anti-sox2 Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95
    Thermo Fisher anti sox2
    Effect of Embryonic CSF (E-CSF) on mitotic activity in SVZ niche NSCs, monitored by nuclear incorporation of BrdU (red). Confocal photomicrographs ( A,B show the SVZ of “ in vitro ” cultured adult mice brain slices (see Figure 1B ). Note the substantial increase in NSCs mitotic activity under the ventricular surface (white arrows) induced by E-CSF (B) , compared with the controls (A) . Confocal photomicrographs (C–E) show the Striatum area of “ in vitro ” cultured adult mice brain slices close to the SVZ (ST in Figure 1B ). Quantification of BrdU positive nuclei in Control (C) , E-CSF (D) and A-CSF (E) treated brain slices was plotted in graph bars (F) and expressed as means ± SD ( n = 20); the significant threshold was set at p ≤ 0.001 (*) according to the one-way ANOVA, post hoc Bonferroni test. The results reveal a statistically significant increase (49%) in the number of BrdU positive NSCs in CSF-E treated brain slices with respect to the controls and A-CSF treated slices; this suggests a specific activation by E-CSF of mitotic activity in NSCs of the SVZ niche. Confocal photomicrographs (G,I) correspond to the striatum area of “ in vitro ” cultured adult mice brain slices, close to SVZ (ST in Figure 1B ). Images show double immunolabeling with antiBrdU (green) and antiSox2 (red) antibody; co-localization of both antibodies was interpreted as NSCs dividing (BrdU positive) but not differentiating <t>(Sox2</t> positive). Quantification of co-labeled BrdU and Sox2 NSCs in Control (G) and E-CSF treated brain slices (I) was plotted in graph bars (H) and expressed as means ± SD ( n = 20); the significant threshold was set at p ≤ 0.001 (*) according to the two-tailed Student’s t -test. The results reveal a statistically significant increase (141%) in the number of NSCs that undergo replication but remain undifferentiated in CSF-E treated brain slices, in comparison with the controls. Scale bar: 25 μm (A–E) and 10 μm (G,I) .
    Anti Sox2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti sox2/product/Thermo Fisher
    Average 95 stars, based on 105 article reviews
    Price from $9.99 to $1999.99
    anti sox2 - by Bioz Stars, 2020-07
    95/100 stars
      Buy from Supplier

    99
    Millipore anti sox2
    Tamoxifen induces <t>SOX2</t> to enhance tamoxifen resistance through TARBP2. ( A , B ) Expression of different stem cell markers after tamoxifen treatment. MCF-7 cells were treated with 2 μM tamoxifen for 48 h and then RNA was isolated to analyze the mRNA expression of stem cell markers by reverse-transcription PCR (qRT-PCR). The experiments were repeated at least 3 times, and ATP5E was used as a positive control for tamoxifen treatment ( A ). * p ≤ 0.05 by t -test. Cells as indicated in ( A ) were collected to analyze protein expression by western blotting ( B ). ( C , D ) Effect of SOX2 expression on tamoxifen sensitivity. MCF-7 cells were transfected with shRNA targeting SOX2 for 48 h and treated with different concentrations of tamoxifen (1, 2, 5, 10, 20 μM) for 72 h. The efficiency of SOX2 knock-down was examined by western blot ( C ), and the proliferation and colony formation were determined by MTT ( D ) and colony formation assays ( E ), respectively. MTT experimental results are given as the means ± SEM from at least three separate experiments that were performed in duplicate or triplicate and analyzed by two-way ANOVA. * p ≤ 0.05, ** p ≤ 0.01. ( F , G ) Tamoxifen downregulated the protein level of SOX2 through TARBP2. MCF-7 cells were transfected with shRNAs targeting TARBP2 for 48 h; 2 μM tamoxifen was then added to the culture medium for 48 h. The cells were harvested to determine the protein expressions by western blot. ( G – I ) TARBP2-regulated protein stability of SOX2 in tamoxifen-treated and resistant cells. Tamoxifen-treated (2 μM for 48 h) MCF-7 ( G ) and MCF-7/TR1 ( H ) cells were treated with 50 μg/mL cycloheximide to block protein synthesis and were then harvested at the indicated time point to analyze the expression of SOX2 by western blotting. ( I ) MCF-7 cells were transfected with the indicated shRNAs targeting TARBP2 for 48 h and treated with 2 μM tamoxifen for 48 h. Cells were add 50 μg/mL cycloheximide and harvested at the indicated time point to analyze the expression of SOX2 by western blotting. The degradation rates were plotted for the average ± SEM of at least three independent experiments and analyzed by two-way ANOVA. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
    Anti Sox2, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1092 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti sox2/product/Millipore
    Average 99 stars, based on 1092 article reviews
    Price from $9.99 to $1999.99
    anti sox2 - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    94
    Abcam mouse anti sox 2 antibody
    Relative expression of miR-34a, b and c in OSCs and U-2OS cell monolayers, detected using SYBR Green reverse transcription-quantitative polymerase chain reaction analysis. (A) Culture of OSCs in serum-free medium on days 3, 10 and 14. (B) Representative photomicrographs reveal the staining of marker antigens <t>Sox-2</t> (red) and Sca-1 (green). (C) Average expression levels of miR-34a, b and c in OSCs, compared with those in monolayer cells. *P
    Mouse Anti Sox 2 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti sox 2 antibody/product/Abcam
    Average 94 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    mouse anti sox 2 antibody - by Bioz Stars, 2020-07
    94/100 stars
      Buy from Supplier

    93
    Boster Bio anti sox2
    <t>SOX2</t> knockdown inhibits the viability, migration and invasion of glioma cell lines. a The relative expression of SOX2 increases in glioma tissues compared with that in normal tissues. The SOX2 expression is higher in high grade (stage III and stage IV) glioma tissues than that in low grade (stage I and stage II) glioma tissues. GAPDH is the internal control. ** P
    Anti Sox2, supplied by Boster Bio, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti sox2/product/Boster Bio
    Average 93 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    anti sox2 - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

    93
    BioLegend pe anti sox2
    <t>SOX2</t> knockdown inhibits the viability, migration and invasion of glioma cell lines. a The relative expression of SOX2 increases in glioma tissues compared with that in normal tissues. The SOX2 expression is higher in high grade (stage III and stage IV) glioma tissues than that in low grade (stage I and stage II) glioma tissues. GAPDH is the internal control. ** P
    Pe Anti Sox2, supplied by BioLegend, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pe anti sox2/product/BioLegend
    Average 93 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    pe anti sox2 - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

    99
    Abcam goat anti sox 2 antibody
    NU6140 treatment compared to nocodazole treatment affects differently the expression of pluripotency markers in 2102Ep carcinoma cells. Expression of pluripotency markers NANOG, OCT4, and <t>SOX2</t> in nocodazole and NU6140 treated 2102Ep cells as detected by using flow cytometric assays. (a) Fixed and permeabilised (methanol permeabilisation) cells were stained with anti-NANOG (PE), anti-OCT4 (Alexa Fluor 647), and anti-SOX2 (PerCp Cy5.5 conjugate) antibodies and with DAPI. For analysis cellular debris and doublets were excluded. (b) Correlation between expression of CDK2 with NANOG and SOX2. Fixed and permeabilised (methanol permeabilisation) cells were stained with anti-CDK2 (Alexa Fluor 488 conjugate), anti-NANOG (PE), and anti-SOX2 (PerCp Cy5.5 conjugate) antibodies and with DAPI. Results are shown as density plots.
    Goat Anti Sox 2 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti sox 2 antibody/product/Abcam
    Average 99 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    goat anti sox 2 antibody - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    96
    Abcam anti sox2
    Spatial analysis of the primitive streak region. (A) Unique DEGs to each E8.5 region (≥1.5-fold change compared with other samples; for unique DEGs in St5, see Table S1). Asterisk indicates that no specific in situ probe could be constructed. (B) Bhmt2 and Ccno expression in the ventral (V) but not dorsal (D) NSB layer. (C) Whole-mount in situ hybridisation for Mnx1 . tg, tail gut; vn, ventral node. (D) <t>Sox2</t> versus T intensity values for all samples. Abbreviations are preceded by embryo age; colours indicates fate. Regions of similar fate are enclosed by a coloured line.
    Anti Sox2, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 1162 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti sox2/product/Abcam
    Average 96 stars, based on 1162 article reviews
    Price from $9.99 to $1999.99
    anti sox2 - by Bioz Stars, 2020-07
    96/100 stars
      Buy from Supplier

    94
    Atlas Antibodies sox2 antibody
    Structure analysis of <t>Sox2-HMG/nucleic</t> acid-binding modes. a 1 H– 15 N HSQC NMR spectrum of Sox2-HMG (black) overlaid with the spectrum of the Sox2-HMG/DNA complex (red). The DNA sequence used in this experiment was derived from the FGF4 enhancer, corresponding to the sequence shown in Supplementary Table 1 . b 1 H– 15 N HSQC NMR spectrum of Sox2-HMG (black) overlaid with the spectrum of the Sox2-HMG/RNA complex (blue). The RNA sequence used in this experiment was the hairpin Loop B/Bulge(0 + 1) (Fig. 3c ).
    Sox2 Antibody, supplied by Atlas Antibodies, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sox2 antibody/product/Atlas Antibodies
    Average 94 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    sox2 antibody - by Bioz Stars, 2020-07
    94/100 stars
      Buy from Supplier

    94
    Millipore rabbit anti sox2
    Ascl1 -expressing cells give rise directly to Sus cells . ( A ) Immunofluorescence (IF) for <t>SOX2</t> and ASCL1 in wild-type OE. White arrowheads indicate SOX2 + ; ASCL1 + cells. ( B ) In Ascl1 GFP/+ OE, apical GFP + cells are SUS4 + (arrowheads). The bottom row shows
    Rabbit Anti Sox2, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1084 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti sox2/product/Millipore
    Average 94 stars, based on 1084 article reviews
    Price from $9.99 to $1999.99
    rabbit anti sox2 - by Bioz Stars, 2020-07
    94/100 stars
      Buy from Supplier

    99
    Abcam rb anti sox2
    Ascl1 -expressing cells give rise directly to Sus cells . ( A ) Immunofluorescence (IF) for <t>SOX2</t> and ASCL1 in wild-type OE. White arrowheads indicate SOX2 + ; ASCL1 + cells. ( B ) In Ascl1 GFP/+ OE, apical GFP + cells are SUS4 + (arrowheads). The bottom row shows
    Rb Anti Sox2, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rb anti sox2/product/Abcam
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    rb anti sox2 - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    86
    BioLegend purified anti sox2
    Ascl1 -expressing cells give rise directly to Sus cells . ( A ) Immunofluorescence (IF) for <t>SOX2</t> and ASCL1 in wild-type OE. White arrowheads indicate SOX2 + ; ASCL1 + cells. ( B ) In Ascl1 GFP/+ OE, apical GFP + cells are SUS4 + (arrowheads). The bottom row shows
    Purified Anti Sox2, supplied by BioLegend, used in various techniques. Bioz Stars score: 86/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/purified anti sox2/product/BioLegend
    Average 86 stars, based on 35 article reviews
    Price from $9.99 to $1999.99
    purified anti sox2 - by Bioz Stars, 2020-07
    86/100 stars
      Buy from Supplier

    93
    Thermo Fisher rat anti sox2
    Western blotting. Protein extractions from 3 LGCA and 3 HGCA EpCAM High (+) and EpCAM Low (-) cell lines were probed for OCT4 (A; 40kDa), <t>SOX2</t> (B; 40-43kDa), NANOG (C; 37-40kDa), KLF4 (D; 54kDa) and c-MYC (E; 42 57kDa). NTERA-2 cells were used as the positive control for all iPSC markers. α-tubulin (F; 50kDa) was used as a loading control. EpCAM High and EpCAM Low cell lines were also probed for their expression of EpCAM (G; bands from ~30-40kDa) and α-SMA (H; 42kDa). HepG2 cells and 3T3 cells were used as the positive control for EpCAM and α-SMA, respectively.
    Rat Anti Sox2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rat anti sox2/product/Thermo Fisher
    Average 93 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    rat anti sox2 - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

    94
    Thermo Fisher rabbit anti sox2
    Proposed model for bFGF signaling in pluripotent (green) vs. differentiated (orange) hiPSCs (for details see discussion). bFGF, basic fibroblast growth factor; OCT4, octamer-binding transcription factor 4; <t>SOX2,</t> sex determining region Y-box 2; GFAP, glial fibrillary acidic protein; RAS, rat sarcoma; PI3K, phosphoinositide 3-kinase; PDK1, 3-phosphoinositidedependent protein kinase; MEK, MAP/ERK kinase; ERK, extracellular signal-regulated kinase; JAK, Janus kinase; STAT3, signal transducer and activator of transcription 3
    Rabbit Anti Sox2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 176 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti sox2/product/Thermo Fisher
    Average 94 stars, based on 176 article reviews
    Price from $9.99 to $1999.99
    rabbit anti sox2 - by Bioz Stars, 2020-07
    94/100 stars
      Buy from Supplier

    92
    Stemgent anti sox2
    Merkel cell differentiation is a temporal maturation process. (A-C) Whole-mount IF (WMIF) staining showing overlap of Merkel cell-specific genes Atoh1 , K8 ( Krt8 ), K20 ( Krt20 ) and <t>Sox2</t> in neonatal (P0) mouse skin. Percentage of overlap is shown in C. Merkel
    Anti Sox2, supplied by Stemgent, used in various techniques. Bioz Stars score: 92/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti sox2/product/Stemgent
    Average 92 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    anti sox2 - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    92
    Santa Cruz Biotechnology anti sox2
    Cyclin K expression positively correlates with proliferation. a Analyses of cyclin K protein expression during murine brain development by immunoblotting. Cyclin K protein expression in embryonic (E) and postnatal (P) murine brains correlated with that of <t>Sox2,</t> a marker of neural progenitor cell proliferation. b Analyses of cyclin K protein expression by immunoblotting during murine liver development. c Cyclin K expression detected by immunochemistry during the process of murine liver regeneration in vivo. 2nd, immunochemistry using secondary antibodies alone. 0 h denotes samples collected immediately after partial hepatectomy. Scale bar, 40 μm. d Comparison of cyclin K by immunoblotting in normal and H1299 cancer cells using equal cell numbers as loading control. HFF, neonatal human foreskin fibroblast. e Cyclin K expression detected by immunochemistry in normal and H1299 cancer cells. HFF, neonatal human foreskin fibroblast. Scale bar, 40 μm. f Time course analyses of cyclin K expression by immunoblotting in HCT116 cells treated with protein synthesis inhibitor cycloheximide (CHX, 50 μg/ml). g Time course analyses of cyclin K expression by immunoblotting in cells treated with proteasome inhibitor MG132 (5 μM) in human normal and HCT116 cancer cells. HFF, neonatal human foreskin fibroblast. Experiments were repeated for three times ( a – c ), and more than three times when cell lines were used ( d – g ). Representative results are shown
    Anti Sox2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti sox2/product/Santa Cruz Biotechnology
    Average 92 stars, based on 1110 article reviews
    Price from $9.99 to $1999.99
    anti sox2 - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    92
    Becton Dickinson anti sox2
    Proliferation in the embryonic cochleae of Lgr4 deficient mice. (A–B′′) In the cochleae from heterozygous and homozygous Lgr4-LacZ mice, CCND1 (green) expression increased in the mid-basal turn, where CCND1 merged with red signal of <t>SOX2</t> (arrows) in the pro-sensory domain. (C–D′′) The proliferation marker, EdU (red), was incorporated into pro-sensory cells (arrows) positive for SOX2 (green) in the mid-basal turn of the cochlea from Lgr4 deficient mice. Cell nuclei were counterstained with DAPI (blue), scale bars indicate 20 μm.
    Anti Sox2, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 91 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti sox2/product/Becton Dickinson
    Average 92 stars, based on 91 article reviews
    Price from $9.99 to $1999.99
    anti sox2 - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    94
    R&D Systems sox2
    eNOS-derived NO scavenging significantly impairs the CSC phenotype in human colon cancer cells. a Formation of tumorspheres from HCT-116 and Caco-2 cells pre-treated for 24 hours with c-PTIO (100 μM) with or without DETANONOate or CSNO (100 μM). Representative images 1–2 weeks after seeding are shown. b Formation of organoids from cells pre-treated for 24 hours with c-PTIO (100 μM) with or without NO donors (100 μM). Representative images 1–2 weeks after seeding are shown. c Immunoblot of β-catenin, Bmi1, Notch1, and <t>Sox2</t> expression in HCT-116 and Caco-2 tumor cells treated for 24 hours with c-PTIO (100 μM) with or without DETANONOate or CSNO (100 μM). d Organoid immunofluorescence of β-catenin from control or Caco-2 organoids treated with c-PTIO (100 μM) for 24 hours. e RT-qPCR of iNOS-induced expression (IL-1β 3 ng/ml, IFN-γ 200 U/ml and TNF-α 75 ng/ml, for 6 hours) from control and iNOS-shRNA expressing HCT-116 cells. f Immunoblot of previously mentioned proteins in iNOS-knockdown HCT-116 cancer cells treated with c-PTIO (100 μM). Stain-free technology was used as loading control in immunoblot experiments. * P
    Sox2, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1352 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sox2/product/R&D Systems
    Average 94 stars, based on 1352 article reviews
    Price from $9.99 to $1999.99
    sox2 - by Bioz Stars, 2020-07
    94/100 stars
      Buy from Supplier

