Journal:

Article Title: Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells D⃞

doi: 10.1091/mbc.E05-06-0572

Figure Lengend Snippet: Morphology of 293T cells treated with NCCIT extract. (A) Untreated 293T and NCCIT cells. (B) 293T cells at indicated time points after exposure to NCCIT (a–e) or 293T (f–j) extract. (C) 293T cells 10 d after exposure to Jurkat extract and cultured under T-cell growth conditions. Dark spots are Dynal Biotech (Montebello, Norway) magnetic beads bearing antibodies against CD3 and CD28 surface antigens and used to promote T-cell expansion. Bars, 30 μm.

Article Snippet: NCCIT, Jurkat (clone E6-1), and 293T cells (American Type Culture Collection, Vanassas, MD) were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal calf serum (FCS), 2 mM l -glutamine, 1 mM sodium pyruvate, and nonessential amino acids (complete RPMI 1640 medium).

Techniques: Cell Culture, Magnetic Beads

Journal:

Article Title: Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells D⃞

doi: 10.1091/mbc.E05-06-0572

Figure Lengend Snippet: Immunofluorescence analysis of Oct4, lamin A/C and B-type lamin expression in 293T cells exposed to NCCIT extract. Untreated NCCIT and 293T cells (A) and 293T cells (B) treated with NCCIT or 293T extract were immunolabeled with antibodies against Oct4, lamin A/C, and B-type lamins (B, 1 wk after extract treatment). Bars, 20 μm. (C) Proportions (mean ± SD) of untreated NCCIT and 293T cells and of extract-treated cells expressing Oct4, lamin A/C, and B-type lamins. Three sets of 200 cells were examined for each marker. *p < 0.05 compared with 293T cells (t test); **p < 0.001 compared with 293T cells and 293T cells treated with 293T extract (t test).

Article Snippet: NCCIT, Jurkat (clone E6-1), and 293T cells (American Type Culture Collection, Vanassas, MD) were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal calf serum (FCS), 2 mM l -glutamine, 1 mM sodium pyruvate, and nonessential amino acids (complete RPMI 1640 medium).

Techniques: Immunofluorescence, Expressing, Immunolabeling, Marker

Journal:

Article Title: Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells D⃞

doi: 10.1091/mbc.E05-06-0572

Figure Lengend Snippet: Quantitative RT-PCR analysis of expression of indicated stem cell genes in 293T cells treated with NCCIT extract

Article Snippet: NCCIT, Jurkat (clone E6-1), and 293T cells (American Type Culture Collection, Vanassas, MD) were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal calf serum (FCS), 2 mM l -glutamine, 1 mM sodium pyruvate, and nonessential amino acids (complete RPMI 1640 medium).

Techniques: Quantitative RT-PCR, Expressing

Journal:

Article Title: Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells D⃞

doi: 10.1091/mbc.E05-06-0572

Figure Lengend Snippet: Oct4 expression in EGFP-labeled 293T cells. (A) 293T cells stably expressing EGFP and a geneticin resistance (GenR) gene were treated with 293T or NCCIT extract and cultured for 2 wk with 700 ng/ml geneticin before immunolabeling with anti-Oct4 antibodies. NCCIT extract was also treated with 500 μg/ml DNAse I before incubating cells (bottom row). Bar, 20 μm. (B) Quantitative RT-PCR analysis of expression of indicated genes in 293T-EGFP-GenR cells 2 wk after incubation in intact or DNAse I-treated NCCIT extract (relative to 293T extract-treated controls). (C) PCR analysis of the presence of SV40 large T antigen in 293T, NCCIT, and extract-treated cells. Ladder is a 123-base pair DNA ladder.

Article Snippet: NCCIT, Jurkat (clone E6-1), and 293T cells (American Type Culture Collection, Vanassas, MD) were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal calf serum (FCS), 2 mM l -glutamine, 1 mM sodium pyruvate, and nonessential amino acids (complete RPMI 1640 medium).

