primary antibodies against nanog  (PeproTech)

Journal: Stem Cells

Article Title: NK-cell cytotoxicity toward pluripotent stem cells and their neural progeny: impacts of activating and inhibitory receptors and KIR/HLA mismatch

doi: 10.1093/stmcls/sxae083

Figure Lengend Snippet: B7H6 expression is maintained in motoneurons differentiated from iPS-derived neural progenitor cells. (A) B7H6 (NCR3LG1) transcription was assessed by qPCR analysis using cDNA from the indicated cells, displaying relative expression using GNB2LI as housekeeping control. The tumor cell lines HCT15 and MDA.MB.231 have been included as B7H6+ and B7H6− controls, respectively. The results are normalized and represent 3 or more independent triplicate experiments. Neurons were generated from iPS-derived NPCs, and their phenotype was documented by (B) phase contrast microscopy demonstrating dendritic morphology, and (C) qPCR analysis for the neuronal markers NeuN (RBFOX3), MAP-2 (MAP2), class III β-tubulin (TuJ1; gene TUBB3), and synaptotagmin (SYT1), as indicated. NPC phenotype was defined by downregulation of the pluripotency marker Oct4 (POU5F1) and concomitant upregulation of nestin (NES). Expression was calculated using β-actin as an internal control. Error bars indicate SD based on 1 triplicate experiment.

Article Snippet: Following permeabilization in 0.1% Triton, the cells were stained with primary antibodies against the pluripotency markers Nanog (clone 7F7.1; 1:400; Millipore), Oct4, and SOX2 (cat. no. 090023 and 090024, respectively; 1:100; StemGent) overnight at 4 °C, washed, incubated for 1 hour at room temperature with Cy2-conjugated or Cy3-conjugated secondary antibodies (1:400; Jackson ImmunoResearch) followed by counterstaining of nuclei with DAPI.

Techniques: Expressing, Derivative Assay, Control, Generated, Microscopy, Marker

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: Identification of surface antigens for GSCs (A) Schematic illustrating the experimental design. Human astrocytes and GSCs (83 and X01) were profiled using multiple antibody libraries. (B) Heatmap showing the top 32 genes of human cell surface antigens, ranked based on the fold change values of fluorescence intensity compared to normal astrocytes or differentiated GSCs. (C and D) High-throughput confocal immunofluorescence (IF) staining images showing staining with the anti-CD97 antibody (C) and quantification of fluorescence in the antibody array (values normalized to those of astrocytes) (D). Scale bar, 50 μm. All error bars represent the mean ± standard error of the mean (SEM; n = 3 independent experiments). (E) Quantitative reverse-transcription PCR (RT-qPCR) and immunoblot (IB) analyses of CD97 expression in astrocytes, GSCs, and serum-differentiated GSCs (83-Diff and X01-Diff). β-actin was used as a loading control. Error bars represent the mean ± standard deviation (SD; n = 3 independent experiments). (F) Flow cytometry analysis of astrocytes, GSCs, and serum-differentiated GSCs (83-Diff and X01-Diff) using a phycoerythrin (PE)-conjugated anti-CD97 antibody. (G) Flow cytometry analysis of CD97 expression on patient-derived GSCs, stained with a PE-conjugated anti-CD97 antibody. (H and I) CD97 protein expression in NT and GBM tissues (H) and the expression in each GBM subtype (I) according to the CPTAC database. NT, nontumor; CL, classical; MES, mesenchymal; NL, neural; PN, proneural. (J) Kaplan-Meier overall survival curves for patients with GBM presenting high and low CD97 protein expression according to the CPTAC database. Groups were divided by optimal cutpoints using “survminer” R package. The p value was determined using the log rank (Mantel-Cox) test. ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗ p < 0.05, t test in (C), (D), (E), and (H). See also Figure S1 .