    94
    Proteintech anti sox2
    Ectopic expression of WIP1 reduces the levels of activated p38 and enhances stemness-related protein expression and CSC properties in NSCLC cells. a Western blotting was used to analyze the expression of WIP1, phospho-p38, p38, <t>SOX2,</t> OCT4, NANOG, and ALDH1A1 in H1299 (left panels) and H460 (right panels) cells transduced with a WIP1-overexpressing plasmid (WIP1) or vector control (pLV). Arrows indicate the positions of p38 isoforms. b Western blotting was used to analyze MK2, phospho-MK2 (Thr222), phospho-MK2 (Thr334), HSP27, and phospho-HSP27 (Ser82) in H460 cells transduced with the WIP1-overexpressing plasmid (WIP1) or vector control (pLV). c , d A sphere formation assay was performed with H1299 (top graphs) and H460 (bottom graphs) cells transduced with the WIP1-overexpressing (WIP1) or vector control (pLV) plasmid. Quantifications of sphere sizes ( d ) and numbers ( e ) are shown in bar graphs. The data are presented as the mean ± SD of three independent experiments. * indicates P
    Anti Sox2, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti sox2/product/Proteintech
    Average 94 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    anti sox2 - by Bioz Stars, 2020-07
    94/100 stars
      Buy from Supplier

    90
    Merck KGaA anti sox2
    Differentiation of ADSCs into cells of neuronal lineage. ( A ) Semi-quantitative RT-PCR analysis for the expression of neurogenic lineage-specific genes sex determining region Y-box 2 (Sox-2), fascin, nestin, tubulinβIII. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used to normalize cDNA concentration for each sample set. Graphs and SD are from nine patients analyzed. The expression of <t>Sox2</t> ( B ) and nestin ( C ) was also verified by immunofluorescence analysis in several independent experiments. Nuclei were labelled with bisbenzidine (DAPI). Original magnification 40x. Ctrl, undifferentiated control; CL, collagenase; FW, fat wash; LF, liquid fraction.
    Anti Sox2, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 90/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti sox2/product/Merck KGaA
    Average 90 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    anti sox2 - by Bioz Stars, 2020-07
    90/100 stars
      Buy from Supplier

    Image Search Results


    Effect of Embryonic CSF (E-CSF) on mitotic activity in SVZ niche NSCs, monitored by nuclear incorporation of BrdU (red). Confocal photomicrographs ( A,B show the SVZ of “ in vitro ” cultured adult mice brain slices (see Figure 1B ). Note the substantial increase in NSCs mitotic activity under the ventricular surface (white arrows) induced by E-CSF (B) , compared with the controls (A) . Confocal photomicrographs (C–E) show the Striatum area of “ in vitro ” cultured adult mice brain slices close to the SVZ (ST in Figure 1B ). Quantification of BrdU positive nuclei in Control (C) , E-CSF (D) and A-CSF (E) treated brain slices was plotted in graph bars (F) and expressed as means ± SD ( n = 20); the significant threshold was set at p ≤ 0.001 (*) according to the one-way ANOVA, post hoc Bonferroni test. The results reveal a statistically significant increase (49%) in the number of BrdU positive NSCs in CSF-E treated brain slices with respect to the controls and A-CSF treated slices; this suggests a specific activation by E-CSF of mitotic activity in NSCs of the SVZ niche. Confocal photomicrographs (G,I) correspond to the striatum area of “ in vitro ” cultured adult mice brain slices, close to SVZ (ST in Figure 1B ). Images show double immunolabeling with antiBrdU (green) and antiSox2 (red) antibody; co-localization of both antibodies was interpreted as NSCs dividing (BrdU positive) but not differentiating (Sox2 positive). Quantification of co-labeled BrdU and Sox2 NSCs in Control (G) and E-CSF treated brain slices (I) was plotted in graph bars (H) and expressed as means ± SD ( n = 20); the significant threshold was set at p ≤ 0.001 (*) according to the two-tailed Student’s t -test. The results reveal a statistically significant increase (141%) in the number of NSCs that undergo replication but remain undifferentiated in CSF-E treated brain slices, in comparison with the controls. Scale bar: 25 μm (A–E) and 10 μm (G,I) .

    Journal: Frontiers in Neuroanatomy

    Article Title: Embryonic Cerebrospinal Fluid Increases Neurogenic Activity in the Brain Ventricular-Subventricular Zone of Adult Mice

    doi: 10.3389/fnana.2017.00124

    Figure Lengend Snippet: Effect of Embryonic CSF (E-CSF) on mitotic activity in SVZ niche NSCs, monitored by nuclear incorporation of BrdU (red). Confocal photomicrographs ( A,B show the SVZ of “ in vitro ” cultured adult mice brain slices (see Figure 1B ). Note the substantial increase in NSCs mitotic activity under the ventricular surface (white arrows) induced by E-CSF (B) , compared with the controls (A) . Confocal photomicrographs (C–E) show the Striatum area of “ in vitro ” cultured adult mice brain slices close to the SVZ (ST in Figure 1B ). Quantification of BrdU positive nuclei in Control (C) , E-CSF (D) and A-CSF (E) treated brain slices was plotted in graph bars (F) and expressed as means ± SD ( n = 20); the significant threshold was set at p ≤ 0.001 (*) according to the one-way ANOVA, post hoc Bonferroni test. The results reveal a statistically significant increase (49%) in the number of BrdU positive NSCs in CSF-E treated brain slices with respect to the controls and A-CSF treated slices; this suggests a specific activation by E-CSF of mitotic activity in NSCs of the SVZ niche. Confocal photomicrographs (G,I) correspond to the striatum area of “ in vitro ” cultured adult mice brain slices, close to SVZ (ST in Figure 1B ). Images show double immunolabeling with antiBrdU (green) and antiSox2 (red) antibody; co-localization of both antibodies was interpreted as NSCs dividing (BrdU positive) but not differentiating (Sox2 positive). Quantification of co-labeled BrdU and Sox2 NSCs in Control (G) and E-CSF treated brain slices (I) was plotted in graph bars (H) and expressed as means ± SD ( n = 20); the significant threshold was set at p ≤ 0.001 (*) according to the two-tailed Student’s t -test. The results reveal a statistically significant increase (141%) in the number of NSCs that undergo replication but remain undifferentiated in CSF-E treated brain slices, in comparison with the controls. Scale bar: 25 μm (A–E) and 10 μm (G,I) .

    Article Snippet: Secondary antibody for Anti Sox2 was Antigoat Ig G-Alexa 594 (1/1000 dilution Invitrogen, Ref. A110 58).

    Techniques: Activity Assay, In Vitro, Cell Culture, Mouse Assay, Activation Assay, Immunolabeling, Labeling, Two Tailed Test

    TrLp is significantly more potent than CLp in causing suppression of CD133(+) and SOX2(+) GL261 stem cells. Treated GL261 cells were stained with antibodies against CD133 and SOX2 and analyzed via flow cytometry. ( A , E , F ) The Vehicle-treated GL261 cells showed an abundance of CD133(+) GBM stem cells (UL quadrant, within the red rectangle). ( A – C , E , F ) Compared to the Vehicle-treated, the CLp-treated cells (UL quadrant, within the red rectangle) showed a 69% suppression of CD133 IF (* p = 1.2 × 10 −3 ) and TrLp-treated cells (UL quadrant, within the red rectangle) showed a 92% suppression of CD133(+) IF (** p = 6.9 × 10 −4 ) ( E , F ); ( F ) TrLp treatment yielded 23% greater suppression of CD133 IF than CLp (Δ p = 1.6 × 10 −3 ); ( D ) 2° antibody staining showed background fluorescence; ( G – I , K , L ) Compared to the Vehicle-treated cells, the CLp-treated cells (UL quadrant, within the red circle) showed 56% suppression of SOX2 IF (* p = 3.2 × 10 −3 ), and the TrLp-treated cells showed 82% suppression of SOX2 IF (** p = 1.0 × 10 −3 ) ( K , L ); ( L ) This inhibition was 26% greater in the TrLp group than in the CLp group (Δ p = 5.6 × 10 −3 ). Data (mean ± S.E.M.) were obtained from Vehicle ( n = 3), CLp ( n = 3) and TrLp ( n = 3); ( J ) 2° antibody-treated samples showed background staining.

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Liposomal TriCurin, A Synergistic Combination of Curcumin, Epicatechin Gallate and Resveratrol, Repolarizes Tumor-Associated Microglia/Macrophages, and Eliminates Glioblastoma (GBM) and GBM Stem Cells

    doi: 10.3390/molecules23010201

    Figure Lengend Snippet: TrLp is significantly more potent than CLp in causing suppression of CD133(+) and SOX2(+) GL261 stem cells. Treated GL261 cells were stained with antibodies against CD133 and SOX2 and analyzed via flow cytometry. ( A , E , F ) The Vehicle-treated GL261 cells showed an abundance of CD133(+) GBM stem cells (UL quadrant, within the red rectangle). ( A – C , E , F ) Compared to the Vehicle-treated, the CLp-treated cells (UL quadrant, within the red rectangle) showed a 69% suppression of CD133 IF (* p = 1.2 × 10 −3 ) and TrLp-treated cells (UL quadrant, within the red rectangle) showed a 92% suppression of CD133(+) IF (** p = 6.9 × 10 −4 ) ( E , F ); ( F ) TrLp treatment yielded 23% greater suppression of CD133 IF than CLp (Δ p = 1.6 × 10 −3 ); ( D ) 2° antibody staining showed background fluorescence; ( G – I , K , L ) Compared to the Vehicle-treated cells, the CLp-treated cells (UL quadrant, within the red circle) showed 56% suppression of SOX2 IF (* p = 3.2 × 10 −3 ), and the TrLp-treated cells showed 82% suppression of SOX2 IF (** p = 1.0 × 10 −3 ) ( K , L ); ( L ) This inhibition was 26% greater in the TrLp group than in the CLp group (Δ p = 5.6 × 10 −3 ). Data (mean ± S.E.M.) were obtained from Vehicle ( n = 3), CLp ( n = 3) and TrLp ( n = 3); ( J ) 2° antibody-treated samples showed background staining.

    Article Snippet: Cells attached to cover-slips in the wells were washed with 10 mM phosphate-buffered saline (PBS), fixed in 4% ( w / v ) paraformaldehyde, washed three times with PBS, blocked in 10% goat serum in PBS plus 0.1% Triton X-100, and then immunostained using anti-SOX2 Ab (mouse IgG) (sc-365823) (1:50), followed by a secondary Ab (Alexa Fluor 568 goat anti-mouse, 1:1000, Invitrogen) [ , ].

    Techniques: Staining, Flow Cytometry, Cytometry, IF-P, Fluorescence, Inhibition

    Tamoxifen induces SOX2 to enhance tamoxifen resistance through TARBP2. ( A , B ) Expression of different stem cell markers after tamoxifen treatment. MCF-7 cells were treated with 2 μM tamoxifen for 48 h and then RNA was isolated to analyze the mRNA expression of stem cell markers by reverse-transcription PCR (qRT-PCR). The experiments were repeated at least 3 times, and ATP5E was used as a positive control for tamoxifen treatment ( A ). * p ≤ 0.05 by t -test. Cells as indicated in ( A ) were collected to analyze protein expression by western blotting ( B ). ( C , D ) Effect of SOX2 expression on tamoxifen sensitivity. MCF-7 cells were transfected with shRNA targeting SOX2 for 48 h and treated with different concentrations of tamoxifen (1, 2, 5, 10, 20 μM) for 72 h. The efficiency of SOX2 knock-down was examined by western blot ( C ), and the proliferation and colony formation were determined by MTT ( D ) and colony formation assays ( E ), respectively. MTT experimental results are given as the means ± SEM from at least three separate experiments that were performed in duplicate or triplicate and analyzed by two-way ANOVA. * p ≤ 0.05, ** p ≤ 0.01. ( F , G ) Tamoxifen downregulated the protein level of SOX2 through TARBP2. MCF-7 cells were transfected with shRNAs targeting TARBP2 for 48 h; 2 μM tamoxifen was then added to the culture medium for 48 h. The cells were harvested to determine the protein expressions by western blot. ( G – I ) TARBP2-regulated protein stability of SOX2 in tamoxifen-treated and resistant cells. Tamoxifen-treated (2 μM for 48 h) MCF-7 ( G ) and MCF-7/TR1 ( H ) cells were treated with 50 μg/mL cycloheximide to block protein synthesis and were then harvested at the indicated time point to analyze the expression of SOX2 by western blotting. ( I ) MCF-7 cells were transfected with the indicated shRNAs targeting TARBP2 for 48 h and treated with 2 μM tamoxifen for 48 h. Cells were add 50 μg/mL cycloheximide and harvested at the indicated time point to analyze the expression of SOX2 by western blotting. The degradation rates were plotted for the average ± SEM of at least three independent experiments and analyzed by two-way ANOVA. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

    Journal: Cancers

    Article Title: TARBP2-Enhanced Resistance during Tamoxifen Treatment in Breast Cancer

    doi: 10.3390/cancers11020210

    Figure Lengend Snippet: Tamoxifen induces SOX2 to enhance tamoxifen resistance through TARBP2. ( A , B ) Expression of different stem cell markers after tamoxifen treatment. MCF-7 cells were treated with 2 μM tamoxifen for 48 h and then RNA was isolated to analyze the mRNA expression of stem cell markers by reverse-transcription PCR (qRT-PCR). The experiments were repeated at least 3 times, and ATP5E was used as a positive control for tamoxifen treatment ( A ). * p ≤ 0.05 by t -test. Cells as indicated in ( A ) were collected to analyze protein expression by western blotting ( B ). ( C , D ) Effect of SOX2 expression on tamoxifen sensitivity. MCF-7 cells were transfected with shRNA targeting SOX2 for 48 h and treated with different concentrations of tamoxifen (1, 2, 5, 10, 20 μM) for 72 h. The efficiency of SOX2 knock-down was examined by western blot ( C ), and the proliferation and colony formation were determined by MTT ( D ) and colony formation assays ( E ), respectively. MTT experimental results are given as the means ± SEM from at least three separate experiments that were performed in duplicate or triplicate and analyzed by two-way ANOVA. * p ≤ 0.05, ** p ≤ 0.01. ( F , G ) Tamoxifen downregulated the protein level of SOX2 through TARBP2. MCF-7 cells were transfected with shRNAs targeting TARBP2 for 48 h; 2 μM tamoxifen was then added to the culture medium for 48 h. The cells were harvested to determine the protein expressions by western blot. ( G – I ) TARBP2-regulated protein stability of SOX2 in tamoxifen-treated and resistant cells. Tamoxifen-treated (2 μM for 48 h) MCF-7 ( G ) and MCF-7/TR1 ( H ) cells were treated with 50 μg/mL cycloheximide to block protein synthesis and were then harvested at the indicated time point to analyze the expression of SOX2 by western blotting. ( I ) MCF-7 cells were transfected with the indicated shRNAs targeting TARBP2 for 48 h and treated with 2 μM tamoxifen for 48 h. Cells were add 50 μg/mL cycloheximide and harvested at the indicated time point to analyze the expression of SOX2 by western blotting. The degradation rates were plotted for the average ± SEM of at least three independent experiments and analyzed by two-way ANOVA. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

    Article Snippet: The primary antibodies used were anti-SOX2 (Millipore, MA, USA; cat. AB5603, 1:50) anti-TARBP2 (Thermo, MA, USA; cat. LF-MA0209, Clone 46D1, 1:600) for 30 min.