Techniques: Expressing, Labeling, Stable Transfection, Cell Culture, Immunolabeling, Quantitative RT-PCR, Incubation

Journal:

Article Title: Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells D⃞

doi: 10.1091/mbc.E05-06-0572

Figure Lengend Snippet: Bisulfite sequencing analysis of DNA methylation changes in extract-treated cells. 293T cells, NCCIT cells, and cells treated with 293T or NCCIT extract were examined for cytosine methylation in underlined CpG dinucleotides within shown genomic regions of the human OCT4 (A), LMNA (B), and LMNB1 (C) genes. Diagrams show localization (nucleotide numbers) of regions examined relative to the ATG translation start (+1).

Article Snippet: NCCIT, Jurkat (clone E6-1), and 293T cells (American Type Culture Collection, Vanassas, MD) were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal calf serum (FCS), 2 mM l -glutamine, 1 mM sodium pyruvate, and nonessential amino acids (complete RPMI 1640 medium).

Techniques: Methylation Sequencing, DNA Methylation Assay, Methylation

Journal:

Article Title: Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells D⃞

doi: 10.1091/mbc.E05-06-0572

Figure Lengend Snippet: Microarray analysis of gene expression in extract-treated 293T cells. (A) Venn diagram identifying “NCCIT-specific” genes (yellow area). Numbers of genes up- or down-regulated more than threefold (relative to input 293T cells) in cells incubated in extract of NCCIT (B), 293T (C), and Jurkat (D) cells (mean ± SD of two [B and C] and four [D] experiments). Yellow bars indicate genes up- or down-regulated in extract-treated cells and shared with NCCIT cells. In B, the likelihood that NCCIT genes are up- or down-regulated by chance rather than by extract treatment is extremely low (p < 10-5 and p < 10-4, respectively; t tests). By contrast, in C and D these probabilities are relatively high (p > 0.07 and p > 0.08, respectively; t tests). (E) Percentage of NCCIT genes up- or down-regulated in extract-treated cells (percentage of total up- or down-regulated genes). (F) Number of NCCIT genes specifically up- or down-regulated by treatment with NCCIT extract, over time. (G) Consistency of gene up- or down-regulation over time after treatment with NCCIT extract. Numbers of up- and down-regulated NCCIT genes in cells exposed to NCCIT extract are shown in green and red. Gray bars represent genes consistently up- or down-regulated at weeks 1 and 2 (gray bars at week 2), weeks 1, 2, and 4 (gray bars at week 4), etc., and shared between the two experiments. (H) Functional class distribution of genes consistently up- or down-regulated over 8 wk in two experiments (gray bars in G). These genes are listed in Table S3.

Article Snippet: NCCIT, Jurkat (clone E6-1), and 293T cells (American Type Culture Collection, Vanassas, MD) were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal calf serum (FCS), 2 mM l -glutamine, 1 mM sodium pyruvate, and nonessential amino acids (complete RPMI 1640 medium).

Techniques: Microarray, Gene Expression, Incubation, Functional Assay

Journal:

Article Title: Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells D⃞

doi: 10.1091/mbc.E05-06-0572

Figure Lengend Snippet: Quantitative RT-PCR analysis of expression of indicated multilineage priming genes in 293T cells treated with NCCIT extract relative to transcript levels in 293T cells exposed to 293T extract. Expression levels were adjusted to those of GAPDH in triplicate samples. Single data points show mean expression level in NCCIT cells.

Article Snippet: NCCIT, Jurkat (clone E6-1), and 293T cells (American Type Culture Collection, Vanassas, MD) were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal calf serum (FCS), 2 mM l -glutamine, 1 mM sodium pyruvate, and nonessential amino acids (complete RPMI 1640 medium).

Techniques: Quantitative RT-PCR, Expressing

Journal:

Article Title: Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells D⃞

doi: 10.1091/mbc.E05-06-0572

Figure Lengend Snippet: Neuronal differentiation of 293T cells treated with 293T or NCCIT extract. (A) Top, induction of differentiation. Suspended aggregates after 2 wk of culture with 10 μM all-trans-retinoic acid. Bottom, differentiation. Cells were plated onto poly-l-lysine-coated coverslips after 2 d of culture in the absence of retinoic acid but with mitotic inhibitors. Note neurite extensions in NCCIT extract-treated cells. (B) NCCIT extract-treated cells either treated with retinoic acid (+RA) or not (-RA) for 3 wk were immunolabeled using antibodies against Oct4 and nestin. Graph shows proportions (mean ± SD) of cells immunolabeled with anti-Oct4 and anti-nestin antibodies (n = 200 cells in each of a triplicate analysis for each treatment). (C) Quantitative RT-PCR analysis of expression of OCT4, NES (nestin), LMNA and LMNB1 in 293T cells treated with 293T or NCCIT extract, in absence (-RA) or presence (+RA) or retinoic acid for 3 wk. (D) NeuN and NF200 immunofluorescence analysis of indicated cell types induced to differentiate as described in A. Insets, DNA labeled with Hoechst 33342. Bars, 400 μm (A, top); 40 μm (A, bottom); 40 μm (B and D).