Article Snippet: The membranes were incubated with primary antibodies against CD97 (Abcam), Nestin (BD Transduction Laboratories), Oct3/4 (Proteintech), Nanog (Sangon biotech), p-S6K S371 (Cell Signaling Technology), p-S6K T389 (Cell Signaling Technology), S6K (Cell Signaling Technology), p -AKT T308 (Cell Signaling Technology), p -AKT S473 (Cell Signaling Technology), AKT (Cell Signaling Technology), ARHGAP1 (Santa Cruz), BZW1 (GeneTex), BZW2 (Bethyl Laboratories), CD133 (Proteintech), CD44 (R&D Systems), SOX2 (R&D Systems), Vinculin (Sigma-Aldrich), and β-actin (Bioworld Technology) overnight at 4°C.

Techniques: Fluorescence, High Throughput Screening Assay, Immunofluorescence, Staining, Ab Array, Reverse Transcription, Quantitative RT-PCR, Western Blot, Expressing, Control, Standard Deviation, Flow Cytometry, Derivative Assay

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: Validation of CD97 as an optimal GSC marker (A) Bubble plot comparing expression of ADGRE5/CD97 and stem cell-related genes on GSCs and GBM tumors using single-cell RNA sequencing datasets from the study by Restall and Richards. (B) Scatterplots to compare the RNA expression level of ADGRE5 and NES according to patients with GBM in the TCGA, CCGA, and REMBRANDT databases. CD97 is encoded by ADGRE5 gene. Nestin is encoded by NES gene. (C) Scatterplots to compare the protein expression level of CD97 and Nestin according to patients with GBM in the CPTAC database. (D) Flow cytometry plots in which CD97-PE was co-stained along with other GSC markers, including Nestin, Oct 3/4, Nanog, CD133 or CD49f, CD15, or CD44 in 83, X01, and GSC772. Each gate on the x and y axis separates positive from negative cells. The numbers in each quadrant indicate the percentages of cells. (E) Limiting dilution assay (LDA) using CD97 high CD49f high , CD97 high CD49f −/low , CD97 −/low CD49f high , and CD97 −/low CD49f −/low GSC 83 cells sorted through flow cytometry. (F–H) Confocal images of CD97 (green) and Nestin (red) co-immunofluorescence staining in brain tissues of the X01-xenograft (F) and human patients with GBM who have Nestin Low (G) and Nestin High (H). Scale bar, 50 μm. Quantification analysis of the percentage of the cell number CD97 + /Nestin + and CD97 − /Nestin + cells. All error bars represent mean ± SD ( n = 3 independent experiments) in (E)–(H). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, t test. n.s, non-significant. See also Figures S2–S5 .

Article Snippet: The membranes were incubated with primary antibodies against CD97 (Abcam), Nestin (BD Transduction Laboratories), Oct3/4 (Proteintech), Nanog (Sangon biotech), p-S6K S371 (Cell Signaling Technology), p-S6K T389 (Cell Signaling Technology), S6K (Cell Signaling Technology), p -AKT T308 (Cell Signaling Technology), p -AKT S473 (Cell Signaling Technology), AKT (Cell Signaling Technology), ARHGAP1 (Santa Cruz), BZW1 (GeneTex), BZW2 (Bethyl Laboratories), CD133 (Proteintech), CD44 (R&D Systems), SOX2 (R&D Systems), Vinculin (Sigma-Aldrich), and β-actin (Bioworld Technology) overnight at 4°C.

Techniques: Biomarker Discovery, Marker, Expressing, RNA Sequencing, RNA Expression, Flow Cytometry, Staining, Limiting Dilution Assay, Immunofluorescence

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: Characterization of CD97 high cells in vitro and in vivo (A) GSC772 cells were stained with an anti-CD97 antibody and analyzed using flow cytometry. The Neg.Ctrl group is shown in gray and the Anti-CD97 group is shown in red. (B–E) RT-qPCR (B) and IB analysis (C) of stemness-related genes, proliferation assays (D), and LDAs (E) in the CD97 high and CD97 −/low subpopulations of GSC772. β-actin was used as a loading control. (F) GSC924 cells were stained with an anti-CD97 antibody and analyzed using flow cytometry as in (A). (G–J) RT-qPCR (G) and IB analysis (H) of stemness-related genes, proliferation assays (I), and LDAs (J) in the CD97 high and CD97 −/low subpopulations of GSC924. GAPDH was used as a loading control. (K) Kaplan-Meier survival curves of mice orthotopically implanted with CD97 high or CD97 −/low GSC772 cells ( n = 8, 2.5 × 10 5 cells/mouse). MST, median survival time. Log rank (Mantel-Cox) test. (L and M) Representative axial MRI (L) and the quantified tumor size (M) of mice bearing orthotopic xenografts of CD97 high or CD97 −/low GSC772 cells. Regions of interest (ROIs) within the tumor are indicated by white polygons. All error bars represent mean ± SD ( n = 3 independent experiments). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, t test in (B)–(J), (L), and (M).

Article Snippet: The membranes were incubated with primary antibodies against CD97 (Abcam), Nestin (BD Transduction Laboratories), Oct3/4 (Proteintech), Nanog (Sangon biotech), p-S6K S371 (Cell Signaling Technology), p-S6K T389 (Cell Signaling Technology), S6K (Cell Signaling Technology), p -AKT T308 (Cell Signaling Technology), p -AKT S473 (Cell Signaling Technology), AKT (Cell Signaling Technology), ARHGAP1 (Santa Cruz), BZW1 (GeneTex), BZW2 (Bethyl Laboratories), CD133 (Proteintech), CD44 (R&D Systems), SOX2 (R&D Systems), Vinculin (Sigma-Aldrich), and β-actin (Bioworld Technology) overnight at 4°C.

Techniques: In Vitro, In Vivo, Staining, Flow Cytometry, Quantitative RT-PCR, Control

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: CD97 regulates GSC tumorigenicity (A–D) RT-qPCR (A) of CD97 expression, IB analysis (B) of GSC intracellular markers, LDAs (C), and cell proliferation assays (D) in three different GSCs (83, X01, and 528 cells) infected with shCtrl, shCD97-1, or shCD97-3 lentivirus. β-actin, GAPDH, and vinculin were used as loading controls. All error bars represent mean ± SD ( n = 3 independent experiments). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, t test. (E) Flow cytometry analysis of Annexin V and PI staining in GSCs 83 infected with shCtrl, shCD97-1, or shCD97-3 lentivirus. (F) Kaplan-Meier survival curves of mice bearing orthotopic xenografts of three GSCs infected with shRNA lentivirus ( n = 5 or 6, 1 × 10 4 for 83 and X01, or 1 × 10 5 for 528 cells/mouse). MST, median survival time. Log rank (Mantel-Cox) test. See also Figures S6–S10 .

Article Snippet: The membranes were incubated with primary antibodies against CD97 (Abcam), Nestin (BD Transduction Laboratories), Oct3/4 (Proteintech), Nanog (Sangon biotech), p-S6K S371 (Cell Signaling Technology), p-S6K T389 (Cell Signaling Technology), S6K (Cell Signaling Technology), p -AKT T308 (Cell Signaling Technology), p -AKT S473 (Cell Signaling Technology), AKT (Cell Signaling Technology), ARHGAP1 (Santa Cruz), BZW1 (GeneTex), BZW2 (Bethyl Laboratories), CD133 (Proteintech), CD44 (R&D Systems), SOX2 (R&D Systems), Vinculin (Sigma-Aldrich), and β-actin (Bioworld Technology) overnight at 4°C.

Techniques: Quantitative RT-PCR, Expressing, Infection, Flow Cytometry, Staining, shRNA

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: Screening of downstream targets of CD97 in GSCs (A) Schematic representation of the experimental strategy for the transcriptome analysis. Heatmap of the hierarchical clustering analysis showing the expression of 131 common downregulated genes in CD97-knockdown GSCs (right). (B) Gene set enrichment analysis (GSEA) plot for the regulation of stem cell population maintenance signature from the Gene Ontology Biological Process (GOBP) category comparing the shCtrl and shCD97 groups. NES, normalized enrichment score. (C) Bubble chart showing the top 10 biological processes of Gene Ontology terms for genes downregulated in CD97-knockdown GSCs. Size, gene number; color, −log 10 ( p value). (D) RT-qPCR of PCBP1, BSG, ARHGAP1, BZW1, BZW2, and DBN1 expression in three different GSCs (83, X01, and 528 cells) infected with shCtrl or shCD97 lentivirus. β-actin was used as a loading control. All error bars represent mean ± SD ( n = 3 independent experiments). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, t test. (E) Kaplan-Meier overall survival curves for patients with GBM presenting the high and low expression of the CD97/ARHGAP1 (left), CD97/BZW1 (middle), and CD97/BZW2 (right) mRNAs according to TCGA GBM database. Log rank (Mantel-Cox) test. (F) Bubble chart showing the top 10 hallmark genes enriched in shCtrl GSCs. Size, gene number; color, −log 2 ( p value). (G) GSEA plot for the PI3K/AKT/mTOR signaling signature from the Hallmark category comparing the shCtrl and shCD97 groups. See also Figure S11 .

Article Snippet: The membranes were incubated with primary antibodies against CD97 (Abcam), Nestin (BD Transduction Laboratories), Oct3/4 (Proteintech), Nanog (Sangon biotech), p-S6K S371 (Cell Signaling Technology), p-S6K T389 (Cell Signaling Technology), S6K (Cell Signaling Technology), p -AKT T308 (Cell Signaling Technology), p -AKT S473 (Cell Signaling Technology), AKT (Cell Signaling Technology), ARHGAP1 (Santa Cruz), BZW1 (GeneTex), BZW2 (Bethyl Laboratories), CD133 (Proteintech), CD44 (R&D Systems), SOX2 (R&D Systems), Vinculin (Sigma-Aldrich), and β-actin (Bioworld Technology) overnight at 4°C.

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Infection, Control

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: mTORC2 inhibition prevents proliferation and self-renewal in GSCs (A) IB analysis of CD97, p-S6K, p-AKT, S6K, AKT, ARHGAP1, BZW1, and BZW2 levels in three different GSCs (83, X01, and 528 cells) infected with shCtrl or shCD97 lentivirus. (B) RT-qPCR of BZW1 expression (left), cell proliferation assay (middle), and LDAs (right) in 83 and X01 GSCs infected with shCtrl and shCD97 lentivirus, followed by BZW1 lentivirus infection. (C–F) IB analysis of p-S6K, p-AKT, S6K, AKT, ARHGAP1, BZW1, and BZW2 levels in three different GSCs (83, X01, and 528 cells) treated with Torin1 48 h (C), AKT inhibitor IV 24 h (D), 24 h rapamycin (E), and JR-AB2-011 24 h (F). (G) Cell proliferation assays (left) and LDAs (right) were performed on 83 and X01 GSCs after treatment with JR-AB2-011. (H) Kaplan-Meier survival curves of mice orthotopically implanted with X01-Luc cells ( n = 5, 1 × 10 5 cells/mouse) and intraperitoneally (i.p.) treated with JR-AB2-011 (4 mg/kg) or vehicle. MST, median survival time. Log rank (Mantel-Cox) test. (I) Schematic representation of the CD97-related signaling pathway regulating the proliferation, self-renewal, and tumor progression of GSCs. Vinculin and GAPDH, and β-actin were used as loading controls in IB, β-actin was used as a loading control in RT-PCR. All error bars represent mean ± SD ( n = 3 independent experiments) in (B) and (G). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗ p < 0.05, t test. See also Figures S12–S15 .

Article Snippet: The membranes were incubated with primary antibodies against CD97 (Abcam), Nestin (BD Transduction Laboratories), Oct3/4 (Proteintech), Nanog (Sangon biotech), p-S6K S371 (Cell Signaling Technology), p-S6K T389 (Cell Signaling Technology), S6K (Cell Signaling Technology), p -AKT T308 (Cell Signaling Technology), p -AKT S473 (Cell Signaling Technology), AKT (Cell Signaling Technology), ARHGAP1 (Santa Cruz), BZW1 (GeneTex), BZW2 (Bethyl Laboratories), CD133 (Proteintech), CD44 (R&D Systems), SOX2 (R&D Systems), Vinculin (Sigma-Aldrich), and β-actin (Bioworld Technology) overnight at 4°C.