    Techniques: Expressing, Isolation, Polymerase Chain Reaction, Quantitative RT-PCR, Positive Control, Western Blot, Transfection, shRNA, MTT Assay, Blocking Assay

    Both SOX2 and TARBP2 expression are elevated in hormone therapy-resistant tumor cells. ( A ) The correlation of SOX2 expression with the overall survival of ER-positive breast cancer patients was analyzed and downloaded using Kaplan-Meier Plotter ( http://kmplot.com/ ). ( B , C ) Association of SOX2 expression and hormone therapy resistance in breast cancer tissues. Representative serial sections of Figure 1 B showed images of SOX2 IHC in primary tumors and tumors in lymph nodes in cases of cancer recurrence ( B ). Scale Bar: 100 uM. Statistics of SOX2 protein expression levels in primary tumors and metastatic tumor cells in cases of cancer recurrence ( C ). ( D ) Resistance mechanism for tamoxifen–induced TARBP2-SOX2 in breast cancer.

    Journal: Cancers

    Article Title: TARBP2-Enhanced Resistance during Tamoxifen Treatment in Breast Cancer

    doi: 10.3390/cancers11020210

    Figure Lengend Snippet: Both SOX2 and TARBP2 expression are elevated in hormone therapy-resistant tumor cells. ( A ) The correlation of SOX2 expression with the overall survival of ER-positive breast cancer patients was analyzed and downloaded using Kaplan-Meier Plotter ( http://kmplot.com/ ). ( B , C ) Association of SOX2 expression and hormone therapy resistance in breast cancer tissues. Representative serial sections of Figure 1 B showed images of SOX2 IHC in primary tumors and tumors in lymph nodes in cases of cancer recurrence ( B ). Scale Bar: 100 uM. Statistics of SOX2 protein expression levels in primary tumors and metastatic tumor cells in cases of cancer recurrence ( C ). ( D ) Resistance mechanism for tamoxifen–induced TARBP2-SOX2 in breast cancer.

    Article Snippet: The primary antibodies used were anti-SOX2 (Millipore, MA, USA; cat. AB5603, 1:50) anti-TARBP2 (Thermo, MA, USA; cat. LF-MA0209, Clone 46D1, 1:600) for 30 min.

    Techniques: Expressing, Immunohistochemistry

    Relative expression of miR-34a, b and c in OSCs and U-2OS cell monolayers, detected using SYBR Green reverse transcription-quantitative polymerase chain reaction analysis. (A) Culture of OSCs in serum-free medium on days 3, 10 and 14. (B) Representative photomicrographs reveal the staining of marker antigens Sox-2 (red) and Sca-1 (green). (C) Average expression levels of miR-34a, b and c in OSCs, compared with those in monolayer cells. *P

    Journal: Molecular Medicine Reports

    Article Title: miR-34a is downregulated in human osteosarcoma stem-like cells and promotes invasion, tumorigenic ability and self-renewal capacity

    doi: 10.3892/mmr.2017.6187

    Figure Lengend Snippet: Relative expression of miR-34a, b and c in OSCs and U-2OS cell monolayers, detected using SYBR Green reverse transcription-quantitative polymerase chain reaction analysis. (A) Culture of OSCs in serum-free medium on days 3, 10 and 14. (B) Representative photomicrographs reveal the staining of marker antigens Sox-2 (red) and Sca-1 (green). (C) Average expression levels of miR-34a, b and c in OSCs, compared with those in monolayer cells. *P

    Article Snippet: Membranes were blocked with 5% BSA (Sigma-Aldrich, Merck Millipore) in TBST (20 mM Tris, 150 mM NaCl, containing 0.3% Tween-20, pH 7.4) for 30 min. Membranes were then incubated with mouse anti-SOX-2 antibody (catalog no. ab171380; 1:1,000; Abcam) or mouse anti β-actin antibody (catalog no. ab8227; 1:1,000; Abcam) in TBST with 1% BSA (Sigma-Aldrich, Merck Millipore) at room temperature for 1 h. Following 3 washes with TBST, the membranes was incubated with anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (catalog no. ab6785; 1:5,000; Abcam) for 1 h at room temperature.

    Techniques: Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction, Staining, Marker

    Sox-2 and Sca-1 are expressed at low levels in osteosarcoma-derived form U-2OS cells. (A) mRNA (left) and protein (right) levels of Sox-2 were analyzed using RT-qPCR and semiquantitative western blot analyses. *P

    Journal: Molecular Medicine Reports

    Article Title: miR-34a is downregulated in human osteosarcoma stem-like cells and promotes invasion, tumorigenic ability and self-renewal capacity

    doi: 10.3892/mmr.2017.6187

    Figure Lengend Snippet: Sox-2 and Sca-1 are expressed at low levels in osteosarcoma-derived form U-2OS cells. (A) mRNA (left) and protein (right) levels of Sox-2 were analyzed using RT-qPCR and semiquantitative western blot analyses. *P

    Article Snippet: Membranes were blocked with 5% BSA (Sigma-Aldrich, Merck Millipore) in TBST (20 mM Tris, 150 mM NaCl, containing 0.3% Tween-20, pH 7.4) for 30 min. Membranes were then incubated with mouse anti-SOX-2 antibody (catalog no. ab171380; 1:1,000; Abcam) or mouse anti β-actin antibody (catalog no. ab8227; 1:1,000; Abcam) in TBST with 1% BSA (Sigma-Aldrich, Merck Millipore) at room temperature for 1 h. Following 3 washes with TBST, the membranes was incubated with anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (catalog no. ab6785; 1:5,000; Abcam) for 1 h at room temperature.

    Techniques: Derivative Assay, Quantitative RT-PCR, Western Blot

    SOX2 knockdown inhibits the viability, migration and invasion of glioma cell lines. a The relative expression of SOX2 increases in glioma tissues compared with that in normal tissues. The SOX2 expression is higher in high grade (stage III and stage IV) glioma tissues than that in low grade (stage I and stage II) glioma tissues. GAPDH is the internal control. ** P

    Journal: Molecular Cancer

    Article Title: Knockdown of long non-coding RNA NEAT1 inhibits glioma cell migration and invasion via modulation of SOX2 targeted by miR-132

    doi: 10.1186/s12943-018-0849-2

    Figure Lengend Snippet: SOX2 knockdown inhibits the viability, migration and invasion of glioma cell lines. a The relative expression of SOX2 increases in glioma tissues compared with that in normal tissues. The SOX2 expression is higher in high grade (stage III and stage IV) glioma tissues than that in low grade (stage I and stage II) glioma tissues. GAPDH is the internal control. ** P

    Article Snippet: Anti-SOX2 (BA3292) and anti-β-actin (BM3873) from Bosterbio (Wuhan, China) were diluted to 1:200 and used as primary antibodies.

    Techniques: Migration, Expressing

    NEAT1 targets miR-132 and miR-132 targets SOX2 . a The relative expression of miR-132 decrease in glioma tissue compared with that in normal tissue. The miR-132 expression is lower in high grade (stage III and stage IV) glioma tissue than that in low grade (stage I and stage II) glioma tissue. U6 is the internal control. * P

    Journal: Molecular Cancer

    Article Title: Knockdown of long non-coding RNA NEAT1 inhibits glioma cell migration and invasion via modulation of SOX2 targeted by miR-132

    doi: 10.1186/s12943-018-0849-2

    Figure Lengend Snippet: NEAT1 targets miR-132 and miR-132 targets SOX2 . a The relative expression of miR-132 decrease in glioma tissue compared with that in normal tissue. The miR-132 expression is lower in high grade (stage III and stage IV) glioma tissue than that in low grade (stage I and stage II) glioma tissue. U6 is the internal control. * P

    Article Snippet: Anti-SOX2 (BA3292) and anti-β-actin (BM3873) from Bosterbio (Wuhan, China) were diluted to 1:200 and used as primary antibodies.

    Techniques: Expressing

    NEAT1 elevates SOX2 expression through targeting miR-132 to promote glioma progression. a The relative mRNA expression of SOX2 in U251 and U87 cells decreases in miR-132 mimics group compared with NC, increases in miR-132 + NEAT1 group compared with miR-132 + pcDNA3.1, and almost remains the same between NC and miR-132 + NEAT1 groups. GAPDH is the internal control. * P

    Journal: Molecular Cancer

    Article Title: Knockdown of long non-coding RNA NEAT1 inhibits glioma cell migration and invasion via modulation of SOX2 targeted by miR-132

    doi: 10.1186/s12943-018-0849-2

    Figure Lengend Snippet: NEAT1 elevates SOX2 expression through targeting miR-132 to promote glioma progression. a The relative mRNA expression of SOX2 in U251 and U87 cells decreases in miR-132 mimics group compared with NC, increases in miR-132 + NEAT1 group compared with miR-132 + pcDNA3.1, and almost remains the same between NC and miR-132 + NEAT1 groups. GAPDH is the internal control. * P

    Article Snippet: Anti-SOX2 (BA3292) and anti-β-actin (BM3873) from Bosterbio (Wuhan, China) were diluted to 1:200 and used as primary antibodies.

    Techniques: Expressing

    Rough distribution of the stem cells in the primary and recurrent pterygium tissue. A. H E staining of the primary and recurrent pterygium tissue. B. Immunohistochemistry staining for stem cell markers SOX2, NESTIN, VIMENTIN and CD44 in the primary pterygium tissues. C. Immunohistochemically staining for stem cell markers SOX2, NESTIN, VIMENTIN and CD44 in the recurrent pterygium tissues. The scale bars are 100 μm.

    Journal: American Journal of Translational Research

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

    doi:

    Figure Lengend Snippet: Rough distribution of the stem cells in the primary and recurrent pterygium tissue. A. H E staining of the primary and recurrent pterygium tissue. B. Immunohistochemistry staining for stem cell markers SOX2, NESTIN, VIMENTIN and CD44 in the primary pterygium tissues. C. Immunohistochemically staining for stem cell markers SOX2, NESTIN, VIMENTIN and CD44 in the recurrent pterygium tissues. The scale bars are 100 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: Staining, Immunohistochemistry

    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:

    NU6140 treatment compared to nocodazole treatment affects differently the expression of pluripotency markers in 2102Ep carcinoma cells. Expression of pluripotency markers NANOG, OCT4, and SOX2 in nocodazole and NU6140 treated 2102Ep cells as detected by using flow cytometric assays. (a) Fixed and permeabilised (methanol permeabilisation) cells were stained with anti-NANOG (PE), anti-OCT4 (Alexa Fluor 647), and anti-SOX2 (PerCp Cy5.5 conjugate) antibodies and with DAPI. For analysis cellular debris and doublets were excluded. (b) Correlation between expression of CDK2 with NANOG and SOX2. Fixed and permeabilised (methanol permeabilisation) cells were stained with anti-CDK2 (Alexa Fluor 488 conjugate), anti-NANOG (PE), and anti-SOX2 (PerCp Cy5.5 conjugate) antibodies and with DAPI. Results are shown as density plots.

    Journal: International Journal of Cell Biology

    Article Title: Assessment of the Potential of CDK2 Inhibitor NU6140 to Influence the Expression of Pluripotency Markers NANOG, OCT4, and SOX2 in 2102Ep and H9 Cells

    doi: 10.1155/2014/280638

    Figure Lengend Snippet: NU6140 treatment compared to nocodazole treatment affects differently the expression of pluripotency markers in 2102Ep carcinoma cells. Expression of pluripotency markers NANOG, OCT4, and SOX2 in nocodazole and NU6140 treated 2102Ep cells as detected by using flow cytometric assays. (a) Fixed and permeabilised (methanol permeabilisation) cells were stained with anti-NANOG (PE), anti-OCT4 (Alexa Fluor 647), and anti-SOX2 (PerCp Cy5.5 conjugate) antibodies and with DAPI. For analysis cellular debris and doublets were excluded. (b) Correlation between expression of CDK2 with NANOG and SOX2. Fixed and permeabilised (methanol permeabilisation) cells were stained with anti-CDK2 (Alexa Fluor 488 conjugate), anti-NANOG (PE), and anti-SOX2 (PerCp Cy5.5 conjugate) antibodies and with DAPI. Results are shown as density plots.

    Article Snippet: Membranes were probed with rabbit anti-NANOG antibodies (Aviva Systems Biology, San Diego, CA, USA), mouse anti-OCT4 antibodies (Santa Cruz Biotechnology), and anti-SOX2 antibodies (Abcam, Cambridge, MA, USA) followed by horseradish peroxidase-conjugated goat anti-rabbit (Cell Signaling) or goat anti-mouse secondary antibodies (LabAs, Tartu, Estonia).

    Techniques: Expressing, Flow Cytometry, Staining

    NU6140 treatment causes changes in the colony structure and differentiation potential of hES cells. hES cells were treated with NU6140 (10 μ M) for 24 h and then cultured in fresh mTeSR1 maintenance medium for a further 3 days with the medium changed daily. Embryoid bodies were formed from hES cells treated with NU6140 or from untreated hES cells. (a) Morphological changes in colony structure after NU6140 treatment and formation of EBs. (b) Fixed and permeabilised hES cells were stained with anti-OCT4 (Alexa Fluor 647 conjugate), anti-NANOG (PE), and anti-SOX2 (PerCp-Cy5.5) antibodies and with DAPI. Other hES cells were stained with anti-GATA4 (NorthernLights, NL-493 conjugate), anti-OTX-2 (NL-557), anti-HAND1 (NL-637) antibodies and with DAPI. (c) Differentiation into three germ cell layers as detected by flow cytometric assay. Fixed (1.6% PFA) and permeabilised cells from dissociated embryoid bodies (EBs) were stained with anti-SOX1 (NL-493), anti-Brachyury (NL-557), anti-SOX17 (NL-637), and anti-OCT4 (PerCp-Cy5.5) antibodies and with DAPI.

    Journal: International Journal of Cell Biology

    Article Title: Assessment of the Potential of CDK2 Inhibitor NU6140 to Influence the Expression of Pluripotency Markers NANOG, OCT4, and SOX2 in 2102Ep and H9 Cells

    doi: 10.1155/2014/280638

    Figure Lengend Snippet: NU6140 treatment causes changes in the colony structure and differentiation potential of hES cells. hES cells were treated with NU6140 (10 μ M) for 24 h and then cultured in fresh mTeSR1 maintenance medium for a further 3 days with the medium changed daily. Embryoid bodies were formed from hES cells treated with NU6140 or from untreated hES cells. (a) Morphological changes in colony structure after NU6140 treatment and formation of EBs. (b) Fixed and permeabilised hES cells were stained with anti-OCT4 (Alexa Fluor 647 conjugate), anti-NANOG (PE), and anti-SOX2 (PerCp-Cy5.5) antibodies and with DAPI. Other hES cells were stained with anti-GATA4 (NorthernLights, NL-493 conjugate), anti-OTX-2 (NL-557), anti-HAND1 (NL-637) antibodies and with DAPI. (c) Differentiation into three germ cell layers as detected by flow cytometric assay. Fixed (1.6% PFA) and permeabilised cells from dissociated embryoid bodies (EBs) were stained with anti-SOX1 (NL-493), anti-Brachyury (NL-557), anti-SOX17 (NL-637), and anti-OCT4 (PerCp-Cy5.5) antibodies and with DAPI.