Article Snippet: NCCIT, Jurkat (clone E6-1), and 293T cells (American Type Culture Collection, Vanassas, MD) were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal calf serum (FCS), 2 mM l -glutamine, 1 mM sodium pyruvate, and nonessential amino acids (complete RPMI 1640 medium).

Techniques: Immunolabeling, Quantitative RT-PCR, Expressing, Immunofluorescence, Labeling

Journal:

Article Title: Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells D⃞

doi: 10.1091/mbc.E05-06-0572

Figure Lengend Snippet: Induction of adipogenic, osteogenic, and endothelial differentiation of 293T cells treated with 293T or NCCIT extract. (A and B) Cells were exposed to 10 μM retinoic acid for 21 d, washed, and cultured in the absence of retinoic acid in adipocyte (A) or osteoblast (B) differentiation medium for 21 d. (A) Cells were stained with Oil-Red-O to reveal lipid droplets. Images on the right are enlargements of two areas framed in white in the adjacent panel (top right-hand quadrant). Arrows point to strongly stained lipid droplets. (B) Cells were stained with Alzarin red to visualize mineralized nodules (arrows). Graph shows mean ± SD number of distinct strongly mineralized nodules (arrows) per unit area (area shown in B). Twenty-four to 27 areas were analyzed within wells of six-well culture plates. *p < 10-6 relative to other treatments (ANOVA). (C) 293T and NCCIT extract-treated cells were passaged onto methylcellulose for 7 d to elicit endothelial differentiation. Note the formation of a track phenotype characteristic of cultured endothelial cells (right). (D) Direct immunofluorescence labeling of CD31 and CD144 surface antigens in extract-treated cells induced to differentiate as described in C. After differentiation, cells were loosened from the methylcellulose semisolid substrate by dilution with PBS and thus lost their elongated phenotype. Bars, 40 μm (A), 200 μm (B and C), and 40 μm (D).

Article Snippet: NCCIT, Jurkat (clone E6-1), and 293T cells (American Type Culture Collection, Vanassas, MD) were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal calf serum (FCS), 2 mM l -glutamine, 1 mM sodium pyruvate, and nonessential amino acids (complete RPMI 1640 medium).

Techniques: Cell Culture, Staining, Immunofluorescence, Labeling

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The component of the m 6 A writer complex VIRMA is implicated in aggressive tumor phenotype, DNA damage response and cisplatin resistance in germ cell tumors

doi: 10.1186/s13046-021-02072-9

Figure Lengend Snippet: Knockdown of VIRMA attenuates the malignant phenotype and enhances sensitivity to cisplatin in vitro. A : CRISPR/Cas9-mediated knockdown of VIRMA in NCCIT cells (~ 50% reduction), leading to decreased protein expression of other members of the writer complex – METTL3, WTAP and METTL14. Results are normalized to ß-actin and expressed as fold-change compared to scramble condition; B : Relative levels of m 6 A, expressed as fold-change compared to scramble condition, both by ELISA kit (top) and dot blot (bottom, normalized to methylene blue); C – Illustration of the m 6 A writer complex and hypothesis related to its disruption upon VIRMA knockdown; D – Tumor cell growth curves in VIRMA knockdown cells compared to scramble condition along 72 h; E – Measurement of tumor cell proliferation by BrdU assay along 72 h. Results are expressed as fold-change compared to scramble condition; F – Measurement of migration capacity. Results are expressed as fold-change compared to scramble condition; G - Measurement of invasion capacity. Results are expressed as fold-change compared to scramble condition; H – Cell viability curves for NCCIT-VIRMA knockdown and scramble cells treated with cisplatin. Results are expressed as percentage cells surviving, normalized to the vehicle. IC 50 concentration is indicated for each condition. * p < 0.05; ** p < 0.01; **** p < 0.0001

Article Snippet: NCCIT cell line (the one showing the highest resistance to cisplatin compared to 2102Ep and NT2, as documented in our previous study [ ]) was chosen to perform VIRMA knockdown by plasmids carrying the CRISPR/Cas9 system containing a guide RNA sequence (available in [ ]) targeting this gene (obtained from GenScript, Piscataway, NJ).

