Journal: The Journal of Neuroscience

Article Title: Semaphorin-5B Controls Spiral Ganglion Neuron Branch Refinement during Development

doi: 10.1523/JNEUROSCI.0113-19.2019

Figure Lengend Snippet: Primary antibodies and tools/software used in this study

Article Snippet: Antibodies used in this study include the following ( ): mouse-anti-Tuj1 (Covance, MMS-435P; 1:500), rabbit-anti-GAP43 (Millipore, AB5220; 1:1000), rabbit-anti-dsRed (Clontech, 632496; 1:2000), rabbit-anti-MyosinVI (MyoVI; Proteus Biosciences, 25-6791; 1:1000), goat-anti-Sox2 (R&D Systems, AF2018; 1:400), goat-anti-MyoVI ( Coate et al., 2015 ), goat-anti-PlexinA1 (R&D Systems, AF4309; 1:100), goat-anti-PlexinA3 (R&D Systems, AF4075; 1:100), and chicken-anti-neurofilament (NF200; Aves Labs, NFH; 1:2000). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Product Category Source/vendor (catalog #) RRID Mouse α-Tuj1 (TUBB3) Antibody BioLegend (801213) AB_2728521 Rabbit α-Gap43 Antibody Millipore (AB5220) AB_2107282 Rabbit α-dsRed Antibody Takara Bio (632496) AB_10013483 Rabbit α-Myo6 Antibody Proteus Biosciences (25-6791) AB_10013626 Goat α-Sox2 Antibody R&D Systems (AF2018) AB_355110 Goat α-Myo6 Antibody Section on Developmental Neuroscience; NIDCD AB_2783873 Goat α-PlxnA1 Antibody R&D Systems (AF4309) AB_10645644 Goat α-PlxnA3 Antibody R&D Systems (AF4075) AB_2166395 Chicken α-NF200 Antibody Aves Labs (NFH) AB_2313552 Human IgG-Fc protein Antibody Jackson ImmunoResearch Labs (009-000-008) AB_2337046 Bitplane Imaris Tools/Software Oxford Instruments SCR_007370 GraphPad Prism Tools/Software GraphPad SCR_002798 Fiji Tools/Software ImageJ SCR_002285 Open in a separate window Primary antibodies and tools/software used in this study

Techniques:

Journal: BMC Neuroscience

Article Title: Sox2 acts as a transcriptional repressor in neural stem cells

doi: 10.1186/1471-2202-15-95

Figure Lengend Snippet: Potential target genes of Sox2-Grg interaction. (A) Scheme of the experiment using microarray analysis to identify genes affected by Sox2 in human NSCs. (B) The relative percentage of genes affected by Sox2 as compared to control cells transfected by GFP alone (only differences of above 1.5-fold were included in both comparisons). (C) Graph representing the most activated (bars to right of midline) and repressed (bars to left of midline) genes. (D) The genes most strongly repressed by Sox2 in our study that were also suggested to be repressed by Sox2 in mouse ES cells (Masui et al. ), or in human ES cells (Greber et al. ). Final column indicates the study in which these were also shown to be direct targets of Sox2.

Article Snippet: Blocking was carried out with 10% BSA in 0.1% Triton X100/PBS for 30 min. Primary antibodies (MYC antibody (9E10), Sox2 antibody (R&D, MAB2018), MAP-2 antibody (Abcam)) and the fluorescent-conjugated secondary antibodies were incubated at room temperature for 1 h. Staining was observed after mounting in mounting medium for fluorescence with DAPI (Vector).

Techniques: Microarray, Control, Transfection

Journal: BMC Neuroscience

Article Title: Sox2 acts as a transcriptional repressor in neural stem cells

doi: 10.1186/1471-2202-15-95

Figure Lengend Snippet: Sox2 interacts with Grg proteins. (A) subcellular localization of overexpressed MYC-tagged Grg3 or Grg5 in COS-7 cells; immunostaining with anti-Sox2 or anti-MYC antibody. Nuclei were stained with DAPI. (B) Co-transfection of Sox2 with either Grg3 or Grg5, caused the subcellular distribution of the Grgs (red) to alter to reflect that of Sox2 (green). (C) Quantification of cytoplasmic Grg5 using Image J software shows effect of Sox2 on Grg5 subcellular distribution was highly significant (p = 0.0023). (D) Immunoprecipitation of overexpressed MYC-tagged Grg3 or Grg5 in human neural stem cells, after treatment with DPS crosslinker, resulted in co-precipitation of endogenous Sox2. Left hand blot was probed for the Grgs using anti-MYC, right hand panel probed for Sox2 using annti-Sox2 antibody. WCE - whole cell extract. Size markers in lanes 3 and 9, sizes shown to right of image. Scale bar in for panel A and B approximately 10 μm.

Article Snippet: Blocking was carried out with 10% BSA in 0.1% Triton X100/PBS for 30 min. Primary antibodies (MYC antibody (9E10), Sox2 antibody (R&D, MAB2018), MAP-2 antibody (Abcam)) and the fluorescent-conjugated secondary antibodies were incubated at room temperature for 1 h. Staining was observed after mounting in mounting medium for fluorescence with DAPI (Vector).

Techniques: Immunostaining, Staining, Cotransfection, Software, Immunoprecipitation

Journal: BMC Neuroscience

Article Title: Sox2 acts as a transcriptional repressor in neural stem cells

doi: 10.1186/1471-2202-15-95

Figure Lengend Snippet: Grgs repress Sox2 transcriptional activation activity. Co-transfected Sox2 caused an approximately 2-fold increase in luciferase activity from the 3xSX promoter (A) or 25-fold increase from the REX promoter (B) in Cos-7 cells, but this increase was significantly inhibited when Grg3 or Grg5 were co-transfected (A,B) . (C) Co-expression of Sox2 with the GFAP-luciferase reporter construct in P19 cells caused a 2-fold decrease in luciferase activity compared to vector alone and the presence of either Grg3 or Grg5 increased this repression (although the difference was only significant in the presence of Grg3). (D) Cotransfected Grg3 or Grg5 caused no significant difference from controls (vector) in luciferase activity from various promoters in P19 cells. *p = <0.05; **p = <0.01.

Article Snippet: Blocking was carried out with 10% BSA in 0.1% Triton X100/PBS for 30 min. Primary antibodies (MYC antibody (9E10), Sox2 antibody (R&D, MAB2018), MAP-2 antibody (Abcam)) and the fluorescent-conjugated secondary antibodies were incubated at room temperature for 1 h. Staining was observed after mounting in mounting medium for fluorescence with DAPI (Vector).