Techniques: Inhibition, Infection, Quantitative RT-PCR, Expressing, Proliferation Assay, Control, Reverse Transcription Polymerase Chain Reaction

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: Antitumor activity of CD97-targeting CAR Th9 cells against GBM (A) Schematic showing the structure of the second-generation lentiviral vectors encoding CD97-CAR, including the 4-1BB and CD3ζ costimulatory domains. TM, transmembrane domain. (B) RT-qPCR analysis of IL-9 expression in CD8 + T cells, CD4 + T cells, and Th9 cells. (C) Flow cytometry analysis of CAR expression detected by the fusion protein EGFP. (D) Expansion kinetics of untreated, Ctrl-CAR, and CD97-CAR T cells in vitro ( n = 3 independent experiments). (E) Antigen-specific in vitro cytotoxicity of CD97-CAR Th9 cells or Ctrl-CAR Th9 cells toward U87-Luc, 83-Luc, or X01-Luc cells, evaluated using a luciferase activity assay. (F) Flow cytometry analysis of CD4 and CD107a expression on CD97-CAR Th9 cells following incubation with 83-Luc and X01-Luc cells. (G) Representative confocal microscopy images showing CD97-CAR Th9 cells or Ctrl-CAR Th9 cells interacting with 83-Luc and X01-Luc cells at an E:T ratio of 1:1 for 24 h. Scale bar, 50 μm. (H and I) Antigen-specific in vitro cytotoxicity of CD97-CAR Th9 cells toward 83-Luc or X01-Luc cells infected with shCD97 or shCtrl lentivirus (H) and against X01-Luc cells at different E:T ratios (I). (J) Schematic experimental design to evaluate the antitumor effects of CD97-targeting CAR Th9 cells. Mice were implanted with X01-Luc cells ( n = 6, 1× 10 5 cells/mouse) and intracranially injected with CD97-CAR or Ctrl-CAR Th9 cells (1.5 × 10 6 cells/mouse) on day 4. (K) Flow cytometry analysis showing the expression of CD4 and CD107a within the tumor two days post-treatment of CD97-CAR Th9 or Ctrl-CAR Th9 cells. (L) Bioluminescence images of mice as in (J). (M) Quantification of bioluminescence intensity of photons emitted from each tumor in the images in (L). Data are presented as mean ± SD, two-way ANOVA at day 24, ∗∗∗∗ p < 0.0001. (N) Kaplan-Meier survival curve of mice as in (J). MST, median survival time. Log rank (Mantel-Cox) test. GAPDH was used as a loading control. All error bars represent mean ± SD ( n = 3 independent experiments) in (B), (D), (E), (H), and (I). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, t test. See also Figures S16–S18 .

Article Snippet: The membranes were incubated with primary antibodies against CD97 (Abcam), Nestin (BD Transduction Laboratories), Oct3/4 (Proteintech), Nanog (Sangon biotech), p-S6K S371 (Cell Signaling Technology), p-S6K T389 (Cell Signaling Technology), S6K (Cell Signaling Technology), p -AKT T308 (Cell Signaling Technology), p -AKT S473 (Cell Signaling Technology), AKT (Cell Signaling Technology), ARHGAP1 (Santa Cruz), BZW1 (GeneTex), BZW2 (Bethyl Laboratories), CD133 (Proteintech), CD44 (R&D Systems), SOX2 (R&D Systems), Vinculin (Sigma-Aldrich), and β-actin (Bioworld Technology) overnight at 4°C.

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Flow Cytometry, In Vitro, Luciferase, Incubation, Confocal Microscopy, Infection, Injection, Control

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: The membranes were incubated with primary antibodies against CD97 (Abcam), Nestin (BD Transduction Laboratories), Oct3/4 (Proteintech), Nanog (Sangon biotech), p-S6K S371 (Cell Signaling Technology), p-S6K T389 (Cell Signaling Technology), S6K (Cell Signaling Technology), p -AKT T308 (Cell Signaling Technology), p -AKT S473 (Cell Signaling Technology), AKT (Cell Signaling Technology), ARHGAP1 (Santa Cruz), BZW1 (GeneTex), BZW2 (Bethyl Laboratories), CD133 (Proteintech), CD44 (R&D Systems), SOX2 (R&D Systems), Vinculin (Sigma-Aldrich), and β-actin (Bioworld Technology) overnight at 4°C.

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

Journal: International Journal of Veterinary Science and Medicine

Article Title: Generation of induced pluripotent stem cells from the Asian bats

doi: 10.1080/23144599.2024.2384835

Figure Lengend Snippet: Immunofluorescence and qPCR characterization of BatiPSC. To identify the cell type most closely resembling iPSCs, we conducted OCT4 (scale bar = 250 µm) and nanog (scale bar = 75 µm) immunofluorescence staining (A) and RT-qPCR (B). The findings demonstrated strong expression of pluripotent stem cell markers, particularly in colonies with a dome-shaped structure. Asterisks were used to indicate statistical significance between the groups (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The cells were subsequently incubated with primary mouse antibodies against Oct4 (P0082-200UL, Sigma-Aldrich), and primary rabbit antibodies against Nanog (RCAB004P-F, ReproCELL, Beltsville, MD, USA) at 4°C overnight.

Techniques: Immunofluorescence, Staining, Quantitative RT-PCR, Expressing

Journal: Frontiers in immunology

Article Title: A novel iPSC-based model of ICF syndrome subtype 2 recapitulates the molecular phenotype of ZBTB24 deficiency.

doi: 10.3389/fimmu.2024.1419748

Figure Lengend Snippet: FIGURE 1 Molecular characterization of pYM-iPSCs. (A) Expression of pluripotency markers hTERT, LIN28, SOX2, DNMT3B, OCT3/4, NANOG and KLF4 transcripts measured by RT-PCR in a representative clone of pYM-iPSCs (C6); (B) Expression of OCT3/4, NANOG and DNMT3B pluripotency markers measured by immunofluorescence in the pYM C6 clone. DAPI, nuclear staining. Bar: 100mm; (C) Dot plot showing the expression of the pluripotency marker SSEA4 in the pYM C6 clone compared to the negative control (CTRL-). PE-CF594-A, conjugated secondary antibody; (D) Karyotype analysis demonstrating chromosomal integrity of the pYM C6 clone; (E) Immunostaining of markers representing the three embryonic germ layers, GATA4 (endoderm), bIII-tubulin (ectoderm) and MF20 (mesoderm) expressed in the in vitro differentiated C6 clone. Bar: 100 mm; (F) ZBTB24, CDCA7, CDC40 and OSTC transcripts level in control (C14 and C22) and pYM (C6 and B9) iPSC clones measured by qPCR. The relative expression compared to the expression of GAPDH is shown for each gene. qPCR data are presented as mean ± SD from independent triplicates. Statistical analyses were performed using a one-side two-sample Student’s t-test compared to control: (*) P-value < 0.05, (***) P-value < 0.001.

Article Snippet: Primary antibodies against NANOG (SCTB, sc-293121), DNMT3B (Diagenode, pAb-076-005), OCT3/4 (SCTB, sc-5279), GATA4 (SCBT, SC25310), bIII-tubulin (Sigma-Aldrich, T8660), MF20 (DSHB, RRID: AB_2147781) were incubated overnight at 4° C. The cells were then incubated with secondary antibodies: AlexaFluor 594 conjugated goat anti-mouse IgG, AlexaFluor 594 Frontiers in Immunology 03 conjugated donkey anti-rabbit IgG (ThermoFisher).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Marker, Negative Control, Immunostaining, In Vitro, Control, Clone Assay