anti-sox2 Search Results


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  • 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 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    95
    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: 95/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    99
    Abcam anti sox2
    Mean ± SEM of Notch1 ( A ), sex determining region Y-box 2 or <t>SOX2</t> ( B ), nestin ( C ) and doublecortin or DCX ( D ) protein expressions in the hippocampus were assessed by immunoblotting. * p
    Anti Sox2, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1161 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    BioLegend pe anti sox2
    Mean ± SEM of Notch1 ( A ), sex determining region Y-box 2 or <t>SOX2</t> ( B ), nestin ( C ) and doublecortin or DCX ( D ) protein expressions in the hippocampus were assessed by immunoblotting. * 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
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    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
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    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
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    Abcam anti sox2 antibody epr3131
    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 ).
    Anti Sox2 Antibody Epr3131, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    Abcam mouse anti sox2
    The number of basal progenitors and mitotic capability of apical progenitors depends on Ythdf2. a Immunostaining of E12.5 and E14.5 sagittal sections with Tbr2 ( green ) and <t>Sox2</t> ( red ) antibodies in wild type, Ythdf2 +/− , and Ythdf2 −/− embryos. VZ ventricular zone, SVZ subventricular zone, IZ intermediate zone, CP cortical plate. Nuclei were counterstained with DAPI. b Percentage of Tbr2 + cells over Tbr2 + /Sox2 + at E12.5 and E14.5. Error bars represent mean ± standard deviation, n = 3 biological and 3 technical replicates. Scale bars, 20 μm. c Immunostaining of E12.5 and E14.5 sagittal sections with Phh3 ( green ) and Sox2 ( red ) antibodies in wild type, Ythdf2 +/− , and Ythdf2 −/− embryos. Nuclei were counterstained with DAPI. d Number of Phh3 + cells per 400 μm of the cortical wall at E12.5/E14.5 from c . Error bars represent mean ± standard deviation, n = 3 biological and 3 technical replicates. * P
    Mouse Anti Sox2, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    86
    BioLegend purified 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.
    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
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    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
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    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
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    90
    Biogenex sox2
    Figure 6. Effect of DPPA3 knockdown in bovine oocytes on cell number and allocation of resulting blastocysts. Representative images of embryos immunostained for <t>SOX2</t> and CDX2 as markers of ICM and TE cells, respectively. Nuclei were counterstained
    Sox2, supplied by Biogenex, used in various techniques. Bioz Stars score: 90/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson sox2
    drNPC cultured in the rSS-PCL scaffold with or without the PRP-based liquid matrix (SPRPix matrix). ( A–C ) SPRPix (PRP + rSS-PCL scaffold); ( A ) Nestin (green), auto-fluorescence of the SPRPix matrix (red). ( B ) Spontaneous neuronal and glial differentiation revealed by MAP2 (green) and GFAP (red). ( C ) Intense proliferation of <t>SOX2-positve</t> (green) drNPCs, and βIII-tubulin-positive (red) neuronal progenitors. ( D ) The same concentration of drNPC cells seeded on rSS-PCL scaffold only. Nestin (green), βIII-tubulin (red). ( E , F ) high magnification examples of drNPCs cultured on rSS-PCL alone ( E ) or on SPRPix ( F ). Bar = 50 μm.
    Sox2, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    scWesterns track NSC lineage commitment during differentiation. ( a ) Immunocytochemistry micrographs of mixed NSC differentiation cultures at days 0 and 6 for stem cell (nestin, NEST; SOX2) and differentiation markers (βIII-tubulin, βIIITUB; glial fibrillary acidic protein, GFAP). Scale bar: 50 µm. ( b ) Micrographs of NSCs in scWestern microwells, fixed and stained as in ( a ). ( c ) Confocal images of fixed and stained stem (NEST+, SOX2+), neuron (βIIITUB+) and astrocyte (GFAP+) cells in a rhodamine-tagged gel (GEL). Scale bar: 10 µm. ( d ) Inverted fluorescence micrographs of scWesterns. SOX2 (Alexa Fluor 555-labeled secondary antibody) and NEST (α and β isoforms, Alexa Fluor 488−) were co-probed in separate blocks as GFAP (Alexa Fluor 555−) and βIIITUB (Alexa Fluor 488−); both block sets were stripped and co-probed for β-tubulin (βTUB, Alexa Fluor 555−) and EGFP (Alexa Fluor 488−). Image sets from each day are the same separation, except EGFP images, which are from the same microwell array row as the corresponding image set. ( e ) Cropped conventional western blots at differentiation days 0 and 6. Full-length blots are presented in Supplementary Fig. 29 . ( f ) scWestern fluorescence normalized by βTUB (arbitrary units). Note arcsinh-transformed scales. Spatial density indicated by contours. scWestern blot NSC marker sample sizes, n = 189, 353, 175, and 274 for time-points at 0, 1, 2, and 6 days, respectively. Differentiation marker sample sizes, n = 178, 253, 303, 280 for time-points at 0, 4, 5, and 6 days, respectively. Data are one of two biological replicates performed.

    Journal: Nature methods

    Article Title: Single-cell western blotting

    doi: 10.1038/nmeth.2992

    Figure Lengend Snippet: scWesterns track NSC lineage commitment during differentiation. ( a ) Immunocytochemistry micrographs of mixed NSC differentiation cultures at days 0 and 6 for stem cell (nestin, NEST; SOX2) and differentiation markers (βIII-tubulin, βIIITUB; glial fibrillary acidic protein, GFAP). Scale bar: 50 µm. ( b ) Micrographs of NSCs in scWestern microwells, fixed and stained as in ( a ). ( c ) Confocal images of fixed and stained stem (NEST+, SOX2+), neuron (βIIITUB+) and astrocyte (GFAP+) cells in a rhodamine-tagged gel (GEL). Scale bar: 10 µm. ( d ) Inverted fluorescence micrographs of scWesterns. SOX2 (Alexa Fluor 555-labeled secondary antibody) and NEST (α and β isoforms, Alexa Fluor 488−) were co-probed in separate blocks as GFAP (Alexa Fluor 555−) and βIIITUB (Alexa Fluor 488−); both block sets were stripped and co-probed for β-tubulin (βTUB, Alexa Fluor 555−) and EGFP (Alexa Fluor 488−). Image sets from each day are the same separation, except EGFP images, which are from the same microwell array row as the corresponding image set. ( e ) Cropped conventional western blots at differentiation days 0 and 6. Full-length blots are presented in Supplementary Fig. 29 . ( f ) scWestern fluorescence normalized by βTUB (arbitrary units). Note arcsinh-transformed scales. Spatial density indicated by contours. scWestern blot NSC marker sample sizes, n = 189, 353, 175, and 274 for time-points at 0, 1, 2, and 6 days, respectively. Differentiation marker sample sizes, n = 178, 253, 303, 280 for time-points at 0, 4, 5, and 6 days, respectively. Data are one of two biological replicates performed.

    Article Snippet: Blots were probed overnight with primary antibodies in the same blocking buffer: rabbit anti-pERK1/2 (1:2,000; see scWestern probing, imaging, and stripping . for product details), rabbit anti-ERK1/2 (1:1,000), mouse anti-ERK1/2 (“ERK #2”, 1:1,000), mouse anti-MASH1 (1:1,000), mouse anti-SRC (1:1,000), goat anti-EphB4 (1:1,000), goat anti-GFP (1:1,000), rabbit anti-pMEK1/2 (1:1,000), rabbit anti-MEK1/2 (1:1,000), goat anti-SOX2 (1:500), mouse anti-nestin (1:1,000), mouse anti-nestin (“NEST #2”, clone: rat-401, 1:2,000), goat anti-GFAP (1:1,000), mouse anti-βIII-tubulin (1:2,000), rabbit anti-β-tubulin (1:500); followed by 1 hour incubation with appropriate horseradish peroxidase-conjugated secondary antibodies: mouse anti-goat HRP (1:5,000, 31400), goat anti-mouse HRP (1:10,000, 32430), goat anti-rabbit HRP (1:10,000, 32460), all from ThermoFisher Scientific.

    Techniques: Immunocytochemistry, Staining, Fluorescence, Labeling, Blocking Assay, Western Blot, Transformation Assay, Marker

    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:

    Mean ± SEM of Notch1 ( A ), sex determining region Y-box 2 or SOX2 ( B ), nestin ( C ) and doublecortin or DCX ( D ) protein expressions in the hippocampus were assessed by immunoblotting. * p

    Journal: Nutrients

    Article Title: Neuroprotective Properties of Asiatic Acid against 5-Fluorouracil Chemotherapy in the Hippocampus in an Adult Rat Model

    doi: 10.3390/nu10081053

    Figure Lengend Snippet: Mean ± SEM of Notch1 ( A ), sex determining region Y-box 2 or SOX2 ( B ), nestin ( C ) and doublecortin or DCX ( D ) protein expressions in the hippocampus were assessed by immunoblotting. * p

    Article Snippet: The blots were incubated overnight at 4 °C with primary antibodies as follows: polyclonal anti-Notch1 (Santa Cruz Biotechnology, Dallas, TX, USA; sc-6014; 1:100), polyclonal anti-DCX (Santa Cruz Biotechnology, Dallas, TX, USA; sc-8066; 1:150), monoclonal anti-nestin (Merck Millipore, MA, USA; MAB353; 1:1000), polyclone al anti-SOX2 (Abcam, Cambridge, UK; ab97959; 1:2000), polyclonal anti-Nrf2 (Abcam, Cambridge, UK; ab31163; 1:1000), and monoclonal mouse anti-GAPDH antibody (Abcam, Cambridge, UK; ab8245; 1:20,000).

    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

    Overexpression of Cdh1 phenocopies the differentiation defect of Mcph1 ‐del neuroprogenitors in vivo FLAG‐Cdc25A was co‐transfected with HA‐EV, or HA‐βTrCP2 or HA‐βTrCP2‐ΔN into 293T cells. The Cdc25A protein level was examined by an anti‐FLAG antibody. β‐actin was used to control the loading. Left panel: Representative images of Sox2 staining at E15.5 wild‐type (control) or Mcph1 ‐del brain section 1 day after IUE with GFP‐EV or GFP‐Cdh1 vectors. n : number of embryos analyzed. Scale bar: 50 μm. Right panel: Quantitation of GFP + Sox2 − cells per total GFP + cells. For each embryo, at least 2,000 GFP + cells were counted. Error bars represent SD. Statistical analysis was performed using one‐way ANOVA. *** P

    Journal: The EMBO Journal

    Article Title: The E3 ubiquitin ligase APC/ CCdh1 degrades MCPH1 after MCPH1‐βTr CP2‐Cdc25A‐mediated mitotic entry to ensure neurogenesis

    doi: 10.15252/embj.201694443

    Figure Lengend Snippet: Overexpression of Cdh1 phenocopies the differentiation defect of Mcph1 ‐del neuroprogenitors in vivo FLAG‐Cdc25A was co‐transfected with HA‐EV, or HA‐βTrCP2 or HA‐βTrCP2‐ΔN into 293T cells. The Cdc25A protein level was examined by an anti‐FLAG antibody. β‐actin was used to control the loading. Left panel: Representative images of Sox2 staining at E15.5 wild‐type (control) or Mcph1 ‐del brain section 1 day after IUE with GFP‐EV or GFP‐Cdh1 vectors. n : number of embryos analyzed. Scale bar: 50 μm. Right panel: Quantitation of GFP + Sox2 − cells per total GFP + cells. For each embryo, at least 2,000 GFP + cells were counted. Error bars represent SD. Statistical analysis was performed using one‐way ANOVA. *** P

    Article Snippet: The sections were incubated with the rabbit anti‐Ki67 (1:200, Neomarkers, Waltham, MA, USA) or rabbit anti‐Sox2 (1:200, # ab97959, Abcam), or with rabbit anti‐pH3‐S10 (1:500, #A301‐844A, Bethyl), at 4°C overnight and washed with PBS.

    Techniques: Over Expression, In Vivo, Transfection, Staining, Quantitation Assay

    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

    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

    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

    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

    The number of basal progenitors and mitotic capability of apical progenitors depends on Ythdf2. a Immunostaining of E12.5 and E14.5 sagittal sections with Tbr2 ( green ) and Sox2 ( red ) antibodies in wild type, Ythdf2 +/− , and Ythdf2 −/− embryos. VZ ventricular zone, SVZ subventricular zone, IZ intermediate zone, CP cortical plate. Nuclei were counterstained with DAPI. b Percentage of Tbr2 + cells over Tbr2 + /Sox2 + at E12.5 and E14.5. Error bars represent mean ± standard deviation, n = 3 biological and 3 technical replicates. Scale bars, 20 μm. c Immunostaining of E12.5 and E14.5 sagittal sections with Phh3 ( green ) and Sox2 ( red ) antibodies in wild type, Ythdf2 +/− , and Ythdf2 −/− embryos. Nuclei were counterstained with DAPI. d Number of Phh3 + cells per 400 μm of the cortical wall at E12.5/E14.5 from c . Error bars represent mean ± standard deviation, n = 3 biological and 3 technical replicates. * P

    Journal: Genome Biology

    Article Title: Ythdf2-mediated m6A mRNA clearance modulates neural development in mice

    doi: 10.1186/s13059-018-1436-y

    Figure Lengend Snippet: The number of basal progenitors and mitotic capability of apical progenitors depends on Ythdf2. a Immunostaining of E12.5 and E14.5 sagittal sections with Tbr2 ( green ) and Sox2 ( red ) antibodies in wild type, Ythdf2 +/− , and Ythdf2 −/− embryos. VZ ventricular zone, SVZ subventricular zone, IZ intermediate zone, CP cortical plate. Nuclei were counterstained with DAPI. b Percentage of Tbr2 + cells over Tbr2 + /Sox2 + at E12.5 and E14.5. Error bars represent mean ± standard deviation, n = 3 biological and 3 technical replicates. Scale bars, 20 μm. c Immunostaining of E12.5 and E14.5 sagittal sections with Phh3 ( green ) and Sox2 ( red ) antibodies in wild type, Ythdf2 +/− , and Ythdf2 −/− embryos. Nuclei were counterstained with DAPI. d Number of Phh3 + cells per 400 μm of the cortical wall at E12.5/E14.5 from c . Error bars represent mean ± standard deviation, n = 3 biological and 3 technical replicates. * P

    Article Snippet: Antibodies The following antibodies were used at the appropriate dilutions: mouse anti-Map2 (M4403, Sigma), rabbit anti-Gfap (Z0334, DAKO), mouse anti-Tuj1 (MAB1195, R & D Systems), rabbit anti-s100-β (ab52642, Abcam), rabbit anti-Dcx (ab18723, Abcam), rabbit anti-Tbr2 (ab23345, Abcam), rabbit anti-phospho-Histone H3 (PHH3; Ser10; 06–570, Millipore), mouse anti-Sox2 (ab79351, Abcam), mouse anti-Nestin (MAB353, Millipore), rabbit anti-Ythdf2 (RN123PW, MBL), mouse anti-anti-β-actin (A1978, Sigma).

    Techniques: Immunostaining, Standard Deviation

    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

    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

    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

    Figure 6. Effect of DPPA3 knockdown in bovine oocytes on cell number and allocation of resulting blastocysts. Representative images of embryos immunostained for SOX2 and CDX2 as markers of ICM and TE cells, respectively. Nuclei were counterstained

    Journal: Epigenetics

    Article Title: DPPA3 prevents cytosine hydroxymethylation of the maternal pronucleus and is required for normal development in bovine embryos

    doi: 10.4161/epi.32087

    Figure Lengend Snippet: Figure 6. Effect of DPPA3 knockdown in bovine oocytes on cell number and allocation of resulting blastocysts. Representative images of embryos immunostained for SOX2 and CDX2 as markers of ICM and TE cells, respectively. Nuclei were counterstained

    Article Snippet: Antibodies used were rabbit polyclonal against SOX2 (BioGenex, AN579) and mouse monoclonal against CDX2 (BioGenex, AM392).

    Techniques:

    drNPC cultured in the rSS-PCL scaffold with or without the PRP-based liquid matrix (SPRPix matrix). ( A–C ) SPRPix (PRP + rSS-PCL scaffold); ( A ) Nestin (green), auto-fluorescence of the SPRPix matrix (red). ( B ) Spontaneous neuronal and glial differentiation revealed by MAP2 (green) and GFAP (red). ( C ) Intense proliferation of SOX2-positve (green) drNPCs, and βIII-tubulin-positive (red) neuronal progenitors. ( D ) The same concentration of drNPC cells seeded on rSS-PCL scaffold only. Nestin (green), βIII-tubulin (red). ( E , F ) high magnification examples of drNPCs cultured on rSS-PCL alone ( E ) or on SPRPix ( F ). Bar = 50 μm.