    Article Snippet: Membranes were probed with rabbit anti-NANOG antibodies (Aviva Systems Biology, San Diego, CA, USA), mouse anti-OCT4 antibodies (Santa Cruz Biotechnology), and anti-SOX2 antibodies (Abcam, Cambridge, MA, USA) followed by horseradish peroxidase-conjugated goat anti-rabbit (Cell Signaling) or goat anti-mouse secondary antibodies (LabAs, Tartu, Estonia).

    Techniques: Cell Culture, Staining, Flow Cytometry

    NU6140 treatment compared to nocodazole treatment affects differently the expression of pluripotency markers in hES cells. (a) Flow cytometric analysis of the expression of pluripotency markers NANOG, OCT4, and SOX2 in hES cells treated with nocodazole, NU6140, and DMSO. Fixed and permeabilised cells were stained with anti-NANOG (PE), anti-OCT4 (Alexa Fluor 647), and anti-SOX2 (PerCp Cy5.5 conjugate) antibodies and with DAPI. For analysis cellular debris and doublets were excluded. (b) Correlation between expression of CDK2 with NANOG and SOX2. Fixed and permeabilised cells were stained with anti-CDK2 (Alexa Fluor 488 conjugate), anti-NANOG (PE), and anti-SOX2 (PerCp Cy5.5 conjugate) antibodies and with DAPI.

    Journal: International Journal of Cell Biology

    Article Title: Assessment of the Potential of CDK2 Inhibitor NU6140 to Influence the Expression of Pluripotency Markers NANOG, OCT4, and SOX2 in 2102Ep and H9 Cells

    doi: 10.1155/2014/280638

    Figure Lengend Snippet: NU6140 treatment compared to nocodazole treatment affects differently the expression of pluripotency markers in hES cells. (a) Flow cytometric analysis of the expression of pluripotency markers NANOG, OCT4, and SOX2 in hES cells treated with nocodazole, NU6140, and DMSO. Fixed and permeabilised cells were stained with anti-NANOG (PE), anti-OCT4 (Alexa Fluor 647), and anti-SOX2 (PerCp Cy5.5 conjugate) antibodies and with DAPI. For analysis cellular debris and doublets were excluded. (b) Correlation between expression of CDK2 with NANOG and SOX2. Fixed and permeabilised cells were stained with anti-CDK2 (Alexa Fluor 488 conjugate), anti-NANOG (PE), and anti-SOX2 (PerCp Cy5.5 conjugate) antibodies and with DAPI.

    Article Snippet: Membranes were probed with rabbit anti-NANOG antibodies (Aviva Systems Biology, San Diego, CA, USA), mouse anti-OCT4 antibodies (Santa Cruz Biotechnology), and anti-SOX2 antibodies (Abcam, Cambridge, MA, USA) followed by horseradish peroxidase-conjugated goat anti-rabbit (Cell Signaling) or goat anti-mouse secondary antibodies (LabAs, Tartu, Estonia).

    Techniques: Expressing, Flow Cytometry, Staining

    Effect of combined treatment on pluripotency markers expression in hES and hEC cells compared to treatments with nocodazole or NU6140 individually. Nocodazole treated cells (10 h) were washed and further treated with 10 μ M NU6140 for 14 h. The coexpression of NANOG/OCT4 and SOX2/OCT4 detected in hEC (a, c) as described in Figure 2 and in hES cells (b, d) as described in Figure 1 . (e) Morphological changes in colony structure of hES cells treated with NU6140 or nocodazole.

    Journal: International Journal of Cell Biology

    Article Title: Assessment of the Potential of CDK2 Inhibitor NU6140 to Influence the Expression of Pluripotency Markers NANOG, OCT4, and SOX2 in 2102Ep and H9 Cells

    doi: 10.1155/2014/280638

    Figure Lengend Snippet: Effect of combined treatment on pluripotency markers expression in hES and hEC cells compared to treatments with nocodazole or NU6140 individually. Nocodazole treated cells (10 h) were washed and further treated with 10 μ M NU6140 for 14 h. The coexpression of NANOG/OCT4 and SOX2/OCT4 detected in hEC (a, c) as described in Figure 2 and in hES cells (b, d) as described in Figure 1 . (e) Morphological changes in colony structure of hES cells treated with NU6140 or nocodazole.

    Article Snippet: Membranes were probed with rabbit anti-NANOG antibodies (Aviva Systems Biology, San Diego, CA, USA), mouse anti-OCT4 antibodies (Santa Cruz Biotechnology), and anti-SOX2 antibodies (Abcam, Cambridge, MA, USA) followed by horseradish peroxidase-conjugated goat anti-rabbit (Cell Signaling) or goat anti-mouse secondary antibodies (LabAs, Tartu, Estonia).

    Techniques: Expressing

    Expression of NSUN2 in the Human Developing Brain and NES Cells (A) DAPI-stained human embryo (6 weeks of gestation) marked for prosencephalon, mesencephalon, and rhombencephalon. Region in square is magnified in (B). Scale bar, 1 mm. (B) Prosencephalon labeled for NSUN2 and SOX1. Region in squares are magnified in (b′) and (b″). Arrows indicate NSUN2-positive cells. Scale bar, 100 μm. (C–F) Bright-field image (C) and immunofluorescence (D–F) of AF22 (upper panels) and Sai1 (lower panels) cells labeled for Nestin (D), SOX2 (E), and βIII-tubulin (F). Scale bar, 50 μm. (G and H) NES cells co-labeled for NSUN2 and Nestin (NES) (G) or SOX1 (H). (I) Differentiation protocol. (J–L) Differentiated AF22 and Sai1 cells (day 15) labeled for Nestin (NES; J), SOX2 (K), and βIII-tubulin (L). Scale bars: 50 μm. (M) Western blot for NSUN2, βIII-tubulin (TUBB3), GFAP, SOX2, and Nestin during differentiation (days). α-Tubulin served as loading control. Nuclei are counterstained with DAPI (A, B, D–F, J–L).

    Journal: Stem Cell Reports

    Article Title: Cytosine-5 RNA Methylation Regulates Neural Stem Cell Differentiation and Motility

    doi: 10.1016/j.stemcr.2016.11.014

    Figure Lengend Snippet: Expression of NSUN2 in the Human Developing Brain and NES Cells (A) DAPI-stained human embryo (6 weeks of gestation) marked for prosencephalon, mesencephalon, and rhombencephalon. Region in square is magnified in (B). Scale bar, 1 mm. (B) Prosencephalon labeled for NSUN2 and SOX1. Region in squares are magnified in (b′) and (b″). Arrows indicate NSUN2-positive cells. Scale bar, 100 μm. (C–F) Bright-field image (C) and immunofluorescence (D–F) of AF22 (upper panels) and Sai1 (lower panels) cells labeled for Nestin (D), SOX2 (E), and βIII-tubulin (F). Scale bar, 50 μm. (G and H) NES cells co-labeled for NSUN2 and Nestin (NES) (G) or SOX1 (H). (I) Differentiation protocol. (J–L) Differentiated AF22 and Sai1 cells (day 15) labeled for Nestin (NES; J), SOX2 (K), and βIII-tubulin (L). Scale bars: 50 μm. (M) Western blot for NSUN2, βIII-tubulin (TUBB3), GFAP, SOX2, and Nestin during differentiation (days). α-Tubulin served as loading control. Nuclei are counterstained with DAPI (A, B, D–F, J–L).

    Article Snippet: Primary antibodies were diluted in blocking solution as follows: 1:200 Sox1 (AF3369; R & D); 1:200 Nsun2 (hmetA); 1:500 Nestin MAB1259; R & D); 1:500 Sox2 (ab75485; Abcam); 1:400 TUBB3 (MAB1195; R & D); 1:400 Pax6 (AB2237; Millipore); 1:500 Tbr1 (ab31940; Abcam); 1:300 Tbr2 (ab23345; Abcam); 1:200 Ctip2 (ab18465; Abcam); 1:500 Satb2 (ab51502; Abcam).

    Techniques: Expressing, Staining, Labeling, Immunofluorescence, Western Blot

    Spatial analysis of the primitive streak region. (A) Unique DEGs to each E8.5 region (≥1.5-fold change compared with other samples; for unique DEGs in St5, see Table S1). Asterisk indicates that no specific in situ probe could be constructed. (B) Bhmt2 and Ccno expression in the ventral (V) but not dorsal (D) NSB layer. (C) Whole-mount in situ hybridisation for Mnx1 . tg, tail gut; vn, ventral node. (D) Sox2 versus T intensity values for all samples. Abbreviations are preceded by embryo age; colours indicates fate. Regions of similar fate are enclosed by a coloured line.

    Journal: Development (Cambridge, England)

    Article Title: Transcriptionally dynamic progenitor populations organised around a stable niche drive axial patterning

    doi: 10.1242/dev.168161

    Figure Lengend Snippet: Spatial analysis of the primitive streak region. (A) Unique DEGs to each E8.5 region (≥1.5-fold change compared with other samples; for unique DEGs in St5, see Table S1). Asterisk indicates that no specific in situ probe could be constructed. (B) Bhmt2 and Ccno expression in the ventral (V) but not dorsal (D) NSB layer. (C) Whole-mount in situ hybridisation for Mnx1 . tg, tail gut; vn, ventral node. (D) Sox2 versus T intensity values for all samples. Abbreviations are preceded by embryo age; colours indicates fate. Regions of similar fate are enclosed by a coloured line.

    Article Snippet: Primary antibodies (supplier, catalogue number and working concentration) were: anti-Timp3-loop1 (Abcam; ab39184; 5 µg/ml), anti-Sox2 (Abcam; ab92494; 1:200), anti-T (R & D; AF2085; 1 mg/ml) and anti-GFP (Abcam; ab13970; 10 mg/ml).

    Techniques: In Situ, Construct, Expressing, Hybridization

    NotoPs are essential for correct axis elongation. (A) DiI labelling of the ventral node or crown at E8.5 (2-5 s; Aa,Ae). (Aa′,Ae′) Magnified view of the ventral node region. (Aa″,Ae″) Red channel showing DiI labelling. The same embryos are shown after 48 h (Ab) or 24 h (Af) ex vivo culture. Ab′ and Af' show magnified views of Ab and Af respectively. (Af″) Red channel showing DiI labelling. DiI was found in the notochord and dorsal gut (Ac,Ad,Ag,Ah). Insets in Ac, Ad, Ag and Ah show magnified views of DiI labelling in notochord and gut. (Ai) DiI labelling per embryo after culture with average anterior limit (±s.d.). The anterior limit in 4/6 crown-labelled embryos was in the presomitic mesoderm. The anterior limit in each of these embryos is denoted as (total somites/embryo) +1. As the presomitic mesoderm contains ∼7 presomites, the average anterior limit may be posterior to s17. Noto, notochord; NT, neural tube; PXM, paraxial mesoderm. Asterisk indicates 2/6 embryos had a minor contribution in the posterior neural tube. (B) Sox2/T immunostained section of embryo labelled with DiI in the crown after 24 h in culture (n embryos ). (D) Sagittal confocal sections through CLE-electroporated (Da), and NSB-electroporated class I (Db) and II (Dc) embryos after 24 h, with the notochord (noto) end shown in yellow. nt, neural tube; som, somite.

    Journal: Development (Cambridge, England)

    Article Title: Transcriptionally dynamic progenitor populations organised around a stable niche drive axial patterning

    doi: 10.1242/dev.168161

    Figure Lengend Snippet: NotoPs are essential for correct axis elongation. (A) DiI labelling of the ventral node or crown at E8.5 (2-5 s; Aa,Ae). (Aa′,Ae′) Magnified view of the ventral node region. (Aa″,Ae″) Red channel showing DiI labelling. The same embryos are shown after 48 h (Ab) or 24 h (Af) ex vivo culture. Ab′ and Af' show magnified views of Ab and Af respectively. (Af″) Red channel showing DiI labelling. DiI was found in the notochord and dorsal gut (Ac,Ad,Ag,Ah). Insets in Ac, Ad, Ag and Ah show magnified views of DiI labelling in notochord and gut. (Ai) DiI labelling per embryo after culture with average anterior limit (±s.d.). The anterior limit in 4/6 crown-labelled embryos was in the presomitic mesoderm. The anterior limit in each of these embryos is denoted as (total somites/embryo) +1. As the presomitic mesoderm contains ∼7 presomites, the average anterior limit may be posterior to s17. Noto, notochord; NT, neural tube; PXM, paraxial mesoderm. Asterisk indicates 2/6 embryos had a minor contribution in the posterior neural tube. (B) Sox2/T immunostained section of embryo labelled with DiI in the crown after 24 h in culture (n embryos ). (D) Sagittal confocal sections through CLE-electroporated (Da), and NSB-electroporated class I (Db) and II (Dc) embryos after 24 h, with the notochord (noto) end shown in yellow. nt, neural tube; som, somite.

    Article Snippet: Primary antibodies (supplier, catalogue number and working concentration) were: anti-Timp3-loop1 (Abcam; ab39184; 5 µg/ml), anti-Sox2 (Abcam; ab92494; 1:200), anti-T (R & D; AF2085; 1 mg/ml) and anti-GFP (Abcam; ab13970; 10 mg/ml).

    Techniques: Ex Vivo

    a Analysis of the pervasiveness of genes that are highly specific to the transformed cells across all eight patients based on differential expression analysis (see “ Methods ”; all genes displayed have eightfold specificity for the transformed cells). The colorbar represents the product of the x - and y -axes. SOX2 is the most pervasively detected gene specific to transformed glioma cells in these eight HGG patients. b Drop-out curve for the total population of transformed cells showing the characteristic sigmoidal shape that indicates how, for the majority of genes, higher expression (counts per thousand or CPT) leads to detection in a higher fraction of cells. Because the detection frequency of SOX2 is close to that of similarly expressed genes, SOX2 is unlikely to be associated with a specific subpopulation of transformed cells and the frequency with which it is expressed among transformed cells is likely to be underestimated by our data. c IHC analysis confirming widespread protein expression of SOX2 in tissue slices from the six of the eight HGG patients in our cohort from which tissue was available for staining. We note that a considerable fraction of unstained nuclei in these specimens appear to be associated with blood vessels

    Journal: Genome Medicine

    Article Title: Single-cell transcriptome analysis of lineage diversity in high-grade glioma

    doi: 10.1186/s13073-018-0567-9

    Figure Lengend Snippet: a Analysis of the pervasiveness of genes that are highly specific to the transformed cells across all eight patients based on differential expression analysis (see “ Methods ”; all genes displayed have eightfold specificity for the transformed cells). The colorbar represents the product of the x - and y -axes. SOX2 is the most pervasively detected gene specific to transformed glioma cells in these eight HGG patients. b Drop-out curve for the total population of transformed cells showing the characteristic sigmoidal shape that indicates how, for the majority of genes, higher expression (counts per thousand or CPT) leads to detection in a higher fraction of cells. Because the detection frequency of SOX2 is close to that of similarly expressed genes, SOX2 is unlikely to be associated with a specific subpopulation of transformed cells and the frequency with which it is expressed among transformed cells is likely to be underestimated by our data. c IHC analysis confirming widespread protein expression of SOX2 in tissue slices from the six of the eight HGG patients in our cohort from which tissue was available for staining. We note that a considerable fraction of unstained nuclei in these specimens appear to be associated with blood vessels

    Article Snippet: We used the following primary antibodies: rabbit anti-CD163 (Abcam, ab182422, 1:50 dilution), rabbit anti-SOX2 (Abcam, ab92494, 1:100 dilution), rabbit anti-TMEM119 (Abcam, ab185333, 1:300 dilution).

    Techniques: Transformation Assay, Expressing, Cycling Probe Technology, Immunohistochemistry, Staining

    a t-SNE projections of the transformed population of cells from each of the eight HGGs from scRNA-Seq. The projections are colored based on the cellular subpopulations identified from unsupervised clustering. b Heatmaps showing the detection frequency of canonical astrocyte, OPC, oligodendrocyte, and neuroblast markers found to be specifically associated with transformed cellular subpopulations shown in a across multiple patients along with SOX2 , which is expressed across all transformed populations. The orange heatmap below each green heatmap shows the average detection frequency of cell cycle control genes found in each subpopulation. Note that some tumors have subpopulations resembling multiple neural lineages (PJ016, PJ018, PJ030, PJ048), while others exhibit a relative loss of neural lineage identity and concomitant reduction in proliferation

    Journal: Genome Medicine

    Article Title: Single-cell transcriptome analysis of lineage diversity in high-grade glioma

    doi: 10.1186/s13073-018-0567-9

    Figure Lengend Snippet: a t-SNE projections of the transformed population of cells from each of the eight HGGs from scRNA-Seq. The projections are colored based on the cellular subpopulations identified from unsupervised clustering. b Heatmaps showing the detection frequency of canonical astrocyte, OPC, oligodendrocyte, and neuroblast markers found to be specifically associated with transformed cellular subpopulations shown in a across multiple patients along with SOX2 , which is expressed across all transformed populations. The orange heatmap below each green heatmap shows the average detection frequency of cell cycle control genes found in each subpopulation. Note that some tumors have subpopulations resembling multiple neural lineages (PJ016, PJ018, PJ030, PJ048), while others exhibit a relative loss of neural lineage identity and concomitant reduction in proliferation

    Article Snippet: We used the following primary antibodies: rabbit anti-CD163 (Abcam, ab182422, 1:50 dilution), rabbit anti-SOX2 (Abcam, ab92494, 1:100 dilution), rabbit anti-TMEM119 (Abcam, ab185333, 1:300 dilution).

    Techniques: Transformation Assay

    Structure analysis of Sox2-HMG/nucleic acid-binding modes. a 1 H– 15 N HSQC NMR spectrum of Sox2-HMG (black) overlaid with the spectrum of the Sox2-HMG/DNA complex (red). The DNA sequence used in this experiment was derived from the FGF4 enhancer, corresponding to the sequence shown in Supplementary Table 1 . b 1 H– 15 N HSQC NMR spectrum of Sox2-HMG (black) overlaid with the spectrum of the Sox2-HMG/RNA complex (blue). The RNA sequence used in this experiment was the hairpin Loop B/Bulge(0 + 1) (Fig. 3c ).

    Journal: Nature Communications

    Article Title: The Sox2 transcription factor binds RNA

    doi: 10.1038/s41467-020-15571-8

    Figure Lengend Snippet: Structure analysis of Sox2-HMG/nucleic acid-binding modes. a 1 H– 15 N HSQC NMR spectrum of Sox2-HMG (black) overlaid with the spectrum of the Sox2-HMG/DNA complex (red). The DNA sequence used in this experiment was derived from the FGF4 enhancer, corresponding to the sequence shown in Supplementary Table 1 . b 1 H– 15 N HSQC NMR spectrum of Sox2-HMG (black) overlaid with the spectrum of the Sox2-HMG/RNA complex (blue). The RNA sequence used in this experiment was the hairpin Loop B/Bulge(0 + 1) (Fig. 3c ).

    Article Snippet: To the remaining supernatant, 5 µg of Sox2 antibody (Atlas Antibodies; validation shown in Supplementary Fig. ) was added and rotated for 2 h at 4 °C.

    Techniques: Binding Assay, Nuclear Magnetic Resonance, Sequencing, Derivative Assay

    Characterization of the base-specific contributions to nucleic acid binding. a The relative binding affinities ( K D,rel ) for DNA and RNA are mapped to the Sox2-HMG surface (PDB ID 1GT0) according to the color scale to the left. b Plotted K D,rel values for DNA and RNA binding colored according to the scale in panel ( a ). The values of K D,rel are presented as the average and s.d. of n = 3 technical replicates. Bars above a gray background correspond to the Sox2-HMG major wing whereas a white background denotes the Sox2-HMG minor wing. P -values for the difference between K D,rel (DNA) and K D,rel (DNA) were calculated by the Student’s t -test assuming non-parametric distribution. All measured binding affinities and p -values for the alanine mutagenesis are provided in Supplementary Table 7 .

    Journal: Nature Communications

    Article Title: The Sox2 transcription factor binds RNA

    doi: 10.1038/s41467-020-15571-8

    Figure Lengend Snippet: Characterization of the base-specific contributions to nucleic acid binding. a The relative binding affinities ( K D,rel ) for DNA and RNA are mapped to the Sox2-HMG surface (PDB ID 1GT0) according to the color scale to the left. b Plotted K D,rel values for DNA and RNA binding colored according to the scale in panel ( a ). The values of K D,rel are presented as the average and s.d. of n = 3 technical replicates. Bars above a gray background correspond to the Sox2-HMG major wing whereas a white background denotes the Sox2-HMG minor wing. P -values for the difference between K D,rel (DNA) and K D,rel (DNA) were calculated by the Student’s t -test assuming non-parametric distribution. All measured binding affinities and p -values for the alanine mutagenesis are provided in Supplementary Table 7 .

    Article Snippet: To the remaining supernatant, 5 µg of Sox2 antibody (Atlas Antibodies; validation shown in Supplementary Fig. ) was added and rotated for 2 h at 4 °C.

    Techniques: Binding Assay, RNA Binding Assay, Mutagenesis

    Sox2 interacts with RNA in mESC. a Schematic of fRIP and UV-RIP-Seq experimental flow. b , c Examples of enriched genes (Pmepa1 and Brd2) identified by both formaldehyde (“CH 2 O”) and UV-based RIP-seq. Each panel describes normalized coverage of sequencing reads (RPM: reads per million reads). d Correlation of enrichment between fRIP and UV-RIP. A dot represents a gene and each axis describes the fold change of IP relative to input in log scale. A color indicates statistical significance of enrichment (FDR-adjusted p -value

    Journal: Nature Communications

    Article Title: The Sox2 transcription factor binds RNA

    doi: 10.1038/s41467-020-15571-8

    Figure Lengend Snippet: Sox2 interacts with RNA in mESC. a Schematic of fRIP and UV-RIP-Seq experimental flow. b , c Examples of enriched genes (Pmepa1 and Brd2) identified by both formaldehyde (“CH 2 O”) and UV-based RIP-seq. Each panel describes normalized coverage of sequencing reads (RPM: reads per million reads). d Correlation of enrichment between fRIP and UV-RIP. A dot represents a gene and each axis describes the fold change of IP relative to input in log scale. A color indicates statistical significance of enrichment (FDR-adjusted p -value

    Article Snippet: To the remaining supernatant, 5 µg of Sox2 antibody (Atlas Antibodies; validation shown in Supplementary Fig. ) was added and rotated for 2 h at 4 °C.

    Techniques: Sequencing

    Nucleic acid binding by Sox2 and Sox2-HMG. a Average binding curves of full-length Sox2 to nucleic acid ligands. Curves are presented as the average of normalized fraction bound with error bars displaying the standard deviation. b Figure legend and measured binding affinity values for panels ( a ) and ( c ). Values are presented as the average and s.e.m. of n = 4 technical replicates with the exception of Sox2-HMG/ ES2 which is the average and s.e.m. of n = 10 technical replicates. c Representative binding curves of the Sox2-HMG domain to nucleic acid ligands. Curves and error bars displayed as in panel ( a ).

    Journal: Nature Communications

    Article Title: The Sox2 transcription factor binds RNA

    doi: 10.1038/s41467-020-15571-8

    Figure Lengend Snippet: Nucleic acid binding by Sox2 and Sox2-HMG. a Average binding curves of full-length Sox2 to nucleic acid ligands. Curves are presented as the average of normalized fraction bound with error bars displaying the standard deviation. b Figure legend and measured binding affinity values for panels ( a ) and ( c ). Values are presented as the average and s.e.m. of n = 4 technical replicates with the exception of Sox2-HMG/ ES2 which is the average and s.e.m. of n = 10 technical replicates. c Representative binding curves of the Sox2-HMG domain to nucleic acid ligands. Curves and error bars displayed as in panel ( a ).

    Article Snippet: To the remaining supernatant, 5 µg of Sox2 antibody (Atlas Antibodies; validation shown in Supplementary Fig. ) was added and rotated for 2 h at 4 °C.

    Techniques: Binding Assay, Standard Deviation

    Deletion analysis of the ES2 lncRNA. a Schematized depiction of the ES2 lncRNA and the segmented transcripts. Apparent binding affinity displayed as the average and s.e.m. of n = 6 technical replicates excluding 1–354 ES2, which is n = 10 technical replicates). b Sfold 44 predicted secondary structure of ES2 , nts 276–354, and minimized loop constructs. c Average binding curve for each Sox2-RNA interaction with error bars displaying the standard deviation of each measurements. K D,app is reported for the first transition and presented as the average and the s.e.m. of n = 6 technical replicates.

    Journal: Nature Communications

    Article Title: The Sox2 transcription factor binds RNA

    doi: 10.1038/s41467-020-15571-8

    Figure Lengend Snippet: Deletion analysis of the ES2 lncRNA. a Schematized depiction of the ES2 lncRNA and the segmented transcripts. Apparent binding affinity displayed as the average and s.e.m. of n = 6 technical replicates excluding 1–354 ES2, which is n = 10 technical replicates). b Sfold 44 predicted secondary structure of ES2 , nts 276–354, and minimized loop constructs. c Average binding curve for each Sox2-RNA interaction with error bars displaying the standard deviation of each measurements. K D,app is reported for the first transition and presented as the average and the s.e.m. of n = 6 technical replicates.

    Article Snippet: To the remaining supernatant, 5 µg of Sox2 antibody (Atlas Antibodies; validation shown in Supplementary Fig. ) was added and rotated for 2 h at 4 °C.

    Techniques: Binding Assay, Construct, Standard Deviation

    Characterizing the nucleic acid-binding modes of Sox2-HMG. a Equilibrium competition binding assays measuring the anisotropy of the labeled nucleic acid as a function of unlabeled competitor. Binding curves presented as the average of n = 3 technical replicates with the error bars reflecting their s.d. b Determination of complex stoichiometry by EMSA. Migration of the radiolabeled nucleic acid species as a function of [Sox2-HMG]:[Ligand]. Bands are denoted as F (free), B1 (bound species 1), and B2 (bound species 2). Quantification provided in Supplementary Fig. 5 . c Analysis of the salt dependence of the Sox2-HMG/nucleic acid interactions. The colored bars show the measured binding affinities as the average of the n = 4 with the standard error reported (black shapes). The calculated slope of each linear regression is provided. d The calculated values of the electrostatic (Δ G el ) and non-electrostatic (Δ G nel ) components to binding the indicated nucleic acid ligand under the standard reaction conditions.

    Journal: Nature Communications

    Article Title: The Sox2 transcription factor binds RNA

    doi: 10.1038/s41467-020-15571-8

    Figure Lengend Snippet: Characterizing the nucleic acid-binding modes of Sox2-HMG. a Equilibrium competition binding assays measuring the anisotropy of the labeled nucleic acid as a function of unlabeled competitor. Binding curves presented as the average of n = 3 technical replicates with the error bars reflecting their s.d. b Determination of complex stoichiometry by EMSA. Migration of the radiolabeled nucleic acid species as a function of [Sox2-HMG]:[Ligand]. Bands are denoted as F (free), B1 (bound species 1), and B2 (bound species 2). Quantification provided in Supplementary Fig. 5 . c Analysis of the salt dependence of the Sox2-HMG/nucleic acid interactions. The colored bars show the measured binding affinities as the average of the n = 4 with the standard error reported (black shapes). The calculated slope of each linear regression is provided. d The calculated values of the electrostatic (Δ G el ) and non-electrostatic (Δ G nel ) components to binding the indicated nucleic acid ligand under the standard reaction conditions.

    Article Snippet: To the remaining supernatant, 5 µg of Sox2 antibody (Atlas Antibodies; validation shown in Supplementary Fig. ) was added and rotated for 2 h at 4 °C.

    Techniques: Binding Assay, Labeling, Migration

    Interrogation of RNA features for high-affinity interaction with Sox2-HMG. a Secondary structure of the Loop B RNA with the designated paired regions (P1 and P2), internal loop (blue dotted line), and terminal loop (green dashed line). Binding assessed to mutations b reducing paired region helix length, n = 8, c reducing the internal loop size, n = 8, d truncation of hairpins, n = 3 e removing the terminal loop, n = 6, f structured RNAs, xpt riboswitch and env4 Cbl riboswitch, n = 4, tRNA Leu , n = 3, and g nucleic acid duplexes with n = 3 technical replicates each. Apparent binding affinity displayed as the average with s.e.m. reported.

    Journal: Nature Communications

    Article Title: The Sox2 transcription factor binds RNA

    doi: 10.1038/s41467-020-15571-8

    Figure Lengend Snippet: Interrogation of RNA features for high-affinity interaction with Sox2-HMG. a Secondary structure of the Loop B RNA with the designated paired regions (P1 and P2), internal loop (blue dotted line), and terminal loop (green dashed line). Binding assessed to mutations b reducing paired region helix length, n = 8, c reducing the internal loop size, n = 8, d truncation of hairpins, n = 3 e removing the terminal loop, n = 6, f structured RNAs, xpt riboswitch and env4 Cbl riboswitch, n = 4, tRNA Leu , n = 3, and g nucleic acid duplexes with n = 3 technical replicates each. Apparent binding affinity displayed as the average with s.e.m. reported.

    Article Snippet: To the remaining supernatant, 5 µg of Sox2 antibody (Atlas Antibodies; validation shown in Supplementary Fig. ) was added and rotated for 2 h at 4 °C.

    Techniques: Binding Assay

    Ascl1 -expressing cells give rise directly to Sus cells . ( A ) Immunofluorescence (IF) for SOX2 and ASCL1 in wild-type OE. White arrowheads indicate SOX2 + ; ASCL1 + cells. ( B ) In Ascl1 GFP/+ OE, apical GFP + cells are SUS4 + (arrowheads). The bottom row shows

    Journal: Development (Cambridge, England)

    Article Title: Activin and GDF11 collaborate in feedback control of neuroepithelial stem cell proliferation and fate

    doi: 10.1242/dev.065870

    Figure Lengend Snippet: Ascl1 -expressing cells give rise directly to Sus cells . ( A ) Immunofluorescence (IF) for SOX2 and ASCL1 in wild-type OE. White arrowheads indicate SOX2 + ; ASCL1 + cells. ( B ) In Ascl1 GFP/+ OE, apical GFP + cells are SUS4 + (arrowheads). The bottom row shows

    Article Snippet: Primary antibodies were rabbit anti-SOX2 (Chemicon, 1:500), rabbit anti-GFP (Molecular Probes, 1:500), mouse anti-cytokeratin 18 (Millipore RGE53, 1:50) and OE SUS cell-specific mouse monoclonal antibody SUS-4 ( ) (1:50).

    Techniques: Expressing, Immunofluorescence

    Increased Sus cells in Gdf11 –/– and ActβB –/– ;Gdf11 –/– OE. ( A ) ISH with the indicated probes indicates increased Sox2 expression and a thicker Sus layer (white arrowhead) in Act β B –/–

    Journal: Development (Cambridge, England)

    Article Title: Activin and GDF11 collaborate in feedback control of neuroepithelial stem cell proliferation and fate

    doi: 10.1242/dev.065870

    Figure Lengend Snippet: Increased Sus cells in Gdf11 –/– and ActβB –/– ;Gdf11 –/– OE. ( A ) ISH with the indicated probes indicates increased Sox2 expression and a thicker Sus layer (white arrowhead) in Act β B –/–

    Article Snippet: Primary antibodies were rabbit anti-SOX2 (Chemicon, 1:500), rabbit anti-GFP (Molecular Probes, 1:500), mouse anti-cytokeratin 18 (Millipore RGE53, 1:50) and OE SUS cell-specific mouse monoclonal antibody SUS-4 ( ) (1:50).