Techniques: Knockdown, In Vitro, CRISPR, Expressing, Enzyme-linked Immunosorbent Assay, Dot Blot, Disruption, BrdU Staining, Migration, Concentration Assay

Journal: PLoS ONE

Article Title: A Data Integration Approach to Mapping OCT4 Gene Regulatory Networks Operative in Embryonic Stem Cells and Embryonal Carcinoma Cells

doi: 10.1371/journal.pone.0010709

Figure Lengend Snippet: A: Bandshifts showing supershifts with OCT4 antibody using NCCIT cells derived- nuclear extracts using two probes in the 5′region of the GADD45G promoter containing an OCT4 motif at positions 9–15 (lane 1–3) and 17–23 (lane 4–6) of 31 nucleotides. Lane 3,6: Nuclear extract plus labelled probe. Lane 2,5: same as lanes 3 and 6 but with the addition of OCT4 antibody (sc-9081). Lane 1,4: same as lanes 3 and 6 but with the addition of a 20-fold increase in unlabelled competitor oligo. B: Multi-species alignment of the selected region chosen for the bandshift assay, the conserved OCT4 binding site is highlighted in red. C: Real time PCR confirmation of the presence of the OCT4 binding site. Position 0 indicates the position shown in the alignment in panel 2B.

Article Snippet: NCCIT cells were grown in high-glucose DMEM supplemented with 10% FCS (Biochrom, Berlin/Germany), 2 mM glutamine, and penicillin/streptomycin on conventional tissue culture plastic surfaces.

Techniques: Derivative Assay, Binding Assay, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: A Data Integration Approach to Mapping OCT4 Gene Regulatory Networks Operative in Embryonic Stem Cells and Embryonal Carcinoma Cells

doi: 10.1371/journal.pone.0010709

Figure Lengend Snippet: De novo motif discovery for genes, identified as OCT4 indirect targets and differentially regulated (2-fold and above) in NCCIT cells but lacking the OCT4 and SOX2 motif within the promoter region analysed. The 4 most significant motifs identified and the potential transcription factor binding sites related to these motifs are displayed. In addition, putative regulated genes harbouring these motifs in their promoter regions shown. Red depicts up-regulated and green down-regulated in response to the ablation of OCT4 activity in ES and EC cells.

Article Snippet: NCCIT cells were grown in high-glucose DMEM supplemented with 10% FCS (Biochrom, Berlin/Germany), 2 mM glutamine, and penicillin/streptomycin on conventional tissue culture plastic surfaces.

Techniques: Binding Assay, Activity Assay

Journal: Frontiers in Genetics

Article Title: Transcript Isoforms of SLC7A11-AS1 Are Associated With Varicocele-Related Male Infertility

doi: 10.3389/fgene.2020.01015

Figure Lengend Snippet: Regulation of SLC7A11 by SLC-AS6-7. (A) Chromosomal organization of SLC7A11 and two SLC7A11-AS1 isoforms, SLC7A11-AS1:6 (SLC-AS6) and SLC7A11-AS1:7 (SLC-AS7). Exons are illustrated as solid boxed lines, whereas introns are represented as barbed lines indicating the direction of transcription. Red dashed boxes indicated overlapping complementary regions between SLC7A11 and SLC-AS6-7. (B) The expression levels of SLC-AS6-7 negatively correlated with SLC7A11. Pearson’s coefficient correlation was implicated for correlation analysis. (C) When SLC-AS6 was overexpressed in NT2 and NCCIT cells, significant downregulation of SLC7A11expression levels was detected by qRT-PCR. Error bars show standard error of mean (SEM) of triplicated experiments (** P < 0.01 and **** P < 0.0001 using independent sample t -test). (D) Overexpression of SLC-AS6 leads to downregulation of SLC7A11 protein level in NT2 and NCCIT cells. