 4′,6-diamidino-2-phenylindole ( DAPI ). The four sets of images show nuclear ( a ), cytoplasmic ( b ), and nucleocytoplasmic

    Article Snippet: These data indicate the anti-Sox10 antibody is specific and is suitable for the experiments described below.

    Techniques: Immunofluorescence, Staining

    ARMCX3 increases the mitochondrial localization of Sox10. A , Neuro-2A cells were co-transfected with either pCMV5-Sox10 (10 μg) and pCMV-Myc-ARMCX3 (10, 20, and 40 μg) or pCMV5-Sox10 (10 μg) and pCMV-Myc-ARMCX3Δ3 (10,

    Journal: The Journal of Biological Chemistry

    Article Title: The Armadillo Repeat-containing Protein, ARMCX3, Physically and Functionally Interacts with the Developmental Regulatory Factor Sox10 *

    doi: 10.1074/jbc.M901177200

    Figure Lengend Snippet: ARMCX3 increases the mitochondrial localization of Sox10. A , Neuro-2A cells were co-transfected with either pCMV5-Sox10 (10 μg) and pCMV-Myc-ARMCX3 (10, 20, and 40 μg) or pCMV5-Sox10 (10 μg) and pCMV-Myc-ARMCX3Δ3 (10,

    Article Snippet: These data indicate the anti-Sox10 antibody is specific and is suitable for the experiments described below.

    Techniques: Transfection

    Mitochondrial localization of ARMCX3 and Sox10. A ) and the corresponding sequences of the murine ARMCX3 proteins, the yeast translocase of the outer membrane of mitochondria70 (

    Journal: The Journal of Biological Chemistry

    Article Title: The Armadillo Repeat-containing Protein, ARMCX3, Physically and Functionally Interacts with the Developmental Regulatory Factor Sox10 *

    doi: 10.1074/jbc.M901177200

    Figure Lengend Snippet: Mitochondrial localization of ARMCX3 and Sox10. A ) and the corresponding sequences of the murine ARMCX3 proteins, the yeast translocase of the outer membrane of mitochondria70 (

    Article Snippet: These data indicate the anti-Sox10 antibody is specific and is suitable for the experiments described below.

    Techniques:

    Co-expression and direct interaction between endogenous Sox10 and ARMCX3. A , RNA was extracted from mouse brain and spinal cord, rat C6 cells, human U87-MG and HeLa cells, and mouse Neuro-2A cells. The RNA was used in multiplex RT-PCR using primers

    Journal: The Journal of Biological Chemistry

    Article Title: The Armadillo Repeat-containing Protein, ARMCX3, Physically and Functionally Interacts with the Developmental Regulatory Factor Sox10 *

    doi: 10.1074/jbc.M901177200

    Figure Lengend Snippet: Co-expression and direct interaction between endogenous Sox10 and ARMCX3. A , RNA was extracted from mouse brain and spinal cord, rat C6 cells, human U87-MG and HeLa cells, and mouse Neuro-2A cells. The RNA was used in multiplex RT-PCR using primers

    Article Snippet: These data indicate the anti-Sox10 antibody is specific and is suitable for the experiments described below.

    Techniques: Expressing, Multiplex Assay, Reverse Transcription Polymerase Chain Reaction

    Identification of ARMCX3 as a Sox10-interacting protein. A , schematic representation of the rat Sox10 protein indicating the HMG box ( top figure ). Numbering refers to amino acids. A cDNA fragment that encodes the first 100 amino acids of Sox10 was

    Journal: The Journal of Biological Chemistry

    Article Title: The Armadillo Repeat-containing Protein, ARMCX3, Physically and Functionally Interacts with the Developmental Regulatory Factor Sox10 *

    doi: 10.1074/jbc.M901177200

    Figure Lengend Snippet: Identification of ARMCX3 as a Sox10-interacting protein. A , schematic representation of the rat Sox10 protein indicating the HMG box ( top figure ). Numbering refers to amino acids. A cDNA fragment that encodes the first 100 amino acids of Sox10 was

    Article Snippet: These data indicate the anti-Sox10 antibody is specific and is suitable for the experiments described below.

    Techniques:

    Sox10-mediated transactivation of the nACh receptor α3 and β4 gene promoters is enhanced by ARMCX3. Neuro-2A cells were transiently transfected with either an α3/luciferase ( panel A ) or a β4/luciferase ( panel B ) reporter

    Journal: The Journal of Biological Chemistry

    Article Title: The Armadillo Repeat-containing Protein, ARMCX3, Physically and Functionally Interacts with the Developmental Regulatory Factor Sox10 *

    doi: 10.1074/jbc.M901177200

    Figure Lengend Snippet: Sox10-mediated transactivation of the nACh receptor α3 and β4 gene promoters is enhanced by ARMCX3. Neuro-2A cells were transiently transfected with either an α3/luciferase ( panel A ) or a β4/luciferase ( panel B ) reporter

    Article Snippet: These data indicate the anti-Sox10 antibody is specific and is suitable for the experiments described below.

    Techniques: Transfection, Luciferase

    Systemic Sox10 activation in SCs following amputation. (A) Scheme of the experiment. (B–H) Caudal fin of adult fish was amputated ( t = 0, blue arrow ), and the axon cytoskeleton ( red ) and immature SCs ( white ) were visualized over time through immunofluorescence staining for the axonal marker, acetylated tubulin ( red ), and the immature SC marker, Sox10 ( white ), respectively. Immunostaining in uncut fin (B) or after amputation (C–H) . (B–G) The upper panel shows the distal part of the amputated fin, and the lower panel shows a more proximal part. (H′) Shows a higher magnification of (H) . Dotted line : amputation plane. Dashed line : distal part of the fin. Scale bars = 50 μ M . R, ray; IR, inter-ray. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

    Journal: Antioxidants & Redox Signaling

    Article Title: Nerves Control Redox Levels in Mature Tissues Through Schwann Cells and Hedgehog Signaling

    doi: 10.1089/ars.2015.6380

    Figure Lengend Snippet: Systemic Sox10 activation in SCs following amputation. (A) Scheme of the experiment. (B–H) Caudal fin of adult fish was amputated ( t = 0, blue arrow ), and the axon cytoskeleton ( red ) and immature SCs ( white ) were visualized over time through immunofluorescence staining for the axonal marker, acetylated tubulin ( red ), and the immature SC marker, Sox10 ( white ), respectively. Immunostaining in uncut fin (B) or after amputation (C–H) . (B–G) The upper panel shows the distal part of the amputated fin, and the lower panel shows a more proximal part. (H′) Shows a higher magnification of (H) . Dotted line : amputation plane. Dashed line : distal part of the fin. Scale bars = 50 μ M . R, ray; IR, inter-ray. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

    Article Snippet: Immunofluorescence and imaging The fins were fixed in 4% paraformaldehyde overnight at 4°C and used for whole-mount immunohistochemistry with antiphospho-histone H3 (No. SC-8656-R; Santa Cruz Biotechnology, Inc., Dallas, TX) to detect proliferative cells, antiacetylated tubulin (No. T7451; Sigma-Aldrich, Saint Louis, MO) to detect axons, anti-GFP to detect GFP in Shh:GFP fish (No. ab13970; Abcam, Cambridge, MA), anti-mCherry (No. 6332543; Clontech Laboratories, Inc., Mountain View, CA) to detect mRFP in sox10:RFP fish, and anti-Sox10 (No. GTX128374; GeneTex, Inc., Irvine, CA).

    Techniques: Activation Assay, Fluorescence In Situ Hybridization, Immunofluorescence, Staining, Marker, Immunostaining

    Activated SCs express Shh and form a specific structure at the tip of the axon. (A) Schematic representation of the intact fin or the fin after amputation. The squares indicate the position of the acquisitions, and the letters refer to the figure panels of this figure. The dorsal part of the caudal fin was denervated ( black arrow ) or the fin was amputated ( blue arrow ). (B) Immunofluorescence staining for GFP in shh:GFP fish ( white ) and axon cytoskeleton ( red ) in uncut fin. (C, D) Immunofluorescence staining for GFP in shh:GFP fish ( green ) and for the axon cytoskeleton ( red ) in control side (-) and denervated part of a nonamputated fin 2 days postdenervation. (E–G) Immunofluorescence staining for GFP in shh:GFP fish ( green ) and Sox10 ( white ) in the whole fin at 0.5 hpa. (F, G) Magnification of (E) . (H, I) Immunofluorescence staining for GFP in shh:GFP fish ( green ), axon cytoskeleton ( red ), and Sox10 ( white ) in a cryosection of a regenerating fin at 12 hpa. (I) Magnification of (H) . Confocal images, 1–3 μ M . Dotted line : amputation plane. Dashed line : distal part of the fin. Scale bars = 50 μ M in (B–H) and 25 μ M in (I) . GFP, green fluorescent protein. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

    Journal: Antioxidants & Redox Signaling

    Article Title: Nerves Control Redox Levels in Mature Tissues Through Schwann Cells and Hedgehog Signaling

    doi: 10.1089/ars.2015.6380

    Figure Lengend Snippet: Activated SCs express Shh and form a specific structure at the tip of the axon. (A) Schematic representation of the intact fin or the fin after amputation. The squares indicate the position of the acquisitions, and the letters refer to the figure panels of this figure. The dorsal part of the caudal fin was denervated ( black arrow ) or the fin was amputated ( blue arrow ). (B) Immunofluorescence staining for GFP in shh:GFP fish ( white ) and axon cytoskeleton ( red ) in uncut fin. (C, D) Immunofluorescence staining for GFP in shh:GFP fish ( green ) and for the axon cytoskeleton ( red ) in control side (-) and denervated part of a nonamputated fin 2 days postdenervation. (E–G) Immunofluorescence staining for GFP in shh:GFP fish ( green ) and Sox10 ( white ) in the whole fin at 0.5 hpa. (F, G) Magnification of (E) . (H, I) Immunofluorescence staining for GFP in shh:GFP fish ( green ), axon cytoskeleton ( red ), and Sox10 ( white ) in a cryosection of a regenerating fin at 12 hpa. (I) Magnification of (H) . Confocal images, 1–3 μ M . Dotted line : amputation plane. Dashed line : distal part of the fin. Scale bars = 50 μ M in (B–H) and 25 μ M in (I) . GFP, green fluorescent protein. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

    Article Snippet: Immunofluorescence and imaging The fins were fixed in 4% paraformaldehyde overnight at 4°C and used for whole-mount immunohistochemistry with antiphospho-histone H3 (No. SC-8656-R; Santa Cruz Biotechnology, Inc., Dallas, TX) to detect proliferative cells, antiacetylated tubulin (No. T7451; Sigma-Aldrich, Saint Louis, MO) to detect axons, anti-GFP to detect GFP in Shh:GFP fish (No. ab13970; Abcam, Cambridge, MA), anti-mCherry (No. 6332543; Clontech Laboratories, Inc., Mountain View, CA) to detect mRFP in sox10:RFP fish, and anti-Sox10 (No. GTX128374; GeneTex, Inc., Irvine, CA).

    Techniques: Immunofluorescence, Staining, Fluorescence In Situ Hybridization

    Proliferative defects in TSC2-SCKO nerves. ( A ) Quantification of glial nuclei in sciatic nerve semithin cross-sections from control and TSC2-SCKO mice at the indicated postnatal ages reveals progressively increasing numbers of developing SCs in the mutant nerves. n = 3 mice per genotype at each age. ( B , Left ) Fluorescence microscopy analysis of immunostained teased fiber preparations from tibial nerves of P28 control and TSC2-SCKO mice using the indicated markers. Note increased numbers of SOX10 + SCs (arrows) associated with axon segments in TSC2-SCKO nerves compared with control. (Scale bars, 10 µm.) ( Right ) Quantification of SOX10 + SCs along TUJ1 + axons ( n = 3 mice per genotype). ( C ) Quantitative immunofluorescence of longitudinal sciatic nerve sections (confocal z -series projections) from control and TSC2-SCKO mice at ages P14 (c-Casp3) and P21 (Ki67, p-H3) to demonstrate increased cell cycling ( Top ; arrows depict Ki67 + cells) and mitotic events ( Middle ; arrows depict p-H3 + cells), and induction of SC apoptosis ( Bottom ; arrows depcit c-Casp3 + cells) in the mutant ( n = 3 mice per genotype at each age). (Scale bars, 50 µm.) ( D , Upper ) Schematic illustrating cyclin expression during cell cycle, and checkpoint control by the restriction (R) point. ( Lower ) Western blots of sciatic nerve lysates from control and TSC2-SCKO mice at age P7 (three mice per group), probed with the indicated antibodies, demonstrating that TSC2-deficient SCs bear elevated levels of cyclin B1 (marker for M-phase) and D1 (marker for G 1 /S-phase). ( E ) Western blots of sciatic nerve lysates from control and TSC2-SCKO mice at age P7 (three mice per group) probed with the indicated antibodies, demonstrating hyperphosphorylation and thus inactivation of Rb in the mutant allowing uncontrolled passage through the G 1 restriction (R) point of the cell cycle.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: mTORC1 promotes proliferation of immature Schwann cells and myelin growth of differentiated Schwann cells

    doi: 10.1073/pnas.1620761114

    Figure Lengend Snippet: Proliferative defects in TSC2-SCKO nerves. ( A ) Quantification of glial nuclei in sciatic nerve semithin cross-sections from control and TSC2-SCKO mice at the indicated postnatal ages reveals progressively increasing numbers of developing SCs in the mutant nerves. n = 3 mice per genotype at each age. ( B , Left ) Fluorescence microscopy analysis of immunostained teased fiber preparations from tibial nerves of P28 control and TSC2-SCKO mice using the indicated markers. Note increased numbers of SOX10 + SCs (arrows) associated with axon segments in TSC2-SCKO nerves compared with control. (Scale bars, 10 µm.) ( Right ) Quantification of SOX10 + SCs along TUJ1 + axons ( n = 3 mice per genotype). ( C ) Quantitative immunofluorescence of longitudinal sciatic nerve sections (confocal z -series projections) from control and TSC2-SCKO mice at ages P14 (c-Casp3) and P21 (Ki67, p-H3) to demonstrate increased cell cycling ( Top ; arrows depict Ki67 + cells) and mitotic events ( Middle ; arrows depict p-H3 + cells), and induction of SC apoptosis ( Bottom ; arrows depcit c-Casp3 + cells) in the mutant ( n = 3 mice per genotype at each age). (Scale bars, 50 µm.) ( D , Upper ) Schematic illustrating cyclin expression during cell cycle, and checkpoint control by the restriction (R) point. ( Lower ) Western blots of sciatic nerve lysates from control and TSC2-SCKO mice at age P7 (three mice per group), probed with the indicated antibodies, demonstrating that TSC2-deficient SCs bear elevated levels of cyclin B1 (marker for M-phase) and D1 (marker for G 1 /S-phase). ( E ) Western blots of sciatic nerve lysates from control and TSC2-SCKO mice at age P7 (three mice per group) probed with the indicated antibodies, demonstrating hyperphosphorylation and thus inactivation of Rb in the mutant allowing uncontrolled passage through the G 1 restriction (R) point of the cell cycle.

    Article Snippet: Following primary antibodies were used for immunofluorescence: p-S6rp(Ser240/244) (Cell Signaling Technologies, #5364), S100-Biotin (Thermo Scientific, #MA5-12966), SOX10 (Cell Signaling Technologies, #89356), TUJ1 (BioLegend #801201), Ki67 (Cell Signaling Technologies, #12202), phospho-Histone H3 (Ser10) (Millipore, #9661), Cleaved-Caspase 3 (Asp175) (Cell Signaling Technologies, #9661), OCT6 (kind gift from John Bermingham, McLaughlin Research Institute for Biomedical Sciences, Great Falls, MT), SOX2 (Active Motif, #39823), Egr2 (Covance #PRB-236P).

    Techniques: Mouse Assay, Mutagenesis, Fluorescence, Microscopy, Immunofluorescence, Expressing, Western Blot, Marker

    Abnormal expression of S100 and SOX10 in TSC2-ablated SCs. ( A ) Confocal immunofluorescence ( z -series projections) of longitudinal frozen sciatic nerve sections from control and TSC2-SCKO mice at age P7 demonstrating increased S100 immunoreactivity in the mutant sample. (Scale bars, 25 µm.) ( B ) Quantitative immunofluorescence analysis of longitudinal frozen sciatic nerve sections (confocal z -series projections) from control and TSC2-SCKO mice at the indicated ages demonstrating significantly increased proportions of SOX10 + SCs in the mutant ( n = 3 mice per genotype). (Scale bars, 25 µm.)

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: mTORC1 promotes proliferation of immature Schwann cells and myelin growth of differentiated Schwann cells

    doi: 10.1073/pnas.1620761114

    Figure Lengend Snippet: Abnormal expression of S100 and SOX10 in TSC2-ablated SCs. ( A ) Confocal immunofluorescence ( z -series projections) of longitudinal frozen sciatic nerve sections from control and TSC2-SCKO mice at age P7 demonstrating increased S100 immunoreactivity in the mutant sample. (Scale bars, 25 µm.) ( B ) Quantitative immunofluorescence analysis of longitudinal frozen sciatic nerve sections (confocal z -series projections) from control and TSC2-SCKO mice at the indicated ages demonstrating significantly increased proportions of SOX10 + SCs in the mutant ( n = 3 mice per genotype). (Scale bars, 25 µm.)

    Article Snippet: Following primary antibodies were used for immunofluorescence: p-S6rp(Ser240/244) (Cell Signaling Technologies, #5364), S100-Biotin (Thermo Scientific, #MA5-12966), SOX10 (Cell Signaling Technologies, #89356), TUJ1 (BioLegend #801201), Ki67 (Cell Signaling Technologies, #12202), phospho-Histone H3 (Ser10) (Millipore, #9661), Cleaved-Caspase 3 (Asp175) (Cell Signaling Technologies, #9661), OCT6 (kind gift from John Bermingham, McLaughlin Research Institute for Biomedical Sciences, Great Falls, MT), SOX2 (Active Motif, #39823), Egr2 (Covance #PRB-236P).

    Techniques: Expressing, Immunofluorescence, Mouse Assay, Mutagenesis

    Morphological and immunophenotypical changes induced by BrdU glial differentiation treatment . A . Morphological changes in SK-N-ER cells (Phase contrast microscopy; 100×). B. GD2 expression levels after 15 days of treatment in I-type cell line SK-N-Be2C. C Calcyclin, ( D ) Sox10 and ( E ) S100 expression after 21 days of BrdU treatment in N-type cell line LA1-55N. F . SA-β-GAL positive cells (arrow) after 21 days of BrdU treatment in the I-type SK-N-ER (Phase contrast; 100×). Control cell images are reported in the small squares. Scale bar: 50 μm.

    Journal: BMC Developmental Biology

    Article Title: Comprehensive characterization of neuroblastoma cell line subtypes reveals bilineage potential similar to neural crest stem cells

    doi: 10.1186/1471-213X-9-12

    Figure Lengend Snippet: Morphological and immunophenotypical changes induced by BrdU glial differentiation treatment . A . Morphological changes in SK-N-ER cells (Phase contrast microscopy; 100×). B. GD2 expression levels after 15 days of treatment in I-type cell line SK-N-Be2C. C Calcyclin, ( D ) Sox10 and ( E ) S100 expression after 21 days of BrdU treatment in N-type cell line LA1-55N. F . SA-β-GAL positive cells (arrow) after 21 days of BrdU treatment in the I-type SK-N-ER (Phase contrast; 100×). Control cell images are reported in the small squares. Scale bar: 50 μm.

    Article Snippet: Endogenous peroxidases were inhibited with H2 O2 for 20 min. Primary antibodies S100 (Novocastra, UK), Sox10 (R & D systems, US) and c-kit (Zymed, US)(Additional file ) were incubated for 15 min-O/N and chromogenically stained with Anti-mouse/rabbit Poly-HRP IHC detection kit (Chemicon, US).

    Techniques: Microscopy, Expressing

    Quantitative RT-PCR analysis of gene expression changes induced by BrdU treatment in NB cell line subtypes . Gene expression changes of GD2 synthase ( A ), calcyclin ( B ) and Sox10 ( C ) in LA1-55N, LA1-5S and SK-N-ER cells. q-RT-PCR was performed by triplicate of two separate differentiation experiments (21 days induction).

    Journal: BMC Developmental Biology

    Article Title: Comprehensive characterization of neuroblastoma cell line subtypes reveals bilineage potential similar to neural crest stem cells

    doi: 10.1186/1471-213X-9-12

    Figure Lengend Snippet: Quantitative RT-PCR analysis of gene expression changes induced by BrdU treatment in NB cell line subtypes . Gene expression changes of GD2 synthase ( A ), calcyclin ( B ) and Sox10 ( C ) in LA1-55N, LA1-5S and SK-N-ER cells. q-RT-PCR was performed by triplicate of two separate differentiation experiments (21 days induction).

    Article Snippet: Endogenous peroxidases were inhibited with H2 O2 for 20 min. Primary antibodies S100 (Novocastra, UK), Sox10 (R & D systems, US) and c-kit (Zymed, US)(Additional file ) were incubated for 15 min-O/N and chromogenically stained with Anti-mouse/rabbit Poly-HRP IHC detection kit (Chemicon, US).

    Techniques: Quantitative RT-PCR, Expressing, Reverse Transcription Polymerase Chain Reaction

    Supporting data for Lin28a and Lin28b loss-of-function analysis. ( a–b ) Transfection strategy for knockdown experiments. HH4 embryos were injected with the control morpholino (blue) on the left and with the Lin28a morpholino (green) on the right ( a ). Following electroporation, embryos were cultured in albumin and incubated until HH9, when they were screened for efficient transfection and analyzed ( b ). ( c ) Dorsal view of a bilaterally electroporated HH9 embryo, showing control MO (left) and Lin28a MO (right). ( d ) Immunohistochemistry for Lin28a showed loss of the protein on the experimental side (downward arrow). ( e–h ) In situ hybridization for neural crest markers FoxD3 and Sox10 following knockdown of Lin28a. ( i–m ) Disruption of Lin28a expression with CRISPR/Cas9. ( i ) Quantitative analysis of Lin28a transcripts following CRISPR-cas9 targeting of the protein through RT-PCR. Immunohistochemistry for Cas9 and Lin28a on transverse sections of embryos electroporated with either Cas9 alone ( j–k ) or with Cas9 vector containing a pair of gRNAs targeting Lin28a. ( l–m ) Dotted lines outline cells expressing Cas9 (arrows). ( n ) RT-PCR for Lin28a, FoxD3 and Sox10 , following knockdown of Lin28a using two different DsiRNAs, confirm that loss of the protein results in reduced expression of these early neural crest markers. ( o ) Downregulation of Lin28b using two independent DsiRNAs followed by quantification of FoxD3 and Sox10 transcripts reveal that Lin28b is not required for neural crest formation. MO: Morpholino, DsiRNA: Dicer-substrate siRNA, gRNA: guide-RNA, HH: Hamburger and Hamilton stages.

    Journal: eLife

    Article Title: Control of neural crest multipotency by Wnt signaling and the Lin28/let-7 axis

    doi: 10.7554/eLife.40556

    Figure Lengend Snippet: Supporting data for Lin28a and Lin28b loss-of-function analysis. ( a–b ) Transfection strategy for knockdown experiments. HH4 embryos were injected with the control morpholino (blue) on the left and with the Lin28a morpholino (green) on the right ( a ). Following electroporation, embryos were cultured in albumin and incubated until HH9, when they were screened for efficient transfection and analyzed ( b ). ( c ) Dorsal view of a bilaterally electroporated HH9 embryo, showing control MO (left) and Lin28a MO (right). ( d ) Immunohistochemistry for Lin28a showed loss of the protein on the experimental side (downward arrow). ( e–h ) In situ hybridization for neural crest markers FoxD3 and Sox10 following knockdown of Lin28a. ( i–m ) Disruption of Lin28a expression with CRISPR/Cas9. ( i ) Quantitative analysis of Lin28a transcripts following CRISPR-cas9 targeting of the protein through RT-PCR. Immunohistochemistry for Cas9 and Lin28a on transverse sections of embryos electroporated with either Cas9 alone ( j–k ) or with Cas9 vector containing a pair of gRNAs targeting Lin28a. ( l–m ) Dotted lines outline cells expressing Cas9 (arrows). ( n ) RT-PCR for Lin28a, FoxD3 and Sox10 , following knockdown of Lin28a using two different DsiRNAs, confirm that loss of the protein results in reduced expression of these early neural crest markers. ( o ) Downregulation of Lin28b using two independent DsiRNAs followed by quantification of FoxD3 and Sox10 transcripts reveal that Lin28b is not required for neural crest formation. MO: Morpholino, DsiRNA: Dicer-substrate siRNA, gRNA: guide-RNA, HH: Hamburger and Hamilton stages.

    Article Snippet: The following primary antibodies were used: anti-Lin28a mouse monoclonal (DSHB, 1:4), anti-Sox10, goat polyclonal (R and D Systems, AF2864, 1:50), anti-Foxd3, rabbit polyclonal (1:200, gift from Patricia Labosky), anti-Pax7, mouse (DSHB AB528428, 1:4), anti-Cas9, rabbit polyclonal (Takara 632607, 1:200) anti-WNT1, rabbit polyclonal (Abcam, ab15251, 1:200), anti-Ctnnb1 (β-catenin) mouse monoclonal (BD Transduction Laboratories, 610154 1:100), anti-Tuj1 (BioLegend, 801202,1:200), anti-pH3(S10) (Abcam, ab47297, 1:200), anti-Caspase3 (R and D Systems, AF835, 1:100) and anti-mCherry, rabbit polyclonal (Abcam, ab167453, 1:200).

    Techniques: Transfection, Injection, Electroporation, Cell Culture, Incubation, Immunohistochemistry, In Situ Hybridization, Expressing, CRISPR, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation

    let-7 miRNAs regulate 3’-UTRs of neural crest genes. ( a ) Position of let-7 binding sites in the 3’-UTRs of neural crest genes assessed in the reporter assay. Red boxes correspond to regions in the UTRs that are complementary to the let-7 seed sequence, while the blue boxes correspond to let-7 compensatory sites in the UTR, which are complementary to a region of the miRNA other than the seed sequence. Direct let-7 targets (Pax7, FoxD3, Myc) have seed sequence complementarity and, in the case of FoxD3, a compensatory binding site. These are absent in the UTRs of Sox10, Zic1, and Sox8. ( b–f ) Representative scatter plots of Sox10, Pax7, Sox8, Zic1 and cMyc UTR-reporter assay, showing the mCherry/GFP intensity ratio of each cell analyzed from the control (gene-UTR) and let-7a mimic transfected (gene-UTR + let7 GOF) halves of the same embryo. Each dot represents a single cell, and the medians and the 99% confidence intervals are overlayed on the scatter plots. ( g–j ) Single cell measurement of FoxD3 protein and NC2 mCherry-PEST reporter construct fluorescence in migrating NC cells. Transverse section of HH12 embryos, showing FoxD3 protein immunostaining ( g ) and FoxD3-NC2 enhancer reporter construct ( h ) in NC cells. Dotted lines show the migrating NC cells. The farthest migrated cells expressing FoxD3-NC2 enhancer (lower dotted region on ( h )) are not positively stained for FoxD3 immunostaining, indicating decreased protein in these cells ( g ) Boxplots quantifying the fluorescent intensity of FoxD3 protein ( i ) NC2-mCherry-PEST reporter construct ( j ) in single neural crest cells as a function of the distance of the cells from the DNT. ‘NEAR’, ‘MID’ and ‘FAR’ corresponds to cells within 0–200 a.u, 201–350 a.u and 350–600 a.u from the DNT. ( k ) Bilateral electroporations of control vs. targeted Cas9 expression vectors were used to disrupt individual let-7 sites in the UTRs of the neural crest genes. UTR- Un-Translated Region, DNT- Dorsal Neural Tube, nt: neural tube, nc: neural crest, a.u- Arbitrary Units (as measured using ImageJ).

    Journal: eLife

    Article Title: Control of neural crest multipotency by Wnt signaling and the Lin28/let-7 axis

    doi: 10.7554/eLife.40556

    Figure Lengend Snippet: let-7 miRNAs regulate 3’-UTRs of neural crest genes. ( a ) Position of let-7 binding sites in the 3’-UTRs of neural crest genes assessed in the reporter assay. Red boxes correspond to regions in the UTRs that are complementary to the let-7 seed sequence, while the blue boxes correspond to let-7 compensatory sites in the UTR, which are complementary to a region of the miRNA other than the seed sequence. Direct let-7 targets (Pax7, FoxD3, Myc) have seed sequence complementarity and, in the case of FoxD3, a compensatory binding site. These are absent in the UTRs of Sox10, Zic1, and Sox8. ( b–f ) Representative scatter plots of Sox10, Pax7, Sox8, Zic1 and cMyc UTR-reporter assay, showing the mCherry/GFP intensity ratio of each cell analyzed from the control (gene-UTR) and let-7a mimic transfected (gene-UTR + let7 GOF) halves of the same embryo. Each dot represents a single cell, and the medians and the 99% confidence intervals are overlayed on the scatter plots. ( g–j ) Single cell measurement of FoxD3 protein and NC2 mCherry-PEST reporter construct fluorescence in migrating NC cells. Transverse section of HH12 embryos, showing FoxD3 protein immunostaining ( g ) and FoxD3-NC2 enhancer reporter construct ( h ) in NC cells. Dotted lines show the migrating NC cells. The farthest migrated cells expressing FoxD3-NC2 enhancer (lower dotted region on ( h )) are not positively stained for FoxD3 immunostaining, indicating decreased protein in these cells ( g ) Boxplots quantifying the fluorescent intensity of FoxD3 protein ( i ) NC2-mCherry-PEST reporter construct ( j ) in single neural crest cells as a function of the distance of the cells from the DNT. ‘NEAR’, ‘MID’ and ‘FAR’ corresponds to cells within 0–200 a.u, 201–350 a.u and 350–600 a.u from the DNT. ( k ) Bilateral electroporations of control vs. targeted Cas9 expression vectors were used to disrupt individual let-7 sites in the UTRs of the neural crest genes. UTR- Un-Translated Region, DNT- Dorsal Neural Tube, nt: neural tube, nc: neural crest, a.u- Arbitrary Units (as measured using ImageJ).

    Article Snippet: The following primary antibodies were used: anti-Lin28a mouse monoclonal (DSHB, 1:4), anti-Sox10, goat polyclonal (R and D Systems, AF2864, 1:50), anti-Foxd3, rabbit polyclonal (1:200, gift from Patricia Labosky), anti-Pax7, mouse (DSHB AB528428, 1:4), anti-Cas9, rabbit polyclonal (Takara 632607, 1:200) anti-WNT1, rabbit polyclonal (Abcam, ab15251, 1:200), anti-Ctnnb1 (β-catenin) mouse monoclonal (BD Transduction Laboratories, 610154 1:100), anti-Tuj1 (BioLegend, 801202,1:200), anti-pH3(S10) (Abcam, ab47297, 1:200), anti-Caspase3 (R and D Systems, AF835, 1:100) and anti-mCherry, rabbit polyclonal (Abcam, ab167453, 1:200).

    Techniques: Binding Assay, Reporter Assay, Sequencing, Transfection, Construct, Fluorescence, Immunostaining, Expressing, Staining

    Expression patterns of Lin28a and Lin28b mRNA and Lin28a protein during early chick development. ( a–f ) Colorimetric in situ hybridization for Lin28a in chick embryos of different developmental stages. Lin28a mRNA is enriched in the neural plate border at HH5 ( a ), in the dorsal neural folds at stage HH7-9 ( b–c ) and in migrating neural crest at stage HH10 ( d ). Transverse sections showing Lin28a expression in pre-migratory and migratory neural crest cells ( e–f ). ( g–j ) Fluorescent in situ hybridization for Lin28a and early neural crest genes Msx1 and Pax7 . At HH6, Lin28a expression overlaps with Msx1 ( g–h ) and at HH7 with Pax7 in the neural plate border (arrowheads) ( i–j ). ( k ) Immunohistochemistry for Lin28a protein, and neural crest markers FoxD3. In HH10 embryos, Lin28a protein (red) is expressed in FoxD3+ (green) neural crest cells ( l–o ). Transverse sections showing the localization of the Lin28a protein in the cytoplasm ( l–m ) of Sox10 +migratory neural crest cells ( n–o ). ( p ) Quantification of Lin28a fluorescence in migratory neural crest cells, showing that levels of Lin28a protein decrease as cells migrate away from the neural tube. ( q ) RT-PCR for Lin28a and Lin28b in FACS sorted neural crest (NC) cells and in whole embryo (WE) at HH8, showed that Lin28a , but not Lin28b , is significantly enriched in neural crest cells. ( r ) Relative expression levels of Lin28b paralog (red line) in FACS sorted neural crest cells at different developmental stages highlight that Lin28b is lowly expressed in neural crest cells and does not recapitulate the expression dynamics of Lin28a. The expression level of Lin28a (blue line) at the same developmental timepoints, shown in Figure 1 , has been included here for comparison. AU: arbitrary units. np: Neural plate, nb: neural plate border, nf: neural fold, nc: neural crest, nt: neural tube.

    Journal: eLife

    Article Title: Control of neural crest multipotency by Wnt signaling and the Lin28/let-7 axis

    doi: 10.7554/eLife.40556

    Figure Lengend Snippet: Expression patterns of Lin28a and Lin28b mRNA and Lin28a protein during early chick development. ( a–f ) Colorimetric in situ hybridization for Lin28a in chick embryos of different developmental stages. Lin28a mRNA is enriched in the neural plate border at HH5 ( a ), in the dorsal neural folds at stage HH7-9 ( b–c ) and in migrating neural crest at stage HH10 ( d ). Transverse sections showing Lin28a expression in pre-migratory and migratory neural crest cells ( e–f ). ( g–j ) Fluorescent in situ hybridization for Lin28a and early neural crest genes Msx1 and Pax7 . At HH6, Lin28a expression overlaps with Msx1 ( g–h ) and at HH7 with Pax7 in the neural plate border (arrowheads) ( i–j ). ( k ) Immunohistochemistry for Lin28a protein, and neural crest markers FoxD3. In HH10 embryos, Lin28a protein (red) is expressed in FoxD3+ (green) neural crest cells ( l–o ). Transverse sections showing the localization of the Lin28a protein in the cytoplasm ( l–m ) of Sox10 +migratory neural crest cells ( n–o ). ( p ) Quantification of Lin28a fluorescence in migratory neural crest cells, showing that levels of Lin28a protein decrease as cells migrate away from the neural tube. ( q ) RT-PCR for Lin28a and Lin28b in FACS sorted neural crest (NC) cells and in whole embryo (WE) at HH8, showed that Lin28a , but not Lin28b , is significantly enriched in neural crest cells. ( r ) Relative expression levels of Lin28b paralog (red line) in FACS sorted neural crest cells at different developmental stages highlight that Lin28b is lowly expressed in neural crest cells and does not recapitulate the expression dynamics of Lin28a. The expression level of Lin28a (blue line) at the same developmental timepoints, shown in Figure 1 , has been included here for comparison. AU: arbitrary units. np: Neural plate, nb: neural plate border, nf: neural fold, nc: neural crest, nt: neural tube.

    Article Snippet: The following primary antibodies were used: anti-Lin28a mouse monoclonal (DSHB, 1:4), anti-Sox10, goat polyclonal (R and D Systems, AF2864, 1:50), anti-Foxd3, rabbit polyclonal (1:200, gift from Patricia Labosky), anti-Pax7, mouse (DSHB AB528428, 1:4), anti-Cas9, rabbit polyclonal (Takara 632607, 1:200) anti-WNT1, rabbit polyclonal (Abcam, ab15251, 1:200), anti-Ctnnb1 (β-catenin) mouse monoclonal (BD Transduction Laboratories, 610154 1:100), anti-Tuj1 (BioLegend, 801202,1:200), anti-pH3(S10) (Abcam, ab47297, 1:200), anti-Caspase3 (R and D Systems, AF835, 1:100) and anti-mCherry, rabbit polyclonal (Abcam, ab167453, 1:200).

    Techniques: Expressing, In Situ Hybridization, Immunohistochemistry, Fluorescence, Reverse Transcription Polymerase Chain Reaction, FACS

    The Lin28/ let-7 axis modulates neural crest progenitor identity in vivo. ( a ) A schematic representation of the let-7 sensor, which consists of several let-7 binding sites downstream of destabilized mCherry fluorescent protein. ( b–c ) Activity of mature let-7 miRNAs increase through neural crest development. ( b ) Boxplots showing mCherry/GFP fluorescence ratio, a readout of let-7 sensor activity, in neural crest cells at different developmental stages. ( c ) RT-PCR for mature let-7 family miRNAs comparing their levels in neural crest cells sorted from HH8 and HH12 embryos. ( d–f ) Loss of Lin28a results in increased activity of mature let-7 miRNAs. ( d ) Whole mount view of an embryo bilaterally injected with control and Lin28a MO. ( e ) Representative image showing let-7 sensor fluorescence in control vs Lin28a MO side of an embryo. Dotted line represents embryo midline. ( f ) RT-PCR for mature let-7 family miRNAs, in the background of Lin28a knockdown. ( g ) Whole mount view of an embryo electroporated with control and let-7a mimic. ( h ) Immunohistochemistry for FoxD3 positive neural crest cells in the presence of let-7a mimic. Dotted line represents embryo midline. ( i ) Quantification of transcript levels of FoxD3 and Sox10 , in presence of increased let-7a. ( j ) Model for modulation of neural crest identity by the Lin28/ let-7 axis. ( k ) Representative dorsal view of an embryo electroporated with control MO (blue) on the left and Lin28a MO (green) co-injected with a Lin28a expression vector (red) on the right. ( l ) Boxplots showing the quantification of FoxD3 and Sox10 fluorescence in epistatic experiments, in which Lin28a Mo was co-electroporated with Lin28a expression vector, mCCHC Lin28a, and a let-7 sponge construct. ( m ) Loss of let-7 activity results in maintenance of multipotency genes in late neural crest cells. RT-PCR for Pax7, FoxD3, Sox5, Myc, Ets1 and Lin28a comparing the expression of these genes in control vs late migratory neural crest cells expressing let-7 sponge construct. Error bars in ( c ), ( f ), ( i ) and ( m ) represent standard error. HH: Hamburger and Hamilton developmental stages, MO: Morpholino. 10.7554/eLife.40556.011 Data for the RT-PCR experiments shown in Figure 3 , and quantitation of FoxD3 and Sox10 intensity in epistasis experiments.

    Journal: eLife

    Article Title: Control of neural crest multipotency by Wnt signaling and the Lin28/let-7 axis

    doi: 10.7554/eLife.40556

    Figure Lengend Snippet: The Lin28/ let-7 axis modulates neural crest progenitor identity in vivo. ( a ) A schematic representation of the let-7 sensor, which consists of several let-7 binding sites downstream of destabilized mCherry fluorescent protein. ( b–c ) Activity of mature let-7 miRNAs increase through neural crest development. ( b ) Boxplots showing mCherry/GFP fluorescence ratio, a readout of let-7 sensor activity, in neural crest cells at different developmental stages. ( c ) RT-PCR for mature let-7 family miRNAs comparing their levels in neural crest cells sorted from HH8 and HH12 embryos. ( d–f ) Loss of Lin28a results in increased activity of mature let-7 miRNAs. ( d ) Whole mount view of an embryo bilaterally injected with control and Lin28a MO. ( e ) Representative image showing let-7 sensor fluorescence in control vs Lin28a MO side of an embryo. Dotted line represents embryo midline. ( f ) RT-PCR for mature let-7 family miRNAs, in the background of Lin28a knockdown. ( g ) Whole mount view of an embryo electroporated with control and let-7a mimic. ( h ) Immunohistochemistry for FoxD3 positive neural crest cells in the presence of let-7a mimic. Dotted line represents embryo midline. ( i ) Quantification of transcript levels of FoxD3 and Sox10 , in presence of increased let-7a. ( j ) Model for modulation of neural crest identity by the Lin28/ let-7 axis. ( k ) Representative dorsal view of an embryo electroporated with control MO (blue) on the left and Lin28a MO (green) co-injected with a Lin28a expression vector (red) on the right. ( l ) Boxplots showing the quantification of FoxD3 and Sox10 fluorescence in epistatic experiments, in which Lin28a Mo was co-electroporated with Lin28a expression vector, mCCHC Lin28a, and a let-7 sponge construct. ( m ) Loss of let-7 activity results in maintenance of multipotency genes in late neural crest cells. RT-PCR for Pax7, FoxD3, Sox5, Myc, Ets1 and Lin28a comparing the expression of these genes in control vs late migratory neural crest cells expressing let-7 sponge construct. Error bars in ( c ), ( f ), ( i ) and ( m ) represent standard error. HH: Hamburger and Hamilton developmental stages, MO: Morpholino. 10.7554/eLife.40556.011 Data for the RT-PCR experiments shown in Figure 3 , and quantitation of FoxD3 and Sox10 intensity in epistasis experiments.

    Article Snippet: The following primary antibodies were used: anti-Lin28a mouse monoclonal (DSHB, 1:4), anti-Sox10, goat polyclonal (R and D Systems, AF2864, 1:50), anti-Foxd3, rabbit polyclonal (1:200, gift from Patricia Labosky), anti-Pax7, mouse (DSHB AB528428, 1:4), anti-Cas9, rabbit polyclonal (Takara 632607, 1:200) anti-WNT1, rabbit polyclonal (Abcam, ab15251, 1:200), anti-Ctnnb1 (β-catenin) mouse monoclonal (BD Transduction Laboratories, 610154 1:100), anti-Tuj1 (BioLegend, 801202,1:200), anti-pH3(S10) (Abcam, ab47297, 1:200), anti-Caspase3 (R and D Systems, AF835, 1:100) and anti-mCherry, rabbit polyclonal (Abcam, ab167453, 1:200).

    Techniques: In Vivo, Binding Assay, Activity Assay, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Injection, Immunohistochemistry, Expressing, Plasmid Preparation, Construct, Quantitation Assay

    Mef2c -F1 is a bona fide Endothelin-responsive enhancer of Mef2c . (A) Schematics of the mouse Mef2c locus showing exons 4-6 (vertical black lines) and the Mef2c -F1 enhancer (green box). The Mef2c F1 Δ and Mef2c -null ( Mef2c Δ ) knockout strategies are indicated. (B) Number of live and dead offspring of each indicated genotype from Mef2c +/F1 Δ × Mef2c +/Δ intercrosses. Note that 23/23 wt and heterozygous (het) offspring survived, whereas only 2/7 Mef2c F1 Δ / Δ survived (Fisher's exact test, P =0.0001). (C-F″) The Mef2c -F1 enhancer is required for ET-1 to induce endogenous MEF2C expression in trunk neural crest (NC) cells. ET-1 induced endogenous MEF2C protein expression in trunk neural crest cells (marked by Sox10 immunofluorescence) in ET-1-treated (D-D″) but not in PBS-treated (C-C″) explants. Note MEF2C expression in skeletal muscle (SkM) in both PBS- and ET-1-treated explants (C′,D′). ET-1 treatment failed to induce endogenous MEF2C protein expression in trunk neural crest cells in Mef2c F1 Δ /F1 Δ explants (F). Note the absence of co-expression of MEF2C and Sox10 in Mef2c F1 Δ /F1 Δ explants treated with either PBS (E-E″) or ET-1 (F-F″). NT, neural tube. Scale bars: 100 µm.

    Journal: Development (Cambridge, England)

    Article Title: Endothelin signaling activates Mef2c expression in the neural crest through a MEF2C-dependent positive-feedback transcriptional pathway

    doi: 10.1242/dev.126391

    Figure Lengend Snippet: Mef2c -F1 is a bona fide Endothelin-responsive enhancer of Mef2c . (A) Schematics of the mouse Mef2c locus showing exons 4-6 (vertical black lines) and the Mef2c -F1 enhancer (green box). The Mef2c F1 Δ and Mef2c -null ( Mef2c Δ ) knockout strategies are indicated. (B) Number of live and dead offspring of each indicated genotype from Mef2c +/F1 Δ × Mef2c +/Δ intercrosses. Note that 23/23 wt and heterozygous (het) offspring survived, whereas only 2/7 Mef2c F1 Δ / Δ survived (Fisher's exact test, P =0.0001). (C-F″) The Mef2c -F1 enhancer is required for ET-1 to induce endogenous MEF2C expression in trunk neural crest (NC) cells. ET-1 induced endogenous MEF2C protein expression in trunk neural crest cells (marked by Sox10 immunofluorescence) in ET-1-treated (D-D″) but not in PBS-treated (C-C″) explants. Note MEF2C expression in skeletal muscle (SkM) in both PBS- and ET-1-treated explants (C′,D′). ET-1 treatment failed to induce endogenous MEF2C protein expression in trunk neural crest cells in Mef2c F1 Δ /F1 Δ explants (F). Note the absence of co-expression of MEF2C and Sox10 in Mef2c F1 Δ /F1 Δ explants treated with either PBS (E-E″) or ET-1 (F-F″). NT, neural tube. Scale bars: 100 µm.

    Article Snippet: Immunolabeling was performed using the following primary antibodies at 1:100 dilutions in PBS with 3% BSA and 0.1% Triton X-100: anti-SOX10 (R & D, AF2864); anti-MEF2C (D80C1, Cell Signaling, #5030); anti-β-galactosidase (Abcam, Ab9361).

    Techniques: Knock-Out, Expressing, Immunofluorescence

    Mef2c -F1 is an Endothelin-responsive neural crest enhancer. (A) Diagram of the Endothelin-responsive cis -regulatory element in the Mef2c locus and the Mef2c -F1- lacZ transgenic reporter construct. (B-E) ET A and ET B are required for Mef2c -F1 Endothelin-responsive enhancer activity in vivo . Mef2c -F1- lacZ transgenic mice were crossed into wild-type (B), Ednra -null (C), Ednrb -null (D) and Ednra; Ednrb double-null (E) backgrounds and were analyzed by X-gal staining at E9.5. (F,G) Bosentan, a dual Endothelin receptor antagonist, inhibited Mef2c -F1 enhancer activity in the neural crest of explanted E9.5 embryos. Trunk neural crest, dashed circles; cranial neural crest, black arrows. (H-L) ET-1 precociously activated the Mef2c -F1 enhancer in transgenic reporter embryo explants as shown by X-gal staining (H,H′, dashed circles) and by immunofluorescence using anti-β-galactosidase antibody (I-K,I′-K′; β-galactosidase is marked by green fluorescence; neural crest cells are marked by Sox10 + red fluorescence). Three independent Mef2c -F1- lacZ transgenic lines displayed nearly identical responses to ET-1 and bosentan treatment. (L,L′) A 300-bp minimal enhancer responds to ET-1 (dashed circles); two independent Mef2c -F1[3-3.3]- lacZ transgenic lines were examined, and both showed a similar ET-1 response. Note the absence of β-galactosidase expression in trunk neural crest cells in PBS-treated explants (H) and the complete overlap of β-galactosidase expression with Sox10 + neural crest cells in ET-1-treated explants (I′-K′). BA, branchial arch; NT, neural tube. Scale bars: 100 µm.

    Journal: Development (Cambridge, England)

    Article Title: Endothelin signaling activates Mef2c expression in the neural crest through a MEF2C-dependent positive-feedback transcriptional pathway

    doi: 10.1242/dev.126391

    Figure Lengend Snippet: Mef2c -F1 is an Endothelin-responsive neural crest enhancer. (A) Diagram of the Endothelin-responsive cis -regulatory element in the Mef2c locus and the Mef2c -F1- lacZ transgenic reporter construct. (B-E) ET A and ET B are required for Mef2c -F1 Endothelin-responsive enhancer activity in vivo . Mef2c -F1- lacZ transgenic mice were crossed into wild-type (B), Ednra -null (C), Ednrb -null (D) and Ednra; Ednrb double-null (E) backgrounds and were analyzed by X-gal staining at E9.5. (F,G) Bosentan, a dual Endothelin receptor antagonist, inhibited Mef2c -F1 enhancer activity in the neural crest of explanted E9.5 embryos. Trunk neural crest, dashed circles; cranial neural crest, black arrows. (H-L) ET-1 precociously activated the Mef2c -F1 enhancer in transgenic reporter embryo explants as shown by X-gal staining (H,H′, dashed circles) and by immunofluorescence using anti-β-galactosidase antibody (I-K,I′-K′; β-galactosidase is marked by green fluorescence; neural crest cells are marked by Sox10 + red fluorescence). Three independent Mef2c -F1- lacZ transgenic lines displayed nearly identical responses to ET-1 and bosentan treatment. (L,L′) A 300-bp minimal enhancer responds to ET-1 (dashed circles); two independent Mef2c -F1[3-3.3]- lacZ transgenic lines were examined, and both showed a similar ET-1 response. Note the absence of β-galactosidase expression in trunk neural crest cells in PBS-treated explants (H) and the complete overlap of β-galactosidase expression with Sox10 + neural crest cells in ET-1-treated explants (I′-K′). BA, branchial arch; NT, neural tube. Scale bars: 100 µm.

    Article Snippet: Immunolabeling was performed using the following primary antibodies at 1:100 dilutions in PBS with 3% BSA and 0.1% Triton X-100: anti-SOX10 (R & D, AF2864); anti-MEF2C (D80C1, Cell Signaling, #5030); anti-β-galactosidase (Abcam, Ab9361).

    Techniques: Transgenic Assay, Construct, Activity Assay, In Vivo, Mouse Assay, Staining, Immunofluorescence, Fluorescence, Expressing

    PEDF induces DCX+/Sox10+ cells in the adult SVZ Saline or PEDF (300ng/ml) was infused into the lateral ventricle of adult wild type mice for 7 days before sacrifice. (A–B) Confocal images of saline-infused SVZ double-immunostained for DCX and Sox10. No DCX+ cells expressed Sox10 in the SVZ. (C–D) Orthogonal (C) and Z-series stack (D) confocal images of PEDF-infused SVZ. PEDF infusion induced DCX+/Sox10+ cells in the SVZ. The Insets in D show magnified images for the area indicated by a rectangle. Scale bar = 20μm.

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

    Article Title: PEDF is a novel oligodendrogenic morphogen acting on the adult SVZ and corpus callosum

    doi: 10.1523/JNEUROSCI.0628-12.2012

    Figure Lengend Snippet: PEDF induces DCX+/Sox10+ cells in the adult SVZ Saline or PEDF (300ng/ml) was infused into the lateral ventricle of adult wild type mice for 7 days before sacrifice. (A–B) Confocal images of saline-infused SVZ double-immunostained for DCX and Sox10. No DCX+ cells expressed Sox10 in the SVZ. (C–D) Orthogonal (C) and Z-series stack (D) confocal images of PEDF-infused SVZ. PEDF infusion induced DCX+/Sox10+ cells in the SVZ. The Insets in D show magnified images for the area indicated by a rectangle. Scale bar = 20μm.

    Article Snippet: Membranes were blocked and then incubated with primary antibodies: Olig1 (1:3000, Millipore), Olig2 (1:3000, R & D), Sox10 (1:1000, Millipore), PDGFrα (1:500, Santa Cruz), and PEDF (1:100, R & D).

    Techniques: Mouse Assay

    PEDF exerts oligodendrogenic effect on adult SVZ GFAP+ neural precursors (A) Experimental flow chart. GFAP:GFP+NG2− and GFAP:GFP+NG2+ single cells were isolated by FACS from primary neurospheres derived from the SVZ of adult GFAP:GFP mice. Secondary neurospheres were then produced from these two FACS-purified populations in the absence or presence of PEDF (50ng/ml). Assays were then performed as indicated. (B–C) qRT-PCR analysis showing that PEDF elevated expression levels of oligodendroglial transcription factors and PDGFrα in both GFP+NG2− and GFP+NG2+ precursor-derived neurospheres. (D) Western blot analysis of Olig1, Olig2, Sox10, and PDGFrα in control and PEDF-treated wild type secondary neurospheres. (E–G) Control and PEDF-treated secondary neurospheres derived from FACS purified GFP+NG2− or GFP+NG2+ subsets were plated in differentiation medium without mitogens or PEDF for 3 days before immunostaining for NG2, O4, or Tuj1. (E and G) Greater numbers of NG2+ and O4+ oligodendroglial cells were produced from both GFP+NG2− and GFP+NG2+ cell-derived neurospheres with PEDF treatment. (F and G) PEDF treatment resulted in diminished neuronal differentiation of GFP+NG2− and GFP+NG2+ cell-derived neurospheres. (G) Representative images of cells differentiated from control or PEDF-treated GFP+NG2− cell-derived neurospheres. Results are means +/− SEM of three or four independent experiments. Scale bar = 25μm. (* P

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

    Article Title: PEDF is a novel oligodendrogenic morphogen acting on the adult SVZ and corpus callosum

    doi: 10.1523/JNEUROSCI.0628-12.2012

    Figure Lengend Snippet: PEDF exerts oligodendrogenic effect on adult SVZ GFAP+ neural precursors (A) Experimental flow chart. GFAP:GFP+NG2− and GFAP:GFP+NG2+ single cells were isolated by FACS from primary neurospheres derived from the SVZ of adult GFAP:GFP mice. Secondary neurospheres were then produced from these two FACS-purified populations in the absence or presence of PEDF (50ng/ml). Assays were then performed as indicated. (B–C) qRT-PCR analysis showing that PEDF elevated expression levels of oligodendroglial transcription factors and PDGFrα in both GFP+NG2− and GFP+NG2+ precursor-derived neurospheres. (D) Western blot analysis of Olig1, Olig2, Sox10, and PDGFrα in control and PEDF-treated wild type secondary neurospheres. (E–G) Control and PEDF-treated secondary neurospheres derived from FACS purified GFP+NG2− or GFP+NG2+ subsets were plated in differentiation medium without mitogens or PEDF for 3 days before immunostaining for NG2, O4, or Tuj1. (E and G) Greater numbers of NG2+ and O4+ oligodendroglial cells were produced from both GFP+NG2− and GFP+NG2+ cell-derived neurospheres with PEDF treatment. (F and G) PEDF treatment resulted in diminished neuronal differentiation of GFP+NG2− and GFP+NG2+ cell-derived neurospheres. (G) Representative images of cells differentiated from control or PEDF-treated GFP+NG2− cell-derived neurospheres. Results are means +/− SEM of three or four independent experiments. Scale bar = 25μm. (* P

    Article Snippet: Membranes were blocked and then incubated with primary antibodies: Olig1 (1:3000, Millipore), Olig2 (1:3000, R & D), Sox10 (1:1000, Millipore), PDGFrα (1:500, Santa Cruz), and PEDF (1:100, R & D).

    Techniques: Flow Cytometry, Isolation, FACS, Derivative Assay, Mouse Assay, Produced, Purification, Quantitative RT-PCR, Expressing, Western Blot, Immunostaining

    PEDF infusion increases the numbers of SVZ cells expressing early oligodendroglial lineage markers or oligodendroglial transcription factors Saline or PEDF (300ng per day) was administered via osmotic pump into the lateral ventricle of adult GFAP:GFP transgenic mice for 7 days. The SVZ tissues were double-immunostained for GFP and markers for early oligodendroglial lineage (PDGFrα or NG2) or oligodendroglial transcription factors (Olig1, Olig2, or Sox10). (A–F) Confocal images of coronal sections of saline- and PEDF-infused SVZ immunolabeled with antibodies against GFP and PDGFrα, NG2, or Olig1. Insets show magnified images of areas indicated by rectangles. Scale bar = 30μm. (G–K) Percentages of dapi+ nuclei labeled with PDGFrα+, PDGFrα+GFP+ and PDGFrα+GFP− (G), NG2+, NG2+GFP+ and NG2+GFP− (H), Olig1+, Olig1+GFP+ and Olig1+GFP− (I), Olig2+, Olig2+GFP+ and Olig2+GFP− (J), and Sox10+, Sox10+GFP+ and Sox10+GFP− (K) in the saline- and PEDF-infused SVZ. PEDF administration increased the proportions of both GFP+ and GFP− cells co-expressing these oligodendroglial markers or transcription factors. Results are means +/− SEM (n = 4–5 brains, * P

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

    Article Title: PEDF is a novel oligodendrogenic morphogen acting on the adult SVZ and corpus callosum

    doi: 10.1523/JNEUROSCI.0628-12.2012

    Figure Lengend Snippet: PEDF infusion increases the numbers of SVZ cells expressing early oligodendroglial lineage markers or oligodendroglial transcription factors Saline or PEDF (300ng per day) was administered via osmotic pump into the lateral ventricle of adult GFAP:GFP transgenic mice for 7 days. The SVZ tissues were double-immunostained for GFP and markers for early oligodendroglial lineage (PDGFrα or NG2) or oligodendroglial transcription factors (Olig1, Olig2, or Sox10). (A–F) Confocal images of coronal sections of saline- and PEDF-infused SVZ immunolabeled with antibodies against GFP and PDGFrα, NG2, or Olig1. Insets show magnified images of areas indicated by rectangles. Scale bar = 30μm. (G–K) Percentages of dapi+ nuclei labeled with PDGFrα+, PDGFrα+GFP+ and PDGFrα+GFP− (G), NG2+, NG2+GFP+ and NG2+GFP− (H), Olig1+, Olig1+GFP+ and Olig1+GFP− (I), Olig2+, Olig2+GFP+ and Olig2+GFP− (J), and Sox10+, Sox10+GFP+ and Sox10+GFP− (K) in the saline- and PEDF-infused SVZ. PEDF administration increased the proportions of both GFP+ and GFP− cells co-expressing these oligodendroglial markers or transcription factors. Results are means +/− SEM (n = 4–5 brains, * P

    Article Snippet: Membranes were blocked and then incubated with primary antibodies: Olig1 (1:3000, Millipore), Olig2 (1:3000, R & D), Sox10 (1:1000, Millipore), PDGFrα (1:500, Santa Cruz), and PEDF (1:100, R & D).

    Techniques: Expressing, Transgenic Assay, Mouse Assay, Immunolabeling, Labeling