    Techniques: In Situ Hybridization, Expressing, Activated Clotting Time Assay

    Increase in SOX2 + and ASCL1 + cells in ActβB –/– OE. Bar charts show cells/mm OE (mean ± s.e.m.; * , P ≤0.05 DT). White asterisk, Bowman's gland. BL, basal lamina. Scale bars: 20 μm.

    Journal: Development (Cambridge, England)

    Article Title: Activin and GDF11 collaborate in feedback control of neuroepithelial stem cell proliferation and fate

    doi: 10.1242/dev.065870

    Figure Lengend Snippet: Increase in SOX2 + and ASCL1 + cells in ActβB –/– OE. Bar charts show cells/mm OE (mean ± s.e.m.; * , P ≤0.05 DT). White asterisk, Bowman's gland. BL, basal lamina. Scale bars: 20 μm.

    Article Snippet: Primary antibodies were rabbit anti-SOX2 (Chemicon, 1:500), rabbit anti-GFP (Molecular Probes, 1:500), mouse anti-cytokeratin 18 (Millipore RGE53, 1:50) and OE SUS cell-specific mouse monoclonal antibody SUS-4 ( ) (1:50).

    Techniques:

    ActβB and GDF11 modulate stem cell fates. ( A-C ) Cells in three marker categories (SOX2 + , black; ASCL1 + , white; SOX2 + ; ASCL1 + , gray) were quantified for (A) total OE, (B) basal stem/progenitor cell compartment and (C) apical sustentacular cell

    Journal: Development (Cambridge, England)

    Article Title: Activin and GDF11 collaborate in feedback control of neuroepithelial stem cell proliferation and fate

    doi: 10.1242/dev.065870

    Figure Lengend Snippet: ActβB and GDF11 modulate stem cell fates. ( A-C ) Cells in three marker categories (SOX2 + , black; ASCL1 + , white; SOX2 + ; ASCL1 + , gray) were quantified for (A) total OE, (B) basal stem/progenitor cell compartment and (C) apical sustentacular cell

    Article Snippet: Primary antibodies were rabbit anti-SOX2 (Chemicon, 1:500), rabbit anti-GFP (Molecular Probes, 1:500), mouse anti-cytokeratin 18 (Millipore RGE53, 1:50) and OE SUS cell-specific mouse monoclonal antibody SUS-4 ( ) (1:50).

    Techniques: Marker

    Western blotting. Protein extractions from 3 LGCA and 3 HGCA EpCAM High (+) and EpCAM Low (-) cell lines were probed for OCT4 (A; 40kDa), SOX2 (B; 40-43kDa), NANOG (C; 37-40kDa), KLF4 (D; 54kDa) and c-MYC (E; 42 57kDa). NTERA-2 cells were used as the positive control for all iPSC markers. α-tubulin (F; 50kDa) was used as a loading control. EpCAM High and EpCAM Low cell lines were also probed for their expression of EpCAM (G; bands from ~30-40kDa) and α-SMA (H; 42kDa). HepG2 cells and 3T3 cells were used as the positive control for EpCAM and α-SMA, respectively.

    Journal: PLoS ONE

    Article Title: Colon adenocarcinoma-derived cells that express induced-pluripotent stem cell markers possess stem cell function

    doi: 10.1371/journal.pone.0232934

    Figure Lengend Snippet: Western blotting. Protein extractions from 3 LGCA and 3 HGCA EpCAM High (+) and EpCAM Low (-) cell lines were probed for OCT4 (A; 40kDa), SOX2 (B; 40-43kDa), NANOG (C; 37-40kDa), KLF4 (D; 54kDa) and c-MYC (E; 42 57kDa). NTERA-2 cells were used as the positive control for all iPSC markers. α-tubulin (F; 50kDa) was used as a loading control. EpCAM High and EpCAM Low cell lines were also probed for their expression of EpCAM (G; bands from ~30-40kDa) and α-SMA (H; 42kDa). HepG2 cells and 3T3 cells were used as the positive control for EpCAM and α-SMA, respectively.

    Article Snippet: Primary antibodies included rabbit anti-OCT4 (cat# A24867, Thermo), rat anti-SOX2 (cat# A24759, Thermo), mouse IgG3 anti-SSEA4 (cat# A24866, Thermo) and mouse IgM anti-TRA-1-60 (cat# A24868, Thermo).

    Techniques: Western Blot, Positive Control, Expressing

    RT-qPCR. RNA was extracted from EpCAM High and EpCAM Low cells lines from 3 LGCA and 3 HGCA cases, and RT-qPCR was carried out to measure the mRNA levels of OCT4 (A), SOX2 (B), NANOG (C), KLF4 (D) and c-MYC (E). Triplicate values are shown by dots (EpCAM Low ) and squares (EpCAM High ), with mean and 95% confidence intervals. Abundance was measured relative to qPCR Human Reference Total RNA (Mediray). LGCA (n = 3); HGCA (n = 3).

    Journal: PLoS ONE

    Article Title: Colon adenocarcinoma-derived cells that express induced-pluripotent stem cell markers possess stem cell function

    doi: 10.1371/journal.pone.0232934

    Figure Lengend Snippet: RT-qPCR. RNA was extracted from EpCAM High and EpCAM Low cells lines from 3 LGCA and 3 HGCA cases, and RT-qPCR was carried out to measure the mRNA levels of OCT4 (A), SOX2 (B), NANOG (C), KLF4 (D) and c-MYC (E). Triplicate values are shown by dots (EpCAM Low ) and squares (EpCAM High ), with mean and 95% confidence intervals. Abundance was measured relative to qPCR Human Reference Total RNA (Mediray). LGCA (n = 3); HGCA (n = 3).

    Article Snippet: Primary antibodies included rabbit anti-OCT4 (cat# A24867, Thermo), rat anti-SOX2 (cat# A24759, Thermo), mouse IgG3 anti-SSEA4 (cat# A24866, Thermo) and mouse IgM anti-TRA-1-60 (cat# A24868, Thermo).

    Techniques: Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    Densitometry performed on western blot. Densitometry data provided semi-quantitative data for protein abundance. The intensity values of all LGCA cell lines (both EpCAM High and EpCAM Low ) and all HGCA cell lines (both EpCAM High and EpCAM Low ) were combined and the average intensity calculated, and these are shown for OCT4 (A), SOX2 (C), NANOG (E), KLF4 (G) and c-MYC (I). The intensity values of all EpCAM Low cell lines (both LGCA and HGCA) and all EpCAM High cell lines (both LGCA and HGCA) were combined and the average intensity calculated, and these are shown for OCT4 (B), SOX2 (D), NANOG (F), KLF4 (H), c-MYC (J), EpCAM (K) and α-SMA (L). Individual intensity values were normalized against the loading control α-tubulin before being combined and averaged. Error bars show standard deviation.

    Journal: PLoS ONE

    Article Title: Colon adenocarcinoma-derived cells that express induced-pluripotent stem cell markers possess stem cell function

    doi: 10.1371/journal.pone.0232934

    Figure Lengend Snippet: Densitometry performed on western blot. Densitometry data provided semi-quantitative data for protein abundance. The intensity values of all LGCA cell lines (both EpCAM High and EpCAM Low ) and all HGCA cell lines (both EpCAM High and EpCAM Low ) were combined and the average intensity calculated, and these are shown for OCT4 (A), SOX2 (C), NANOG (E), KLF4 (G) and c-MYC (I). The intensity values of all EpCAM Low cell lines (both LGCA and HGCA) and all EpCAM High cell lines (both LGCA and HGCA) were combined and the average intensity calculated, and these are shown for OCT4 (B), SOX2 (D), NANOG (F), KLF4 (H), c-MYC (J), EpCAM (K) and α-SMA (L). Individual intensity values were normalized against the loading control α-tubulin before being combined and averaged. Error bars show standard deviation.

    Article Snippet: Primary antibodies included rabbit anti-OCT4 (cat# A24867, Thermo), rat anti-SOX2 (cat# A24759, Thermo), mouse IgG3 anti-SSEA4 (cat# A24866, Thermo) and mouse IgM anti-TRA-1-60 (cat# A24868, Thermo).

    Techniques: Western Blot, Standard Deviation

    Representative immunofluorescence immunocytochemical images. EpCAM Low (A) and EpCAM High (B) cells from low-grade colon adenocarcinoma (LGCA)-derived primary cell lines, and EpCAM Low (C) and EpCAM High (D) cells from high-grade colon adenocarcinoma (HGCA)-derived primary cell lines showing expression of SOX2 (green) and TRA-1-60 (red). LGCA (n = 3); HGCA (n = 3). Positive control NTERA-2 (E) and CaCo2 (F) cells were stained for SOX2 (green) and TRA-1-60 (red) (B). Original magnification: 400x; scale bar = 20 μm.

    Journal: PLoS ONE

    Article Title: Colon adenocarcinoma-derived cells that express induced-pluripotent stem cell markers possess stem cell function

    doi: 10.1371/journal.pone.0232934

    Figure Lengend Snippet: Representative immunofluorescence immunocytochemical images. EpCAM Low (A) and EpCAM High (B) cells from low-grade colon adenocarcinoma (LGCA)-derived primary cell lines, and EpCAM Low (C) and EpCAM High (D) cells from high-grade colon adenocarcinoma (HGCA)-derived primary cell lines showing expression of SOX2 (green) and TRA-1-60 (red). LGCA (n = 3); HGCA (n = 3). Positive control NTERA-2 (E) and CaCo2 (F) cells were stained for SOX2 (green) and TRA-1-60 (red) (B). Original magnification: 400x; scale bar = 20 μm.

    Article Snippet: Primary antibodies included rabbit anti-OCT4 (cat# A24867, Thermo), rat anti-SOX2 (cat# A24759, Thermo), mouse IgG3 anti-SSEA4 (cat# A24866, Thermo) and mouse IgM anti-TRA-1-60 (cat# A24868, Thermo).

    Techniques: Immunofluorescence, Derivative Assay, Expressing, Positive Control, Staining

    Proposed model for bFGF signaling in pluripotent (green) vs. differentiated (orange) hiPSCs (for details see discussion). bFGF, basic fibroblast growth factor; OCT4, octamer-binding transcription factor 4; SOX2, sex determining region Y-box 2; GFAP, glial fibrillary acidic protein; RAS, rat sarcoma; PI3K, phosphoinositide 3-kinase; PDK1, 3-phosphoinositidedependent protein kinase; MEK, MAP/ERK kinase; ERK, extracellular signal-regulated kinase; JAK, Janus kinase; STAT3, signal transducer and activator of transcription 3

    Journal: Cell Communication and Signaling : CCS

    Article Title: bFGF-mediated pluripotency maintenance in human induced pluripotent stem cells is associated with NRAS-MAPK signaling

    doi: 10.1186/s12964-018-0307-1

    Figure Lengend Snippet: Proposed model for bFGF signaling in pluripotent (green) vs. differentiated (orange) hiPSCs (for details see discussion). bFGF, basic fibroblast growth factor; OCT4, octamer-binding transcription factor 4; SOX2, sex determining region Y-box 2; GFAP, glial fibrillary acidic protein; RAS, rat sarcoma; PI3K, phosphoinositide 3-kinase; PDK1, 3-phosphoinositidedependent protein kinase; MEK, MAP/ERK kinase; ERK, extracellular signal-regulated kinase; JAK, Janus kinase; STAT3, signal transducer and activator of transcription 3

    Article Snippet: The following antibodies were applied for immunoblotting: mouse anti-γ-tubulin (Sigma-Aldrich, T5326); mouse anti-OCT4 (Santa Cruz, sc-5279); rabbit anti-SOX2 (Invitrogen, PA1–16968); goat anti-NANOG (R & D systems, AF1997); mouse anti-SSEA4 (Millipore, MAB4304); mouse anti-α-SMA (DAKO, M0851); rabbit anti-GFAP (DAKO, Z0334); rabbit anti-MEK1/2 (#9126), rabbit anti-ERK1/2 (#9102), rabbit anti-RSK (#9355), rabbit anti-AKT (#9272), rabbit anti-p-MEK1/2 (S217/S221, #9154), rabbit anti-p-ERK1/2 (T202/T204, #9106), rabbit anti-p-p90RSK (T573, #9346), rabbit anti-p-AKT (S473, # 4060 and T308, #2965), rabbit anti-FOXO1 (#2880), rabbit anti-p-FOXO1 (S256, #9461), rabbit anti-S6 kinase (#2708), rabbit anti-p-p70 S6 kinase (T389, #9205), rabbit anti-p38 (#8690), rabbit anti-p-p38 (T180/Y182, #9211), rabbit anti-JNK (#9252), rabbit anti-p-JNK (T183/Y185, #9251), mouse anti-STAT3 (#9139S) and rabbit anti-p-STAT3 (Y705, #9145S) all from Cell Signaling.

    Techniques: Binding Assay

    Long-term maintenance of undifferentiated hiPSCs. a Confocal imaging showed the expression of pluripotency markers (OCT4, SOX2, NANOG and SSEA4) and the absence of differentiation markers GFAP and α-SMA as ectodermal and mesodermal markers, respectively. Cell nuclei were stained with DAPI (blue). Scale bars, 10 μm. b Flow cytometry confirmed expression of OCT4, SSEA4 and SOX2 in hiPSCs with more than 98% of positive cells. c qPCR analysis for undifferentiated stem cell markers ( POU5F1 , SOX2 and NANOG ) and early commitment to differentiation markers ( BRACHYURY , PAX6 and AFP ). GAPDH was used as an internal control. d Immunoblot analysis showing the specificity of antibodies and expression of markers. HeLa cells were used as negative control, HFF and astrocytes were used as positive control for α-SMA and GFAP, respectively

    Journal: Cell Communication and Signaling : CCS

    Article Title: bFGF-mediated pluripotency maintenance in human induced pluripotent stem cells is associated with NRAS-MAPK signaling

    doi: 10.1186/s12964-018-0307-1

    Figure Lengend Snippet: Long-term maintenance of undifferentiated hiPSCs. a Confocal imaging showed the expression of pluripotency markers (OCT4, SOX2, NANOG and SSEA4) and the absence of differentiation markers GFAP and α-SMA as ectodermal and mesodermal markers, respectively. Cell nuclei were stained with DAPI (blue). Scale bars, 10 μm. b Flow cytometry confirmed expression of OCT4, SSEA4 and SOX2 in hiPSCs with more than 98% of positive cells. c qPCR analysis for undifferentiated stem cell markers ( POU5F1 , SOX2 and NANOG ) and early commitment to differentiation markers ( BRACHYURY , PAX6 and AFP ). GAPDH was used as an internal control. d Immunoblot analysis showing the specificity of antibodies and expression of markers. HeLa cells were used as negative control, HFF and astrocytes were used as positive control for α-SMA and GFAP, respectively

    Article Snippet: The following antibodies were applied for immunoblotting: mouse anti-γ-tubulin (Sigma-Aldrich, T5326); mouse anti-OCT4 (Santa Cruz, sc-5279); rabbit anti-SOX2 (Invitrogen, PA1–16968); goat anti-NANOG (R & D systems, AF1997); mouse anti-SSEA4 (Millipore, MAB4304); mouse anti-α-SMA (DAKO, M0851); rabbit anti-GFAP (DAKO, Z0334); rabbit anti-MEK1/2 (#9126), rabbit anti-ERK1/2 (#9102), rabbit anti-RSK (#9355), rabbit anti-AKT (#9272), rabbit anti-p-MEK1/2 (S217/S221, #9154), rabbit anti-p-ERK1/2 (T202/T204, #9106), rabbit anti-p-p90RSK (T573, #9346), rabbit anti-p-AKT (S473, # 4060 and T308, #2965), rabbit anti-FOXO1 (#2880), rabbit anti-p-FOXO1 (S256, #9461), rabbit anti-S6 kinase (#2708), rabbit anti-p-p70 S6 kinase (T389, #9205), rabbit anti-p38 (#8690), rabbit anti-p-p38 (T180/Y182, #9211), rabbit anti-JNK (#9252), rabbit anti-p-JNK (T183/Y185, #9251), mouse anti-STAT3 (#9139S) and rabbit anti-p-STAT3 (Y705, #9145S) all from Cell Signaling.