Techniques: Activation Assay, Activity Assay, Transfection, Luciferase, Expressing, Construct, Plasmid Preparation

Journal: BMC Neuroscience

Article Title: Sox2 acts as a transcriptional repressor in neural stem cells

doi: 10.1186/1471-2202-15-95

Figure Lengend Snippet: Mapping the Grg interacting region of Sox2. Schematic representation of C-terminal deletions and their effect on the ability of Sox2 to cause MYC-tagged Grg5 to translocate to the nucleus. The reduction for d1, d2, and d3 was much greater (p < 0.001) than for d4, d5 and d6 (P = 0.01-0.05).

Article Snippet: Blocking was carried out with 10% BSA in 0.1% Triton X100/PBS for 30 min. Primary antibodies (MYC antibody (9E10), Sox2 antibody (R&D, MAB2018), MAP-2 antibody (Abcam)) and the fluorescent-conjugated secondary antibodies were incubated at room temperature for 1 h. Staining was observed after mounting in mounting medium for fluorescence with DAPI (Vector).

Techniques:

Journal: BMC Neuroscience

Article Title: Sox2 acts as a transcriptional repressor in neural stem cells

doi: 10.1186/1471-2202-15-95

Figure Lengend Snippet: The Sox2 M203–209 mutant does not interact with Grgs and fails to repress a reporter construct. (A) Unlike WT Sox2, the Sox2 M203–209 mutant was unable to translocate Grg5 into the nucleus of co-transfected Cos-7 cells suggesting loss of interaction. Counting the proportion of cells exhibiting altered distribution of MYC-Grg in cells co-expressing Sox2 revealed a highly significant reduction of about 50% (p = 0.0002). (B) Immune precipitation of MYC-Grg5 co-transfected with WT Sox2 or with the Sox2 M203–209 mutant, showing that the mutant failed to be co-precipitated with Grg5. (C) In an in vitro pull down assay, immunoprecipitation of GST-fused Grg1 or Grg5 reproducibly pulled-down 4–5 fold less Sox2 M203–209 than it did WT Sox2 (D) The Sox2 M203–209 mutant retained the ability to activate luciferase expression driven by the 3xSX promoter in Cos-7 cells (the difference between this and the level induced by WT Sox2 was not statistically significant). (E) The ability of co-transfected Sox2 M203–209 mutant to induce luciferase expression driven by the REX promoter in P19 cells was very similar to that induced by WT Sox2. (F) Unlike WT Sox2, the Sox2 M203–209 mutant failed to repress luciferase expression driven from the GFAP promoter, and the addition of Grg3 or Grg5 had no effect on this. *p = <0.05; **p = <0.01; ns – not significant Scale bar approximately 20 μm.

Article Snippet: Blocking was carried out with 10% BSA in 0.1% Triton X100/PBS for 30 min. Primary antibodies (MYC antibody (9E10), Sox2 antibody (R&D, MAB2018), MAP-2 antibody (Abcam)) and the fluorescent-conjugated secondary antibodies were incubated at room temperature for 1 h. Staining was observed after mounting in mounting medium for fluorescence with DAPI (Vector).

Techniques: Mutagenesis, Construct, Transfection, Expressing, In Vitro, Pull Down Assay, Immunoprecipitation, Luciferase

Journal: BMC Neuroscience

Article Title: Sox2 acts as a transcriptional repressor in neural stem cells

doi: 10.1186/1471-2202-15-95

Figure Lengend Snippet: A Grg binding mutant of Sox2 fails to inhibit NSC differentiation. (A) Validation of the repression of NNMT, SOX8, SERPINE1, HELLS, IGFBP2, IGFBP3 and GFAP expression using qPCR. P-values shown for 5 PCR replicates *p = <0.05; **p = <0.01. (B) Graph showing the percentage of NSCs extending MAP2 positive processes after 5 days in differentiation medium. This was inhibited by WY Sox2 but not by the Sox2 M203–209 mutant. (C) representative examples of cells after transfection; white lines indicate length of cellular extensions. (D) Model for mechanism of Sox2 action during NSC development.

Article Snippet: Blocking was carried out with 10% BSA in 0.1% Triton X100/PBS for 30 min. Primary antibodies (MYC antibody (9E10), Sox2 antibody (R&D, MAB2018), MAP-2 antibody (Abcam)) and the fluorescent-conjugated secondary antibodies were incubated at room temperature for 1 h. Staining was observed after mounting in mounting medium for fluorescence with DAPI (Vector).

Techniques: Binding Assay, Mutagenesis, Biomarker Discovery, Expressing, Transfection

Journal: Development (Cambridge, England)

Article Title: Sox2 and Lef-1 interact with Pitx2 to regulate incisor development and stem cell renewal.

doi: 10.1242/dev.138883

Figure Lengend Snippet: Fig. 1. Loss of Sox2 in murine embryos causes tooth arrest. (A) Schematic profile of the adult mouse incisor (taken from Biehs et al., 2013 with modifications). The mouse lower incisor comprises a major portion of the mandible. Boxed region depicts the LaCL containing progenitor cells in the stellate reticulum (SR) and the inner (IEE) and outer (OEE) enamel epithelium. Ameloblasts (Am) only appear on the labial side and cause asymmetrical deposition of enamel on labial surface. Dentin (De), produced by odontoblasts (Od), is deposited on both labial and lingual side. DM, dental mesenchyme; En, enamel; SI, stratum intermedium; TA, transient amplifying. (B-G) Hematoxylin and Eosin staining of E12.5, E14.5 and E16.5 embryos (sagittal sections). At E12.5, the tooth bud in Sox2cKO

Article Snippet: DNA/protein complexes were immunoprecipitated with 5 μg Pitx2 antibody (Pitx2 antibody, Capra Sciences, PA-1023) or Sox2 antibody (R&D Systems, AF2018).

Techniques: Produced, Staining

Journal: Development (Cambridge, England)

Article Title: Sox2 and Lef-1 interact with Pitx2 to regulate incisor development and stem cell renewal.

doi: 10.1242/dev.138883

Figure Lengend Snippet: Fig. 2. Deletion of Sox2 in adult mice inhibits incisor regeneration. (A) Tamoxifen treatment, tooth length reduction and histological analysis time line in Sox2F/F control and Rosa26Cre-ERT/Sox2F/F mice. (B) Images of P29 and P34 mouse incisors after tamoxifen treatment and cutting of the left lower incisor. First column: mouse incisors after cutting half of the left lower incisor. Second column: mouse incisors 5 days after shortening. Third column: microcomputed tomography (μCT) analysis of shortened incisors after 5 days of recovery. Fourth column: Sox2 expression visualized by immunofluorescence (IF) in tamoxifen- treated Rosa26CreERT/Sox2F/F and control mice. (C) Quantification data (mean±s.e.m.) of the relative growth rate of the shortened incisor in Sox2F/F and Rosa26CreERT /Sox2F/F mice after treatment with tamoxifen. Scale bars: 5 mm (μCT images); 100 µm (IF images).

Article Snippet: DNA/protein complexes were immunoprecipitated with 5 μg Pitx2 antibody (Pitx2 antibody, Capra Sciences, PA-1023) or Sox2 antibody (R&D Systems, AF2018).

Techniques: Control, Tomography, Expressing, Immunofluorescence

Journal: Development (Cambridge, England)

Article Title: Sox2 and Lef-1 interact with Pitx2 to regulate incisor development and stem cell renewal.

doi: 10.1242/dev.138883

Figure Lengend Snippet: Fig. 3. Sox2 regulates dental epithelial stem cell proliferation and differentiation. (A-B″) Immunofluorescence staining of the proliferation marker Ki67 in sagittal sections of E16.5 Sox2F/F and Sox2cKO mouse incisors. (A′,B′) High magnification view of red boxed regions in A and B to show the proliferation in the LaCL (outlined). (A″,B″) High magnification view of white boxed regions in A and B to show the proliferation in the LiCL (outlined). (C) Quantification of the ratio of Ki67-positive cells to total cells in the cervical loops. Mean±s.e.m., n=3. (D-F) Progenitor cell differentiation measured using two different labels (CldU and IdU), which were injected and measured after 24 h and 1 h, respectively. The red label-retaining cells (CldU) mark differentiated cells and the green label-retaining cells (IdU) mark recently mitotic cells. The arrows indicate the region of neural vascular bundles. The LaCLs are highlighted by yellow dashed boxes. (D′-D‴,E′-E‴) Higher magnifications of the white boxed areas in D and E, respectively, highlight the progenitor cells at the distal tip of the incisors. DI, distal; PR, proximal. (F) Quantification of CldU+ cells in the epithelial tissue of the white boxed region shown in D′ and E′. Mean±s.e.m., n=3. Scale bars: 100 μm.

Article Snippet: DNA/protein complexes were immunoprecipitated with 5 μg Pitx2 antibody (Pitx2 antibody, Capra Sciences, PA-1023) or Sox2 antibody (R&D Systems, AF2018).

Techniques: Immunofluorescence, Staining, Marker, Cell Differentiation, Injection

Journal: Development (Cambridge, England)

Article Title: Sox2 and Lef-1 interact with Pitx2 to regulate incisor development and stem cell renewal.

doi: 10.1242/dev.138883

Figure Lengend Snippet: Fig. 4. Sox2 and Lef-1 expression domains are juxtaposed in the dental placode and oral epithelium. (A) Schematic of an E11.5 mouse head shows upper and lower dental placodes. (B,C) Sox2 and Lef-1 immunofluorescence staining in E11.5 wild-type (WT) embryos. (D) Merged image of Sox2 and Lef-1 staining in E11.5 WT showing the juxtaposed expression domains of these two factors. (E) Higher magnification of the boxed region in D. Arrows indicate position of the lower and upper incisor dental placodes. (F) Double immunofluorescence staining of Sox2 (red) and Lef-1 (green) in E12.5 WT embryos, showing specific expression domains of each factor in the developing incisor tooth buds. Dashed lines mark boundary between dental epithelium and mesenchyme. (G,K) Hematoxylin and Eosin staining of E11.5 Sox2F/F and Sox2cKO embryos; arrows point to the LI and UI dental placodes. (H-J,L-N) Sox2 expression is absent in the Sox2cKO embryos; however, Lef-1 expression is not affected compared with controls (higher magnification of boxed regions in G and K). Arrows in I and M highlight the Lef-1 expression. DL, dental lamina; LI, lower incisor; Md, mandible; Mx, Maxilla; UI, upper incisor. Scale bars: 100 μm.

Article Snippet: DNA/protein complexes were immunoprecipitated with 5 μg Pitx2 antibody (Pitx2 antibody, Capra Sciences, PA-1023) or Sox2 antibody (R&D Systems, AF2018).

Techniques: Expressing, Immunofluorescence, Staining, Double Immunofluorescence Staining

Journal: Development (Cambridge, England)

Article Title: Sox2 and Lef-1 interact with Pitx2 to regulate incisor development and stem cell renewal.

doi: 10.1242/dev.138883

Figure Lengend Snippet: Fig. 5. Conditional overexpression of Lef-1 (COEL) results in tusk-like incisors, creation of a new stem cell compartment in the LaCL and increased dental epithelial stem cell proliferation. (A) Schematic of the Lef-1 conditional overexpression cassette used to generate the COEL mouse. (B,C) Heads showing overgrown incisors from 3-month-old Lef-1cKI (Lef-1 conditional knock-in, control) and Pitx2Cre/Lef-1cKI/cKI (Lef-1 overexpression, COEL) mice. (D,E) μCT analysis of the mouse heads shown in B and C. Both the upper and lower incisors are overgrown in COEL mice. (F,G) Immunofluorescence staining of Sox2 in the LaCL of P1 control and COEL incisors. Note the extra stem cell compartment in the LaCL of COEL mice. (H-K) 3D composites of the LI LaCL comparing WT (Lef-1cKI) with the COEL LI at P2. (L) BrdU labeling of the E18.5 control and COEL incisors. (M) Quantification of the percentage of BrdU+ cells in the E18.5 LaCL of control and COEL incisors. Mean±s.e.m., n=3. (N) BrdU labeling of P1 control and COEL lower incisors. Note the lack of proliferative cells in the extra stem cell compartment (arrow). (O) Quantification of BrdU-positive compared with BrdU-negative cells as a ratio of all cells in the LaCL. Mean±s.e.m., n=3. *P<0.05. Scale bars: 100 μm.

Article Snippet: DNA/protein complexes were immunoprecipitated with 5 μg Pitx2 antibody (Pitx2 antibody, Capra Sciences, PA-1023) or Sox2 antibody (R&D Systems, AF2018).

Techniques: Over Expression, Knock-In, Control, Immunofluorescence, Staining, Labeling

Journal: Development (Cambridge, England)

Article Title: Sox2 and Lef-1 interact with Pitx2 to regulate incisor development and stem cell renewal.

doi: 10.1242/dev.138883

Figure Lengend Snippet: Fig. 6. Overexpression of Lef-1 partially rescues tooth arrest in Sox2cKO embryos. (A,B) Hematoxylin and Eosin staining of sagittal sections of E18.5 Sox2F/F/Lef-1cKI (control) and Pitx2Cre/Sox2F/F/Lef-1cKI (rescue) mandibles and maxilla. Differentiation of the labial dental epithelial cells (Am and Si) and adjacent odontoblast (Od) cells in the rescue incisors is highlighted in the blue boxes (higher magnification shown below). Black boxes show the LaCL is present in rescue embryos but much smaller than in control incisors (higher magnification shown below). Am, ameloblast; Od, odontoblast; Si, stratum intermedium. (C-D′) Amelogenin immunofluorescence staining in E18.5 control and rescue incisors revealed that amelogenin expression was switched to the lingual side in the rescue incisor compared with the labial expression in the control. Boxed regions in C and D are shown at higher magnification in C′ and D′, respectively. (E,F) Sox2 and Lef-1 double immunostaining in E18.5 control and rescue incisors. Dental epithelial cells are outlined. (G) Proliferation as determined by the marker Ki67 was reduced in the Sox2cKO E16.5 LaCL (outlined) but was restored in the rescue embryos compared with controls. (H) Proliferation of the cells in the LaCL was quantified by comparison of Ki67-stained (proliferating) cells versus DAPI-stained (total) cells. Mean±s.e.m., n=3. Scale bars: 100 μm.

Article Snippet: DNA/protein complexes were immunoprecipitated with 5 μg Pitx2 antibody (Pitx2 antibody, Capra Sciences, PA-1023) or Sox2 antibody (R&D Systems, AF2018).

Techniques: Over Expression, Staining, Control, Immunofluorescence, Expressing, Double Immunostaining, Marker, Comparison

Journal: Development (Cambridge, England)

Article Title: Sox2 and Lef-1 interact with Pitx2 to regulate incisor development and stem cell renewal.

doi: 10.1242/dev.138883

Figure Lengend Snippet: Fig. 7. Model for the roles of Pitx2, Sox2 and Lef-1 in regulating DESC maintenance and lower incisor development. (A) Pitx2 (blue) is expressed throughout the dental placode and oral epithelium at E11.5, whereas Sox2 (red) and Lef-1 (green) expression occurs in separate domains, with Sox2 posterior to Lef-1 in the dental placode. (B) Pitx2 is expressed with Sox2 in the LI LaCL at E16.5. However, Pitx2 is also highly expressed in the transient amplifying cells of the lower incisor that give rise to the ameloblasts. Lef-1 is mostly expressed in the dental mesenchyme adjacent to the dental epithelium at this stage. (C) In the Pitx2Cre/Sox2F/F embryos at E16.5, Pitx2 is present in the LaCL but Sox2 and Lef-1 are absent, leading to the failure to maintain the stem cell niche. (D) In the Pitx2Cre/Sox2F/F/Lef-1cKI rescue embryos after E14.5, Pitx2 and Lef-1 can function to produce dental epithelial stem cells that generate an incisor. (E) It has been shown that Fgf can activate both Pitx2 and Sox2 expression in early stages of tooth development. Biochemical assays in this report show that Pitx2 activates Sox2, Lef-1 and Pitx2 to maintain their expression. However, Sox2 directly interacts with Pitx2 to repress Pitx2 transcriptional activity and modulates Sox2, Lef-1 and Pitx2 expression levels to coordinate dental epithelial stem cell renewal, proliferation and differentiation of progenitor cells.

Article Snippet: DNA/protein complexes were immunoprecipitated with 5 μg Pitx2 antibody (Pitx2 antibody, Capra Sciences, PA-1023) or Sox2 antibody (R&D Systems, AF2018).

Techniques: Expressing, Activity Assay

Journal: bioRxiv

Article Title: Epigenomic profiling discovers trans-lineage SOX2 partnerships driving tumor heterogeneity in lung squamous cell carcinoma

doi: 10.1101/646034

Figure Lengend Snippet: Super-enhancer profiling identifies a novel subtype of LUSC. (A) Unsupervised hierarchical clustering of 13 LUSC cell lines using super-enhancer scores near transcriptional regulator genes. (B) Copy number of SOX2 and mRNA expression of SOX2 , TP63 and POU3F2 in LUSC cell lines from CCLE. (C) Super-enhancer plots using H3K27ac scores in the small subset of LUSC cell lines; LK2 cells ( left ) and NCI-H520 cells ( right ). (D) Genome view tracks of H3K27ac signal at loci of SOX2 ( left ), TP63 ( middle ) and POU3F2 ( right ) in LUSC cell lines. (E) Protein expression of Brn2, p63, Sox2 and vinculin as a loading control in LUSC cell lines.

Article Snippet: After centrifugation to remove insoluble debris, lysates were immunoblotted with use of an anti-Brn2 antibody (Santa Cruz Biotechnology, sc-393324 or Cell Signaling Technology, #12137), anti-p63 antibody (Santa Cruz Biotechnology, sc-8344, sc-8431 or Cell Signaling Technology, #13109), anti-Sox2 antibody (R&D, AF2018 or Cell Signaling Technology, #3579), anti-ErbB4 antibody (Cell Signaling Technology, #4795), anti-phospho-ErbB4 antibody (Cell Signaling Technology, #4757), anti-ErbB3 antibody (Cell Signaling Technology, #12708), anti-phospho-ErbB3 antibody (Cell Signaling Technology, #4791), anti-EGFR antibody (Millipore, #06-847), anti-phospho-EGFR antibody (Abcam, ab40815) anti-phospho-Akt antibody (Cell Signaling Technology, #4060) and anti-phospho-Erk1/2 antibody (Cell Signaling Technology, #4370), anti-β-Actin antibody (Sigma), or anti-vinculin (Sigma).

Techniques: Expressing

Journal: bioRxiv

Article Title: Epigenomic profiling discovers trans-lineage SOX2 partnerships driving tumor heterogeneity in lung squamous cell carcinoma

doi: 10.1101/646034

Figure Lengend Snippet: Brn2 is expressed in a subset of human primary LUSC tumors. (A) Expression of POU3F2 in 501 TCGA LUSC tumor tissues. The red dashed line (TPM=1) shows the cutoff to separate samples into POU3F2 high and low samples. (B) Box plots of POU3F2 expression in TP63 -high and low LUSC tumors from TCGA, SCLC tumors from publicly available datasets and normal brain tissues (hypothalamus) from GTEx. (C) Immunohistochemical staining of Brn2 and Sox2 and H&E staining in Brn2-positive and negative human LUSC tumors. Representative images are shown (original images, ×400).

Article Snippet: After centrifugation to remove insoluble debris, lysates were immunoblotted with use of an anti-Brn2 antibody (Santa Cruz Biotechnology, sc-393324 or Cell Signaling Technology, #12137), anti-p63 antibody (Santa Cruz Biotechnology, sc-8344, sc-8431 or Cell Signaling Technology, #13109), anti-Sox2 antibody (R&D, AF2018 or Cell Signaling Technology, #3579), anti-ErbB4 antibody (Cell Signaling Technology, #4795), anti-phospho-ErbB4 antibody (Cell Signaling Technology, #4757), anti-ErbB3 antibody (Cell Signaling Technology, #12708), anti-phospho-ErbB3 antibody (Cell Signaling Technology, #4791), anti-EGFR antibody (Millipore, #06-847), anti-phospho-EGFR antibody (Abcam, ab40815) anti-phospho-Akt antibody (Cell Signaling Technology, #4060) and anti-phospho-Erk1/2 antibody (Cell Signaling Technology, #4370), anti-β-Actin antibody (Sigma), or anti-vinculin (Sigma).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Epigenomic profiling discovers trans-lineage SOX2 partnerships driving tumor heterogeneity in lung squamous cell carcinoma

doi: 10.1101/646034

Figure Lengend Snippet: DNp63 overexpression in the ‘neural’ LUSC cells suppresses Brn2 expression and induces phenotypic changes. (A) Protein expression of p63, Brn2, Sox2 and vinculin as a loading control in parental, GFP-overexpressed and DNp63-overex-pressed LK2 cells. (B) Phase-contrast microphotographs of GFP-overexpressed and DNp63-overexpressed LK2 cells. Bar = 100 μm. (C) Cell growth of GFP-overexpressed and DNp63-overexpressed LK2 cells. Mean ± SD of sextuplicates are shown. **, P <0.001 vs. GFP-overexpressed LK2 cells, t -test. (D) Tumor growth of GFP-overexpressed and DNp63-overexpressed LK2 cells in vivo . Mean ± SD of tetraplicates are shown. **, P <0.001 vs. GFP-overexpressed xenografts, t -test. Xenograft tumors resected 19 days after inoculation are shown in the right picture. (E) H&E staining and immunohistochemical staining of Brn2 and p63 in GFP-overexpressed or DNp63-overexpressed LK2 xenograft. Original Images, ×400.

Article Snippet: After centrifugation to remove insoluble debris, lysates were immunoblotted with use of an anti-Brn2 antibody (Santa Cruz Biotechnology, sc-393324 or Cell Signaling Technology, #12137), anti-p63 antibody (Santa Cruz Biotechnology, sc-8344, sc-8431 or Cell Signaling Technology, #13109), anti-Sox2 antibody (R&D, AF2018 or Cell Signaling Technology, #3579), anti-ErbB4 antibody (Cell Signaling Technology, #4795), anti-phospho-ErbB4 antibody (Cell Signaling Technology, #4757), anti-ErbB3 antibody (Cell Signaling Technology, #12708), anti-phospho-ErbB3 antibody (Cell Signaling Technology, #4791), anti-EGFR antibody (Millipore, #06-847), anti-phospho-EGFR antibody (Abcam, ab40815) anti-phospho-Akt antibody (Cell Signaling Technology, #4060) and anti-phospho-Erk1/2 antibody (Cell Signaling Technology, #4370), anti-β-Actin antibody (Sigma), or anti-vinculin (Sigma).

Techniques: Over Expression, Expressing, In Vivo, Staining, Immunohistochemical staining

Journal: bioRxiv

Article Title: Epigenomic profiling discovers trans-lineage SOX2 partnerships driving tumor heterogeneity in lung squamous cell carcinoma

doi: 10.1101/646034

Figure Lengend Snippet: Brn2 and Sox2 interact and co-localize at genetic loci in the ‘neural’ subset of LUSC. (A) Expression of endogenous Brn2 and Sox2 in LK2 cells, determined by immunofluorescence with anti-Sox2 ( green ) and anti-Brn2 ( red ) antibodies, respectively. DAPI staining (nuclei; blue ) and merged images are also shown. Original magnification, ×200. Scale bar, 100 μm. (B) Sox2-Brn2 interaction, shown by co-immunoprecipitation of Sox2 using an antibody against endogenous Brn2 ( top ) and co-immunoprecipitation of Brn2 using an antibody against endogenous Sox2 ( bottom ) in LK2 and NCI-H520 cells. (C) Heatmap depicting global analysis of ChIP-seq signals for Brn2 and Sox2 in ‘neural’ NCI-H520 and LK2 cells and those for Sox2 in ‘classical’ HCC95, KNS62 and HCC2814 cells at all the peak loci. ChIP-seq signal intensity is shown by color shading. (D) Genome view tracks of Brn2 and Sox2 ChIP-seq signals in NCI-H520 and LK2 cells and Sox2 ChIP signals in HCC95, KNS62 and HCC2814 cells at loci of POU3F2 ( left ) and TP63 ( right ).

Article Snippet: After centrifugation to remove insoluble debris, lysates were immunoblotted with use of an anti-Brn2 antibody (Santa Cruz Biotechnology, sc-393324 or Cell Signaling Technology, #12137), anti-p63 antibody (Santa Cruz Biotechnology, sc-8344, sc-8431 or Cell Signaling Technology, #13109), anti-Sox2 antibody (R&D, AF2018 or Cell Signaling Technology, #3579), anti-ErbB4 antibody (Cell Signaling Technology, #4795), anti-phospho-ErbB4 antibody (Cell Signaling Technology, #4757), anti-ErbB3 antibody (Cell Signaling Technology, #12708), anti-phospho-ErbB3 antibody (Cell Signaling Technology, #4791), anti-EGFR antibody (Millipore, #06-847), anti-phospho-EGFR antibody (Abcam, ab40815) anti-phospho-Akt antibody (Cell Signaling Technology, #4060) and anti-phospho-Erk1/2 antibody (Cell Signaling Technology, #4370), anti-β-Actin antibody (Sigma), or anti-vinculin (Sigma).

Techniques: Expressing, Immunofluorescence, Staining, Immunoprecipitation, ChIP-sequencing

Journal: bioRxiv

Article Title: Epigenomic profiling discovers trans-lineage SOX2 partnerships driving tumor heterogeneity in lung squamous cell carcinoma

doi: 10.1101/646034

Figure Lengend Snippet: DNp63 induces a classical squamous-cell transcriptional program in the ‘neural’ LUSC cells. (A) Heatmap showing 399 differentially expressed genes (287 upregulated and 112 downregulated genes) between control LK2 cells (stable GFP-overexpressed cells and doxycycline-inducible DNp63-overexpressing cells without doxycycline in duplicate) and DNp63-overexpressed LK2 cells (stable DNp63-overexpressed cells and doxycycline-inducible DNp63-overexpressing cells with 2 μg/ml doxycycline in duplicate), sorted by fold change. Color scheme represents Z-score distribution. (B) Gene ontology analyses for the differentially up-regulated ( top ) and down-regulated ( bottom ) genes. Enriched functions for these genes are identified based on Fisher’s exact test against GO terms curated in MSigDB. (C) Average ChIP-seq signals for all Sox2 peaks ( top ) and Sox2 peaks exclusively found in ‘classical’ HCC95, KNS62 and HCC2814 cells ( bottom ) in GFP-overexpressed and DNp63-overexpressed LK2 cells. (D) Genome view tracks of Sox2 ChIP-seq signals in GFP-overexpressed and DNp63-overexpressed LK2 cells, HCC95, KNS62 and HCC2814 cells and p63 ChIP signals in DNp63-overexpressed LK2 cells and HCC95 cells at loci of TP63 ( top ) and CTDSPL ( bottom ).

Article Snippet: After centrifugation to remove insoluble debris, lysates were immunoblotted with use of an anti-Brn2 antibody (Santa Cruz Biotechnology, sc-393324 or Cell Signaling Technology, #12137), anti-p63 antibody (Santa Cruz Biotechnology, sc-8344, sc-8431 or Cell Signaling Technology, #13109), anti-Sox2 antibody (R&D, AF2018 or Cell Signaling Technology, #3579), anti-ErbB4 antibody (Cell Signaling Technology, #4795), anti-phospho-ErbB4 antibody (Cell Signaling Technology, #4757), anti-ErbB3 antibody (Cell Signaling Technology, #12708), anti-phospho-ErbB3 antibody (Cell Signaling Technology, #4791), anti-EGFR antibody (Millipore, #06-847), anti-phospho-EGFR antibody (Abcam, ab40815) anti-phospho-Akt antibody (Cell Signaling Technology, #4060) and anti-phospho-Erk1/2 antibody (Cell Signaling Technology, #4370), anti-β-Actin antibody (Sigma), or anti-vinculin (Sigma).

Techniques: ChIP-sequencing

Journal: BMC Clinical Pathology

Article Title: Autologous anti-SOX2 antibody responses reflect intensity but not frequency of antigen expression in small cell lung cancer

doi: 10.1186/1472-6890-14-24

Figure Lengend Snippet: Clinical features and SOX2 antibody and protein staining characteristics of SCLC patients

Article Snippet: Mouse anti-human SOX2 monoclonal antibody (R&D Systems, Minneapolis, MN, USA) was used as a positive control.

Techniques: Staining

Journal: BMC Clinical Pathology

Article Title: Autologous anti-SOX2 antibody responses reflect intensity but not frequency of antigen expression in small cell lung cancer

doi: 10.1186/1472-6890-14-24

Figure Lengend Snippet: SOX2 antibody and protein staining characteristics of SCLC patients

Article Snippet: Mouse anti-human SOX2 monoclonal antibody (R&D Systems, Minneapolis, MN, USA) was used as a positive control.

Techniques: Staining

Journal: BMC Clinical Pathology

Article Title: Autologous anti-SOX2 antibody responses reflect intensity but not frequency of antigen expression in small cell lung cancer

doi: 10.1186/1472-6890-14-24

Figure Lengend Snippet: SOX2 antibody correlates with clinical stage

Article Snippet: Mouse anti-human SOX2 monoclonal antibody (R&D Systems, Minneapolis, MN, USA) was used as a positive control.

Techniques:

Journal: BMC Clinical Pathology

Article Title: Autologous anti-SOX2 antibody responses reflect intensity but not frequency of antigen expression in small cell lung cancer

doi: 10.1186/1472-6890-14-24

Figure Lengend Snippet: SOX2 antibody correlates with intensity of SOX2 protein expression

Article Snippet: Mouse anti-human SOX2 monoclonal antibody (R&D Systems, Minneapolis, MN, USA) was used as a positive control.

Techniques:

Journal: Biology Open

Article Title: In vivo genetic manipulation of cortical progenitors in gyrencephalic carnivores using in utero electroporation

doi: 10.1242/bio.20123160

Figure Lengend Snippet: In utero electroporation was performed at E37, and sections were prepared at E40. The sections were immunostained with anti-Sox2, anti-Pax6 and anti-Tbr2 antibodies. The cerebral cortex is shown in A . The areas within the white boxes are magnified and are shown in B . CP, cortical plate; OSVZ, outer subventricular zone; ISVZ, inner subventricular zone; VZ, ventricular zone. Scale bars: 200 µm (A) and 100 µm (B).

Article Snippet: Sections were made using a cryostat, permeabilized with 0.1–0.5% Triton X-100/PBS, and incubated overnight with primary antibodies, which included anti-Sox2 antibody (R&D Systems), anti-Pax6 antibody (Covance), anti-Tbr2 antibody (Abcam) and anti-neurofilament-M antibody (Chemicon).

Techniques: In Utero, Electroporation

Journal: Biology Open

Article Title: In vivo genetic manipulation of cortical progenitors in gyrencephalic carnivores using in utero electroporation

doi: 10.1242/bio.20123160

Figure Lengend Snippet: In utero electroporation was performed at E35, and coronal sections were prepared at P0 and were stained with Hoechst 33342. Many GFP-positive cells were distributed throughout the cortex ( A ). The areas within the white boxes in A are magnified and are shown in B . The areas within the white boxes in B are shown in C . Note that the morphology of GFP-positive cells was clearly visible even without immunostaining. ( D , E ) The sections were immunostained with anti-Sox2 antibody (D) and anti-Pax6 antibody (E), and high magnification images of the OSVZ are shown. The GFP-positive cells (arrowheads) expressed Sox2 (D) and Pax6 (E), and had basal fibers but not apical fibers, suggesting that these cell are oRG cells. GFP-positive fibers running tangentially in the inner OSVZ were also visible (arrow). *Lateral ventricle. CP, cortical plate; OSVZ, outer subventricular zone; ISVZ, inner subventricular zone; VZ, ventricular zone. Scale bars: 1 mm (A), 500 µm (B), 100 µm (C) and 20 µm (D,E).

Article Snippet: Sections were made using a cryostat, permeabilized with 0.1–0.5% Triton X-100/PBS, and incubated overnight with primary antibodies, which included anti-Sox2 antibody (R&D Systems), anti-Pax6 antibody (Covance), anti-Tbr2 antibody (Abcam) and anti-neurofilament-M antibody (Chemicon).

Techniques: In Utero, Electroporation, Staining, Immunostaining

Journal: Cancer research

Article Title: Epigenomic profiling discovers trans-lineage SOX2 partnerships driving tumor heterogeneity in lung squamous cell carcinoma

doi: 10.1158/0008-5472.CAN-19-2132

Figure Lengend Snippet: Super-enhancer profiling identifies a novel subtype of LUSC. A, Unsupervised hierarchical clustering of 13 LUSC cell lines using super-enhancer (SE) signals near transcriptional regulator genes. SE signals were normalized to linear scores using the sum of squares of the values with GenePatten. Duplicate data are shown for LK2 and NCI-H520 cells. B, Copy number of SOX2 and mRNA expression of SOX2, TP63 and POU3F2 in LUSC cell lines from CCLE. C, Super-enhancer plots using H3K27ac scores in the small subset of LUSC cell lines; LK2 cells (left) and NCI-H520 cells (right). D, Genome view tracks of H3K27ac signal at loci of SOX2 (left), TP63 (middle) and POU3F2 (right) in LUSC cell lines. Normalized signals are shown in a range from 0 to 50 for all the tracks. E, Protein expression of Brn2, p63, Sox2 and vinculin as a loading control in LUSC cell lines.

Article Snippet: After centrifugation to remove insoluble debris, lysates were immunoblotted with use of an anti-Brn2 antibody (Santa Cruz Biotechnology, sc-393324 or Cell Signaling Technology, #12137), anti-p63 antibody (Santa Cruz Biotechnology, sc-8344, sc-8431 or Cell Signaling Technology, #13109), anti-Sox2 antibody (R&D, AF2018 or Cell Signaling Technology, #3579), anti-ErbB4 antibody (Cell Signaling Technology, #4795), anti-phospho-ErbB4 antibody (Cell Signaling Technology, #4757), anti-ErbB3 antibody (Cell Signaling Technology, #12708), anti-phospho-ErbB3 antibody (Cell Signaling Technology, #4791), anti-EGFR antibody (Millipore, #06-847), anti-phospho-EGFR antibody (Abcam, ab40815) anti-phospho-Akt antibody (Cell Signaling Technology, #4060) and anti-phospho-Erk1/2 antibody (Cell Signaling Technology, #4370), anti-Ascl1 antibody (Santa Cruz, sc-374104), anti-SYP antibody (Santa Cruz, sc-17750), anti-β-Actin antibody (Sigma), or anti-vinculin (Sigma).

Techniques: Expressing

Journal: Cancer research

Article Title: Epigenomic profiling discovers trans-lineage SOX2 partnerships driving tumor heterogeneity in lung squamous cell carcinoma

doi: 10.1158/0008-5472.CAN-19-2132

Figure Lengend Snippet: Brn2 is expressed in a subset of human primary LUSC tumors. A, Expression of POU3F2 in 501 TCGA LUSC tumor tissues. The red dashed line (TPM=1) shows the cutoff to separate samples into POU3F2 high and low samples. B, Box plots of POU3F2 expression in TP63 high and low LUSC tumors from TCGA, SCLC tumors from publicly available datasets and normal brain tissues (hypothalamus) from GTEx. C, Immunohistochemical staining of Brn2 and Sox2 and H&E staining in Brn2-positive and negative human LUSC tumors. Representative images are shown (original images, ×400). D, Scatter plots of expression of SOX2 and POU3F2 in TP63-low LUSC tumors from TCGA.

Article Snippet: After centrifugation to remove insoluble debris, lysates were immunoblotted with use of an anti-Brn2 antibody (Santa Cruz Biotechnology, sc-393324 or Cell Signaling Technology, #12137), anti-p63 antibody (Santa Cruz Biotechnology, sc-8344, sc-8431 or Cell Signaling Technology, #13109), anti-Sox2 antibody (R&D, AF2018 or Cell Signaling Technology, #3579), anti-ErbB4 antibody (Cell Signaling Technology, #4795), anti-phospho-ErbB4 antibody (Cell Signaling Technology, #4757), anti-ErbB3 antibody (Cell Signaling Technology, #12708), anti-phospho-ErbB3 antibody (Cell Signaling Technology, #4791), anti-EGFR antibody (Millipore, #06-847), anti-phospho-EGFR antibody (Abcam, ab40815) anti-phospho-Akt antibody (Cell Signaling Technology, #4060) and anti-phospho-Erk1/2 antibody (Cell Signaling Technology, #4370), anti-Ascl1 antibody (Santa Cruz, sc-374104), anti-SYP antibody (Santa Cruz, sc-17750), anti-β-Actin antibody (Sigma), or anti-vinculin (Sigma).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cancer research

Article Title: Epigenomic profiling discovers trans-lineage SOX2 partnerships driving tumor heterogeneity in lung squamous cell carcinoma

doi: 10.1158/0008-5472.CAN-19-2132

Figure Lengend Snippet: Brn2 and Sox2 interact and co-localize at genetic loci in the ‘neural’ subset of LUSC. A, Expression of endogenous Brn2 and Sox2 in LK2 cells, determined by immunofluorescence with anti-Sox2 (green) and anti-Brn2 (red) antibodies, respectively. DAPI staining (nuclei; blue) and merged images are also shown. Original magnification, ×200. Scale bar, 100 μm. B, Sox2-Brn2 interaction, shown by co-immunoprecipitation of Sox2 using an antibody against endogenous Brn2 (top) and co-immunoprecipitation of Brn2 using an antibody against endogenous Sox2 (bottom) in LK2 and NCI-H520 cells. C, Heatmap depicting global analysis of ChIP-seq signals for Brn2 and Sox2 in ‘neural’ NCI-H520 and LK2 cells and those for Sox2 in ‘classical’ HCC95, KNS62 and HCC2814 cells at all the peak loci. ChIP-seq signal intensity is shown by color shading. D, Genome view tracks of Brn2, Sox2 and H3K27ac ChIP-seq signals in NCI-H520 and LK2 cells, and Sox2 and H3K27ac ChIP signals in HCC95, KNS62 and HCC2814 cells at loci of POU3F2 (left) and TP63 (right).

Article Snippet: After centrifugation to remove insoluble debris, lysates were immunoblotted with use of an anti-Brn2 antibody (Santa Cruz Biotechnology, sc-393324 or Cell Signaling Technology, #12137), anti-p63 antibody (Santa Cruz Biotechnology, sc-8344, sc-8431 or Cell Signaling Technology, #13109), anti-Sox2 antibody (R&D, AF2018 or Cell Signaling Technology, #3579), anti-ErbB4 antibody (Cell Signaling Technology, #4795), anti-phospho-ErbB4 antibody (Cell Signaling Technology, #4757), anti-ErbB3 antibody (Cell Signaling Technology, #12708), anti-phospho-ErbB3 antibody (Cell Signaling Technology, #4791), anti-EGFR antibody (Millipore, #06-847), anti-phospho-EGFR antibody (Abcam, ab40815) anti-phospho-Akt antibody (Cell Signaling Technology, #4060) and anti-phospho-Erk1/2 antibody (Cell Signaling Technology, #4370), anti-Ascl1 antibody (Santa Cruz, sc-374104), anti-SYP antibody (Santa Cruz, sc-17750), anti-β-Actin antibody (Sigma), or anti-vinculin (Sigma).

Techniques: Expressing, Immunofluorescence, Staining, Immunoprecipitation, ChIP-sequencing

Journal: Cancer research

Article Title: Epigenomic profiling discovers trans-lineage SOX2 partnerships driving tumor heterogeneity in lung squamous cell carcinoma

doi: 10.1158/0008-5472.CAN-19-2132

Figure Lengend Snippet: DNp63 overexpression in the ‘neural’ LUSC cells suppresses Brn2 expression and induces phenotypic changes. A, Protein expression of p63, Brn2, Sox2 and vinculin as a loading control in parental, GFP-overexpressed and DNp63-overexpressed LK2 cells. B, Phase-contrast microphotographs of GFP-overexpressed and DNp63-overexpressed LK2 cells. Bar = 100 μm. C, Cell growth of GFP-overexpressed and DNp63-overexpressed LK2 cells. Mean ± SD of sextuplicates are shown. **, P<0.001 vs. GFP-overexpressed LK2 cells, t-test. D, Tumor growth of GFP-overexpressed and DNp63-overexpressed LK2 cells in vivo. Mean ± SD of tetraplicates are shown. **, P<0.001 vs. GFP-overexpressed xenografts, t-test. Xenograft tumors resected 19 days after inoculation are shown in the right picture. E, H&E staining and immunohistochemical staining of Brn2 and p63 in GFP-overexpressed or DNp63-overexpressed LK2 xenograft. Original Images, ×400.

Article Snippet: After centrifugation to remove insoluble debris, lysates were immunoblotted with use of an anti-Brn2 antibody (Santa Cruz Biotechnology, sc-393324 or Cell Signaling Technology, #12137), anti-p63 antibody (Santa Cruz Biotechnology, sc-8344, sc-8431 or Cell Signaling Technology, #13109), anti-Sox2 antibody (R&D, AF2018 or Cell Signaling Technology, #3579), anti-ErbB4 antibody (Cell Signaling Technology, #4795), anti-phospho-ErbB4 antibody (Cell Signaling Technology, #4757), anti-ErbB3 antibody (Cell Signaling Technology, #12708), anti-phospho-ErbB3 antibody (Cell Signaling Technology, #4791), anti-EGFR antibody (Millipore, #06-847), anti-phospho-EGFR antibody (Abcam, ab40815) anti-phospho-Akt antibody (Cell Signaling Technology, #4060) and anti-phospho-Erk1/2 antibody (Cell Signaling Technology, #4370), anti-Ascl1 antibody (Santa Cruz, sc-374104), anti-SYP antibody (Santa Cruz, sc-17750), anti-β-Actin antibody (Sigma), or anti-vinculin (Sigma).

Techniques: Over Expression, Expressing, In Vivo, Staining, Immunohistochemical staining

Journal: Cancer research

Article Title: Epigenomic profiling discovers trans-lineage SOX2 partnerships driving tumor heterogeneity in lung squamous cell carcinoma

doi: 10.1158/0008-5472.CAN-19-2132

Figure Lengend Snippet: DNp63 induces a classical squamous-cell transcriptional program in the ‘neural’ LUSC cells. A, Heatmap showing 399 differentially expressed genes (287 up-regulated and 112 down-regulated genes) between control LK2 cells (stable GFP-overexpressed cells and doxycycline-inducible DNp63-overexpressing cells without doxycycline in duplicate) and DNp63-overexpressed LK2 cells (stable DNp63-overexpressed cells and doxycycline-inducible DNp63-overexpressing cells with 2 μg/ml doxycycline in duplicate), sorted by fold change. Color scheme represents Z-score distribution. B, Gene ontology analyses for the differentially up-regulated (top) and down-regulated (bottom) genes upon DNp63 overexpression in LK2 cells. Enriched functions for these genes are identified based on Fisher’s exact test against GO terms curated in MSigDB. C, Average ChIP-seq signals for all Sox2 peaks (top) and Sox2 peaks exclusively found in ‘classical’ HCC95, KNS62 and HCC2814 cells (bottom) in GFP-overexpressed and DNp63-overexpressed LK2 cells. D, Genome view tracks of Sox2 ChIP-seq signals in GFP-overexpressed and DNp63-overexpressed LK2 cells, HCC95, KNS62 and HCC2814 cells and p63 ChIP signals in DNp63-overexpressed LK2 cells and HCC95 cells at loci of TP63 (top) and CTDSPL (bottom).

Article Snippet: After centrifugation to remove insoluble debris, lysates were immunoblotted with use of an anti-Brn2 antibody (Santa Cruz Biotechnology, sc-393324 or Cell Signaling Technology, #12137), anti-p63 antibody (Santa Cruz Biotechnology, sc-8344, sc-8431 or Cell Signaling Technology, #13109), anti-Sox2 antibody (R&D, AF2018 or Cell Signaling Technology, #3579), anti-ErbB4 antibody (Cell Signaling Technology, #4795), anti-phospho-ErbB4 antibody (Cell Signaling Technology, #4757), anti-ErbB3 antibody (Cell Signaling Technology, #12708), anti-phospho-ErbB3 antibody (Cell Signaling Technology, #4791), anti-EGFR antibody (Millipore, #06-847), anti-phospho-EGFR antibody (Abcam, ab40815) anti-phospho-Akt antibody (Cell Signaling Technology, #4060) and anti-phospho-Erk1/2 antibody (Cell Signaling Technology, #4370), anti-Ascl1 antibody (Santa Cruz, sc-374104), anti-SYP antibody (Santa Cruz, sc-17750), anti-β-Actin antibody (Sigma), or anti-vinculin (Sigma).

Techniques: Over Expression, ChIP-sequencing

Journal: Cell Reports Medicine

Article Title: CD97 maintains tumorigenicity of glioblastoma stem cells via mTORC2 signaling and is targeted by CAR Th9 cells

doi: 10.1016/j.xcrm.2024.101844

Figure Lengend Snippet:

Article Snippet: Human/Mouse/Rat SOX2 Antibody , R&D Systems , Cat# AF2018; RRID: AB_355110.

Techniques: Produced, Virus, Plasmid Preparation, Recombinant, Purification, Cell Culture, Cell Isolation, Reporter Gene Assay, cDNA Synthesis, Apoptosis Assay, Cytotoxicity Assay, Gene Expression, shRNA, Sequencing, Amplification, Software, Microscopy, Western Blot