    Journal: Scientific Reports

    Article Title: Tissue Engineered Neural Constructs Composed of Neural Precursor Cells, Recombinant Spidroin and PRP for Neural Tissue Regeneration

    doi: 10.1038/s41598-019-39341-9

    Figure Lengend Snippet: drNPC cultured in the rSS-PCL scaffold with or without the PRP-based liquid matrix (SPRPix matrix). ( A–C ) SPRPix (PRP + rSS-PCL scaffold); ( A ) Nestin (green), auto-fluorescence of the SPRPix matrix (red). ( B ) Spontaneous neuronal and glial differentiation revealed by MAP2 (green) and GFAP (red). ( C ) Intense proliferation of SOX2-positve (green) drNPCs, and βIII-tubulin-positive (red) neuronal progenitors. ( D ) The same concentration of drNPC cells seeded on rSS-PCL scaffold only. Nestin (green), βIII-tubulin (red). ( E , F ) high magnification examples of drNPCs cultured on rSS-PCL alone ( E ) or on SPRPix ( F ). Bar = 50 μm.

    Article Snippet: The spinal cord and brain sections were stained using antibodies to Nestin (R & D), SOX2 (BD Biosciences), βIII-tubulin (R & D), MAP2 (Sigma-Aldrich), GFAP (DAKO), NF-200 (Sigma-Aldrich,) macroH2A.1 (ab183041, Abcam), human-specific anti-mitochondria antibody (ab92824, Abcam), and STEM-121 (Y40410, TakaraBio), anti- CD3, CD31 and CD68 (Roche Diagnostics) (all as 5 μg/ml solutions in PBS).

    Techniques: Cell Culture, Fluorescence, Concentration Assay

    Immunophenotyping of drNPC. ( A ) Cytofluorometry of drNPC marker expression. 1. Negative control (isotypic immunoglobulins) 2. βIII-tubulin 3. MAP2 and 4. SOX2. ( B – F ) Immunocytochemical analysis for different markers ( B ) anti-βIII-tubulin. Bar = 100 µm ( C , D ) Undifferentiated drNPC coexpress βIII-tubulin (green) and GFAP (red) ( C ), as well as Nestin (green) and SOX2 (red) ( D ). ( E , F) Differentiation of drNPC: MAP2-positive neurons (green) and GFAP-positive astrocytes (red) ( E ) NF200-positve (red) and SOX2 negative neurons ( F ). In panels (B–F), the blue channel corresponds to the Hoechst stained cell nuclei. In panels С–F Bar = 50 µm. Laser scanning confocal microscopy using Nikon A1 device.

    Journal: Scientific Reports

    Article Title: Tissue Engineered Neural Constructs Composed of Neural Precursor Cells, Recombinant Spidroin and PRP for Neural Tissue Regeneration

    doi: 10.1038/s41598-019-39341-9

    Figure Lengend Snippet: Immunophenotyping of drNPC. ( A ) Cytofluorometry of drNPC marker expression. 1. Negative control (isotypic immunoglobulins) 2. βIII-tubulin 3. MAP2 and 4. SOX2. ( B – F ) Immunocytochemical analysis for different markers ( B ) anti-βIII-tubulin. Bar = 100 µm ( C , D ) Undifferentiated drNPC coexpress βIII-tubulin (green) and GFAP (red) ( C ), as well as Nestin (green) and SOX2 (red) ( D ). ( E , F) Differentiation of drNPC: MAP2-positive neurons (green) and GFAP-positive astrocytes (red) ( E ) NF200-positve (red) and SOX2 negative neurons ( F ). In panels (B–F), the blue channel corresponds to the Hoechst stained cell nuclei. In panels С–F Bar = 50 µm. Laser scanning confocal microscopy using Nikon A1 device.

    Article Snippet: The spinal cord and brain sections were stained using antibodies to Nestin (R & D), SOX2 (BD Biosciences), βIII-tubulin (R & D), MAP2 (Sigma-Aldrich), GFAP (DAKO), NF-200 (Sigma-Aldrich,) macroH2A.1 (ab183041, Abcam), human-specific anti-mitochondria antibody (ab92824, Abcam), and STEM-121 (Y40410, TakaraBio), anti- CD3, CD31 and CD68 (Roche Diagnostics) (all as 5 μg/ml solutions in PBS).

    Techniques: Marker, Expressing, Negative Control, Staining, Confocal Microscopy