    Techniques: Imaging, Expressing, Staining, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Negative Control, Positive Control

    The critical role of bFGF for maintaining hiPSC pluripotency. a Phase contrast images of hiPSCs cultured under four different conditions, CM-100, CM-5, CM-0 and non-CM for 6 days. Undifferentiated hiPSCs formed compact colonies (CM-100), while without bFGF supplementation hiPSCs spread and flattened at day 6 (CM-0 and non-CM). Scale bar, 50 μm. b qPCR analysis showed the downregulation of pluripotency markers POU5F1 , SOX2 and NANOG in cells cultured in CM-0 and non-CM in comparison to control group (CM-100). All expression values were normalized to GAPDH . Results from three separate experiments, each carried out in triplicate, are shown as mean ± SD (ANOVA; * p

    Journal: Cell Communication and Signaling : CCS

    Article Title: bFGF-mediated pluripotency maintenance in human induced pluripotent stem cells is associated with NRAS-MAPK signaling

    doi: 10.1186/s12964-018-0307-1

    Figure Lengend Snippet: The critical role of bFGF for maintaining hiPSC pluripotency. a Phase contrast images of hiPSCs cultured under four different conditions, CM-100, CM-5, CM-0 and non-CM for 6 days. Undifferentiated hiPSCs formed compact colonies (CM-100), while without bFGF supplementation hiPSCs spread and flattened at day 6 (CM-0 and non-CM). Scale bar, 50 μm. b qPCR analysis showed the downregulation of pluripotency markers POU5F1 , SOX2 and NANOG in cells cultured in CM-0 and non-CM in comparison to control group (CM-100). All expression values were normalized to GAPDH . Results from three separate experiments, each carried out in triplicate, are shown as mean ± SD (ANOVA; * p

    Article Snippet: The following antibodies were applied for immunoblotting: mouse anti-γ-tubulin (Sigma-Aldrich, T5326); mouse anti-OCT4 (Santa Cruz, sc-5279); rabbit anti-SOX2 (Invitrogen, PA1–16968); goat anti-NANOG (R & D systems, AF1997); mouse anti-SSEA4 (Millipore, MAB4304); mouse anti-α-SMA (DAKO, M0851); rabbit anti-GFAP (DAKO, Z0334); rabbit anti-MEK1/2 (#9126), rabbit anti-ERK1/2 (#9102), rabbit anti-RSK (#9355), rabbit anti-AKT (#9272), rabbit anti-p-MEK1/2 (S217/S221, #9154), rabbit anti-p-ERK1/2 (T202/T204, #9106), rabbit anti-p-p90RSK (T573, #9346), rabbit anti-p-AKT (S473, # 4060 and T308, #2965), rabbit anti-FOXO1 (#2880), rabbit anti-p-FOXO1 (S256, #9461), rabbit anti-S6 kinase (#2708), rabbit anti-p-p70 S6 kinase (T389, #9205), rabbit anti-p38 (#8690), rabbit anti-p-p38 (T180/Y182, #9211), rabbit anti-JNK (#9252), rabbit anti-p-JNK (T183/Y185, #9251), mouse anti-STAT3 (#9139S) and rabbit anti-p-STAT3 (Y705, #9145S) all from Cell Signaling.

    Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Expressing

    Merkel cell differentiation is a temporal maturation process. (A-C) Whole-mount IF (WMIF) staining showing overlap of Merkel cell-specific genes Atoh1 , K8 ( Krt8 ), K20 ( Krt20 ) and Sox2 in neonatal (P0) mouse skin. Percentage of overlap is shown in C. Merkel

    Journal: Development (Cambridge, England)

    Article Title: Embryonic maturation of epidermal Merkel cells is controlled by a redundant transcription factor network

    doi: 10.1242/dev.112169

    Figure Lengend Snippet: Merkel cell differentiation is a temporal maturation process. (A-C) Whole-mount IF (WMIF) staining showing overlap of Merkel cell-specific genes Atoh1 , K8 ( Krt8 ), K20 ( Krt20 ) and Sox2 in neonatal (P0) mouse skin. Percentage of overlap is shown in C. Merkel

    Article Snippet: ; 1:20,000); anti-Keratin 5(generous gift of Elaine Fuchs, The Rockefeller University, NY, USA; 1:500); anti-K8 (DSHB, TROMA-I; 1:500); anti-K18 (Abcam, ab668; 1:100); anti-Isl1 (Abcam, ab109517; 1:400); anti-Isl1/2 (DSHB, 39.4D5; 1:100); anti-Isl2 (DSHB, 67.4E12; 1:500); anti-Rab3C (Abcam, ab3336; 1:250); anti-NF200 (Abcam, ab93340; 1:250); anti-Sox2 (Stemgent, 09-0024; 1:150); anti-K20 (Dako, M7019; 1:70); anti-AcCasp3 (R & D Systems, AF835; 1:250); anti-GFP (Abcam, ab13970; 1:1000); anti-K10 (Covance, PRB-145P; 1:500); anti-Loricrin (Covance, PRB-159P; 1:250).

    Techniques: Cell Differentiation, Staining

    Atoh1 is essential for Merkel cell specification, whereas Sox2 is required for late-phase Merkel cell differentiation. (A-D) IF staining of WT and Atoh1cKO back skin (A,B) and whisker follicles (C,D) at P0 (A,C) and E15 (B,D) shows a complete loss of

    Journal: Development (Cambridge, England)

    Article Title: Embryonic maturation of epidermal Merkel cells is controlled by a redundant transcription factor network

    doi: 10.1242/dev.112169

    Figure Lengend Snippet: Atoh1 is essential for Merkel cell specification, whereas Sox2 is required for late-phase Merkel cell differentiation. (A-D) IF staining of WT and Atoh1cKO back skin (A,B) and whisker follicles (C,D) at P0 (A,C) and E15 (B,D) shows a complete loss of

    Article Snippet: ; 1:20,000); anti-Keratin 5(generous gift of Elaine Fuchs, The Rockefeller University, NY, USA; 1:500); anti-K8 (DSHB, TROMA-I; 1:500); anti-K18 (Abcam, ab668; 1:100); anti-Isl1 (Abcam, ab109517; 1:400); anti-Isl1/2 (DSHB, 39.4D5; 1:100); anti-Isl2 (DSHB, 67.4E12; 1:500); anti-Rab3C (Abcam, ab3336; 1:250); anti-NF200 (Abcam, ab93340; 1:250); anti-Sox2 (Stemgent, 09-0024; 1:150); anti-K20 (Dako, M7019; 1:70); anti-AcCasp3 (R & D Systems, AF835; 1:250); anti-GFP (Abcam, ab13970; 1:1000); anti-K10 (Covance, PRB-145P; 1:500); anti-Loricrin (Covance, PRB-159P; 1:250).

    Techniques: Cell Differentiation, Staining, Whisker Assay

    Sox2 and Isl1 cooperate to orchestrate Merkel cell differentiation. (A) IF analysis of WT, Isl1cKO, Sox2cKO and Isl1 Sox2 2cKO mice, showing a further reduction in the number of K8+ (Krt8+) cells present in the 2cKO epidermis. (B) WMIF analysis confirmation

    Journal: Development (Cambridge, England)

    Article Title: Embryonic maturation of epidermal Merkel cells is controlled by a redundant transcription factor network

    doi: 10.1242/dev.112169

    Figure Lengend Snippet: Sox2 and Isl1 cooperate to orchestrate Merkel cell differentiation. (A) IF analysis of WT, Isl1cKO, Sox2cKO and Isl1 Sox2 2cKO mice, showing a further reduction in the number of K8+ (Krt8+) cells present in the 2cKO epidermis. (B) WMIF analysis confirmation

    Article Snippet: ; 1:20,000); anti-Keratin 5(generous gift of Elaine Fuchs, The Rockefeller University, NY, USA; 1:500); anti-K8 (DSHB, TROMA-I; 1:500); anti-K18 (Abcam, ab668; 1:100); anti-Isl1 (Abcam, ab109517; 1:400); anti-Isl1/2 (DSHB, 39.4D5; 1:100); anti-Isl2 (DSHB, 67.4E12; 1:500); anti-Rab3C (Abcam, ab3336; 1:250); anti-NF200 (Abcam, ab93340; 1:250); anti-Sox2 (Stemgent, 09-0024; 1:150); anti-K20 (Dako, M7019; 1:70); anti-AcCasp3 (R & D Systems, AF835; 1:250); anti-GFP (Abcam, ab13970; 1:1000); anti-K10 (Covance, PRB-145P; 1:500); anti-Loricrin (Covance, PRB-159P; 1:250).

    Techniques: Cell Differentiation, Mouse Assay

    Cyclin K expression positively correlates with proliferation. a Analyses of cyclin K protein expression during murine brain development by immunoblotting. Cyclin K protein expression in embryonic (E) and postnatal (P) murine brains correlated with that of Sox2, a marker of neural progenitor cell proliferation. b Analyses of cyclin K protein expression by immunoblotting during murine liver development. c Cyclin K expression detected by immunochemistry during the process of murine liver regeneration in vivo. 2nd, immunochemistry using secondary antibodies alone. 0 h denotes samples collected immediately after partial hepatectomy. Scale bar, 40 μm. d Comparison of cyclin K by immunoblotting in normal and H1299 cancer cells using equal cell numbers as loading control. HFF, neonatal human foreskin fibroblast. e Cyclin K expression detected by immunochemistry in normal and H1299 cancer cells. HFF, neonatal human foreskin fibroblast. Scale bar, 40 μm. f Time course analyses of cyclin K expression by immunoblotting in HCT116 cells treated with protein synthesis inhibitor cycloheximide (CHX, 50 μg/ml). g Time course analyses of cyclin K expression by immunoblotting in cells treated with proteasome inhibitor MG132 (5 μM) in human normal and HCT116 cancer cells. HFF, neonatal human foreskin fibroblast. Experiments were repeated for three times ( a – c ), and more than three times when cell lines were used ( d – g ). Representative results are shown

    Journal: Nature Communications

    Article Title: Cyclin K regulates prereplicative complex assembly to promote mammalian cell proliferation

    doi: 10.1038/s41467-018-04258-w

    Figure Lengend Snippet: Cyclin K expression positively correlates with proliferation. a Analyses of cyclin K protein expression during murine brain development by immunoblotting. Cyclin K protein expression in embryonic (E) and postnatal (P) murine brains correlated with that of Sox2, a marker of neural progenitor cell proliferation. b Analyses of cyclin K protein expression by immunoblotting during murine liver development. c Cyclin K expression detected by immunochemistry during the process of murine liver regeneration in vivo. 2nd, immunochemistry using secondary antibodies alone. 0 h denotes samples collected immediately after partial hepatectomy. Scale bar, 40 μm. d Comparison of cyclin K by immunoblotting in normal and H1299 cancer cells using equal cell numbers as loading control. HFF, neonatal human foreskin fibroblast. e Cyclin K expression detected by immunochemistry in normal and H1299 cancer cells. HFF, neonatal human foreskin fibroblast. Scale bar, 40 μm. f Time course analyses of cyclin K expression by immunoblotting in HCT116 cells treated with protein synthesis inhibitor cycloheximide (CHX, 50 μg/ml). g Time course analyses of cyclin K expression by immunoblotting in cells treated with proteasome inhibitor MG132 (5 μM) in human normal and HCT116 cancer cells. HFF, neonatal human foreskin fibroblast. Experiments were repeated for three times ( a – c ), and more than three times when cell lines were used ( d – g ). Representative results are shown

    Article Snippet: Following commercial antibodies were used: anti-FLAG M2 (Sigma, a8562, 1:20,000 dilution), anti-Sox2 (Santa Cruz, sc-20088, 1:500 dilution), cyclin D1 (sc-753, 1:500 dilution), cyclin E1 (sc-198, 1:500 dilution), cyclin A2 (sc-596, 1:1000 dilution), cyclin B1 (sc-245, 1:1000 dilution), CDK2 (sc-163, 1:5000 dilution), CDK9 (sc-98491, 1:2000 dilution), CDK12 (sc-81834, 1:500 dilution), ORC6 (sc-390490, 1:500 dilution), MCM4 (sc-22779, 1:5000 dilution), MCM7 (sc-9966, 1:1000 dilution), CDT1 (sc-28262, 1:1000 dilution), CDC6 (sc-9964, 1:500 dilution), PCNA (sc-56, 1:5000 dilution), Geminin (sc-13015, 1:500 dilution), p53 (sc-6243, 1:500 dilution), SMARCA4 (sc-25931, 1:1000 dilution) and p21 (sc-397, 1:500 dilution), anti-Pol II (Covance, 8WG16, MPY-127R, 1:2500 dilution) and Ser5-CTD (Covance, H5, MPY-129R, 1:2500 dilution), anti-GAPDH (KangChen, KC-5G5, 1:20,000 dilution), anti-MCM2 (Zen BioScience, 220023, 1:5000 dilution) and SF3B1 (612452, 1:2500 dilution), anti-MYC (Abcam, ab32072, 1:10,000 dilution), H2B (ab1790, 1:2500 dilution) and H3.1 (P30266M, 1:100,000 dilution), anti-CDC6 pSer54 (HuaAn Biotechnology, ET1612-96, 1:2000 dilution), anti-phospho-T/S-Pro (Abcam, ab9344, 1:1000 dilution).

    Techniques: Expressing, Marker, In Vivo

    Proliferation in the embryonic cochleae of Lgr4 deficient mice. (A–B′′) In the cochleae from heterozygous and homozygous Lgr4-LacZ mice, CCND1 (green) expression increased in the mid-basal turn, where CCND1 merged with red signal of SOX2 (arrows) in the pro-sensory domain. (C–D′′) The proliferation marker, EdU (red), was incorporated into pro-sensory cells (arrows) positive for SOX2 (green) in the mid-basal turn of the cochlea from Lgr4 deficient mice. Cell nuclei were counterstained with DAPI (blue), scale bars indicate 20 μm.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: LGR4 and LGR5 Regulate Hair Cell Differentiation in the Sensory Epithelium of the Developing Mouse Cochlea

    doi: 10.3389/fncel.2016.00186

    Figure Lengend Snippet: Proliferation in the embryonic cochleae of Lgr4 deficient mice. (A–B′′) In the cochleae from heterozygous and homozygous Lgr4-LacZ mice, CCND1 (green) expression increased in the mid-basal turn, where CCND1 merged with red signal of SOX2 (arrows) in the pro-sensory domain. (C–D′′) The proliferation marker, EdU (red), was incorporated into pro-sensory cells (arrows) positive for SOX2 (green) in the mid-basal turn of the cochlea from Lgr4 deficient mice. Cell nuclei were counterstained with DAPI (blue), scale bars indicate 20 μm.

    Article Snippet: The following primary antibodies were used: anti-myosin7a (1:700; rabbit, Proteus Bioscience, 25-6790), anti-myosin7a (1:200; mouse, Developmental Studies Hybridoma Bank, 138-1-c), anti-SOX2 (1:200; goat, Santa Cruz Biotechnology, s-17320), anti-SOX2 (1:50, mouse, Millipore, MAB4343), anti-SOX2 (1:250, BD Pharmingen, 561469) anti-acetylated tubulin (ACTBA) (1:50; mouse, Sigma–Aldrich, T7451), anti-prestin (1:700; goat, Santa Cruz Biotechnology, N-20, sc-22692), anti-LGR4 (1:200; rabbit, Sigma–Aldrich, HPA030267, test of the antibody specificity shown in Supplementary Figure ), anti-jagged1 (JAG1) (1:50; goat, Santa Cruz Biotechnology, C-20, sc-6011), anti-cyclinD1 (CCND1) (1:100; rabbit, Thermo Fisher Scientific, SP4, MA5-14512), anti-β-tubulin class III (TUJ1, 1:200; mouse, Covance, MMS-435P), anti-p75 NGF receptor (1:400; rabbit, Sigma–Aldrich, N3908), anti-MYO6 (1:300, Abcam, ab11096).

    Techniques: Mouse Assay, Expressing, Marker

    The distribution of LGR4 in the developing mouse cochlea. (A) At E14.5, strong LGR4 staining was detected in the cochlear duct and spiral ganglion cells (white arrowhead). LGR4 was detected in the pro-sensory domain, where it overlapped with SOX2 (white arrow), and non-sensory domains lateral and medial to the pro-sensory domain. (B) At E17, LGR4 (green) was detected in IHCs and OHCs (asterisks) and their surrounding supporting cells. Hair cells were labeled with phalloidin (F-actin) (red). (C) At P1, LGR4 expression was observed in the sensory epithelium (arrowhead) and spiral ganglion cells (arrow), from which projected TUJ1-positive nerves (red). (D) At P7, LGR4 was observed in hair cells (asterisks) that co-expressed Prestin (PRES) and in the surrounding supporting cells: Deiters’ cells (arrowheads) and IPCs and OPCs. (E–G) At P21, LGR4 was down-regulated in the OHCs and pillar cells, but was still expressed in the IHCs (arrowhead), Deiters’ cells (arrows), which were also positive for acetylated tubulin (ACTBA), and phalangeal processes of Deiters’ cells (arrowheads). LGR4 signals were also observed in the spiral ganglion cells. (H) At P42, LGR4 was detected in Deiters’ cells and their phalangeal processes. Cell nuclei were counterstained with DAPI (blue), asterisks indicate hair cells, scale bars indicate 20 μm in the picture (B,D,E,G,H) and 50 μm in (A,C,F) . (I,J) Cochlear cryosections from Lgr4-LacZ mice at the age of P21 were stained for β-galactosidase activity. Expression was seen in Deiters’ cells (black arrow) and their phalangeal processes (gray arrow), IHCs (black arrowhead), and spiral ganglion cells.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: LGR4 and LGR5 Regulate Hair Cell Differentiation in the Sensory Epithelium of the Developing Mouse Cochlea

    doi: 10.3389/fncel.2016.00186

    Figure Lengend Snippet: The distribution of LGR4 in the developing mouse cochlea. (A) At E14.5, strong LGR4 staining was detected in the cochlear duct and spiral ganglion cells (white arrowhead). LGR4 was detected in the pro-sensory domain, where it overlapped with SOX2 (white arrow), and non-sensory domains lateral and medial to the pro-sensory domain. (B) At E17, LGR4 (green) was detected in IHCs and OHCs (asterisks) and their surrounding supporting cells. Hair cells were labeled with phalloidin (F-actin) (red). (C) At P1, LGR4 expression was observed in the sensory epithelium (arrowhead) and spiral ganglion cells (arrow), from which projected TUJ1-positive nerves (red). (D) At P7, LGR4 was observed in hair cells (asterisks) that co-expressed Prestin (PRES) and in the surrounding supporting cells: Deiters’ cells (arrowheads) and IPCs and OPCs. (E–G) At P21, LGR4 was down-regulated in the OHCs and pillar cells, but was still expressed in the IHCs (arrowhead), Deiters’ cells (arrows), which were also positive for acetylated tubulin (ACTBA), and phalangeal processes of Deiters’ cells (arrowheads). LGR4 signals were also observed in the spiral ganglion cells. (H) At P42, LGR4 was detected in Deiters’ cells and their phalangeal processes. Cell nuclei were counterstained with DAPI (blue), asterisks indicate hair cells, scale bars indicate 20 μm in the picture (B,D,E,G,H) and 50 μm in (A,C,F) . (I,J) Cochlear cryosections from Lgr4-LacZ mice at the age of P21 were stained for β-galactosidase activity. Expression was seen in Deiters’ cells (black arrow) and their phalangeal processes (gray arrow), IHCs (black arrowhead), and spiral ganglion cells.

    Article Snippet: The following primary antibodies were used: anti-myosin7a (1:700; rabbit, Proteus Bioscience, 25-6790), anti-myosin7a (1:200; mouse, Developmental Studies Hybridoma Bank, 138-1-c), anti-SOX2 (1:200; goat, Santa Cruz Biotechnology, s-17320), anti-SOX2 (1:50, mouse, Millipore, MAB4343), anti-SOX2 (1:250, BD Pharmingen, 561469) anti-acetylated tubulin (ACTBA) (1:50; mouse, Sigma–Aldrich, T7451), anti-prestin (1:700; goat, Santa Cruz Biotechnology, N-20, sc-22692), anti-LGR4 (1:200; rabbit, Sigma–Aldrich, HPA030267, test of the antibody specificity shown in Supplementary Figure ), anti-jagged1 (JAG1) (1:50; goat, Santa Cruz Biotechnology, C-20, sc-6011), anti-cyclinD1 (CCND1) (1:100; rabbit, Thermo Fisher Scientific, SP4, MA5-14512), anti-β-tubulin class III (TUJ1, 1:200; mouse, Covance, MMS-435P), anti-p75 NGF receptor (1:400; rabbit, Sigma–Aldrich, N3908), anti-MYO6 (1:300, Abcam, ab11096).

    Techniques: Staining, Labeling, Expressing, Mouse Assay, Activity Assay

    Proliferation in the cochleae of E14.5 Lgr5-eGFP embryos. (A–B′′) SOX2-positive pro-sensory cells (green, arrow) did not incorporate EdU (red). Cell nuclei were counterstained with DAPI (blue), scale bars indicate 25 μm.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: LGR4 and LGR5 Regulate Hair Cell Differentiation in the Sensory Epithelium of the Developing Mouse Cochlea

    doi: 10.3389/fncel.2016.00186

    Figure Lengend Snippet: Proliferation in the cochleae of E14.5 Lgr5-eGFP embryos. (A–B′′) SOX2-positive pro-sensory cells (green, arrow) did not incorporate EdU (red). Cell nuclei were counterstained with DAPI (blue), scale bars indicate 25 μm.

    Article Snippet: The following primary antibodies were used: anti-myosin7a (1:700; rabbit, Proteus Bioscience, 25-6790), anti-myosin7a (1:200; mouse, Developmental Studies Hybridoma Bank, 138-1-c), anti-SOX2 (1:200; goat, Santa Cruz Biotechnology, s-17320), anti-SOX2 (1:50, mouse, Millipore, MAB4343), anti-SOX2 (1:250, BD Pharmingen, 561469) anti-acetylated tubulin (ACTBA) (1:50; mouse, Sigma–Aldrich, T7451), anti-prestin (1:700; goat, Santa Cruz Biotechnology, N-20, sc-22692), anti-LGR4 (1:200; rabbit, Sigma–Aldrich, HPA030267, test of the antibody specificity shown in Supplementary Figure ), anti-jagged1 (JAG1) (1:50; goat, Santa Cruz Biotechnology, C-20, sc-6011), anti-cyclinD1 (CCND1) (1:100; rabbit, Thermo Fisher Scientific, SP4, MA5-14512), anti-β-tubulin class III (TUJ1, 1:200; mouse, Covance, MMS-435P), anti-p75 NGF receptor (1:400; rabbit, Sigma–Aldrich, N3908), anti-MYO6 (1:300, Abcam, ab11096).

    Techniques:

    eNOS-derived NO scavenging significantly impairs the CSC phenotype in human colon cancer cells. a Formation of tumorspheres from HCT-116 and Caco-2 cells pre-treated for 24 hours with c-PTIO (100 μM) with or without DETANONOate or CSNO (100 μM). Representative images 1–2 weeks after seeding are shown. b Formation of organoids from cells pre-treated for 24 hours with c-PTIO (100 μM) with or without NO donors (100 μM). Representative images 1–2 weeks after seeding are shown. c Immunoblot of β-catenin, Bmi1, Notch1, and Sox2 expression in HCT-116 and Caco-2 tumor cells treated for 24 hours with c-PTIO (100 μM) with or without DETANONOate or CSNO (100 μM). d Organoid immunofluorescence of β-catenin from control or Caco-2 organoids treated with c-PTIO (100 μM) for 24 hours. e RT-qPCR of iNOS-induced expression (IL-1β 3 ng/ml, IFN-γ 200 U/ml and TNF-α 75 ng/ml, for 6 hours) from control and iNOS-shRNA expressing HCT-116 cells. f Immunoblot of previously mentioned proteins in iNOS-knockdown HCT-116 cancer cells treated with c-PTIO (100 μM). Stain-free technology was used as loading control in immunoblot experiments. * P

    Journal: BMC Biology

    Article Title: A role for endothelial nitric oxide synthase in intestinal stem cell proliferation and mesenchymal colorectal cancer

    doi: 10.1186/s12915-017-0472-5

    Figure Lengend Snippet: eNOS-derived NO scavenging significantly impairs the CSC phenotype in human colon cancer cells. a Formation of tumorspheres from HCT-116 and Caco-2 cells pre-treated for 24 hours with c-PTIO (100 μM) with or without DETANONOate or CSNO (100 μM). Representative images 1–2 weeks after seeding are shown. b Formation of organoids from cells pre-treated for 24 hours with c-PTIO (100 μM) with or without NO donors (100 μM). Representative images 1–2 weeks after seeding are shown. c Immunoblot of β-catenin, Bmi1, Notch1, and Sox2 expression in HCT-116 and Caco-2 tumor cells treated for 24 hours with c-PTIO (100 μM) with or without DETANONOate or CSNO (100 μM). d Organoid immunofluorescence of β-catenin from control or Caco-2 organoids treated with c-PTIO (100 μM) for 24 hours. e RT-qPCR of iNOS-induced expression (IL-1β 3 ng/ml, IFN-γ 200 U/ml and TNF-α 75 ng/ml, for 6 hours) from control and iNOS-shRNA expressing HCT-116 cells. f Immunoblot of previously mentioned proteins in iNOS-knockdown HCT-116 cancer cells treated with c-PTIO (100 μM). Stain-free technology was used as loading control in immunoblot experiments. * P

    Article Snippet: Primary antibodies recognizing Notch1 (Abcam, Cambridge, UK, 1:2000, lot GR215257-19, RRID AB_881725), Bmi1 (Abcam, 1:5000), β-catenin (BD, 1:2000) and Sox2 (R & D Systems, Minneapolis, MN, USA 1:1000, lot KOY0212101, RRID AB_355110) were used.

    Techniques: Derivative Assay, Expressing, Immunofluorescence, Quantitative RT-PCR, shRNA, Staining

    Ectopic expression of WIP1 reduces the levels of activated p38 and enhances stemness-related protein expression and CSC properties in NSCLC cells. a Western blotting was used to analyze the expression of WIP1, phospho-p38, p38, SOX2, OCT4, NANOG, and ALDH1A1 in H1299 (left panels) and H460 (right panels) cells transduced with a WIP1-overexpressing plasmid (WIP1) or vector control (pLV). Arrows indicate the positions of p38 isoforms. b Western blotting was used to analyze MK2, phospho-MK2 (Thr222), phospho-MK2 (Thr334), HSP27, and phospho-HSP27 (Ser82) in H460 cells transduced with the WIP1-overexpressing plasmid (WIP1) or vector control (pLV). c , d A sphere formation assay was performed with H1299 (top graphs) and H460 (bottom graphs) cells transduced with the WIP1-overexpressing (WIP1) or vector control (pLV) plasmid. Quantifications of sphere sizes ( d ) and numbers ( e ) are shown in bar graphs. The data are presented as the mean ± SD of three independent experiments. * indicates P

    Journal: Signal Transduction and Targeted Therapy

    Article Title: WIP1 promotes cancer stem cell properties by inhibiting p38 MAPK in NSCLC

    doi: 10.1038/s41392-020-0126-x

    Figure Lengend Snippet: Ectopic expression of WIP1 reduces the levels of activated p38 and enhances stemness-related protein expression and CSC properties in NSCLC cells. a Western blotting was used to analyze the expression of WIP1, phospho-p38, p38, SOX2, OCT4, NANOG, and ALDH1A1 in H1299 (left panels) and H460 (right panels) cells transduced with a WIP1-overexpressing plasmid (WIP1) or vector control (pLV). Arrows indicate the positions of p38 isoforms. b Western blotting was used to analyze MK2, phospho-MK2 (Thr222), phospho-MK2 (Thr334), HSP27, and phospho-HSP27 (Ser82) in H460 cells transduced with the WIP1-overexpressing plasmid (WIP1) or vector control (pLV). c , d A sphere formation assay was performed with H1299 (top graphs) and H460 (bottom graphs) cells transduced with the WIP1-overexpressing (WIP1) or vector control (pLV) plasmid. Quantifications of sphere sizes ( d ) and numbers ( e ) are shown in bar graphs. The data are presented as the mean ± SD of three independent experiments. * indicates P

    Article Snippet: The antibodies used in this study included anti-WIP1 (Abcam, USA, ab31270), anti-p38 (Abcam, ab32142), anti-phospho-p38 MAPK (Cell Signaling Technology, USA, #4511), anti-MAPKAPK-2 (Cell Signaling Technology, #3042), anti-phospho-MAPKAPK-2 (Thr222) (Cell Signaling Technology, #3316), anti-phospho-MAPKAPK-2 (Thr334) (Cell Signaling Technology, #3041), anti-HSP27 (Cell Signaling Technology, #2402), anti-phospho-HSP27 (Ser82) (Cell Signaling Technology, #9709), anti-SOX2 (Proteintech, USA, 11064-1), anti-OCT4 (Proteintech, 11263-1), anti-NANOG (Proteintech, 14295-1), anti-ALDH1A1 (Santa Cruz, USA, sc-374149), and anti-β-actin (Santa Cruz, sc-47778).

    Techniques: Expressing, Western Blot, Transduction, Plasmid Preparation, Tube Formation Assay

    Differentiation of ADSCs into cells of neuronal lineage. ( A ) Semi-quantitative RT-PCR analysis for the expression of neurogenic lineage-specific genes sex determining region Y-box 2 (Sox-2), fascin, nestin, tubulinβIII. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used to normalize cDNA concentration for each sample set. Graphs and SD are from nine patients analyzed. The expression of Sox2 ( B ) and nestin ( C ) was also verified by immunofluorescence analysis in several independent experiments. Nuclei were labelled with bisbenzidine (DAPI). Original magnification 40x. Ctrl, undifferentiated control; CL, collagenase; FW, fat wash; LF, liquid fraction.

    Journal: Scientific Reports

    Article Title: Maximizing non-enzymatic methods for harvesting adipose-derived stem from lipoaspirate: technical considerations and clinical implications for regenerative surgery

    doi: 10.1038/s41598-017-10710-6

    Figure Lengend Snippet: Differentiation of ADSCs into cells of neuronal lineage. ( A ) Semi-quantitative RT-PCR analysis for the expression of neurogenic lineage-specific genes sex determining region Y-box 2 (Sox-2), fascin, nestin, tubulinβIII. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used to normalize cDNA concentration for each sample set. Graphs and SD are from nine patients analyzed. The expression of Sox2 ( B ) and nestin ( C ) was also verified by immunofluorescence analysis in several independent experiments. Nuclei were labelled with bisbenzidine (DAPI). Original magnification 40x. Ctrl, undifferentiated control; CL, collagenase; FW, fat wash; LF, liquid fraction.

    Article Snippet: Cells were then incubated with the following primary antibodies: anti-PPARγ, anti-SREBP1, anti-RUNX2, anti-Mitf, anti-Tyrosinase, anti-TRP2 (Santa Cruz Biotechnology, USA), anti-Sox2, anti-Nestin antibody (Merck Millipore, Germany), for 1 hour.

    Techniques: Quantitative RT-PCR, Expressing, Concentration Assay, Immunofluorescence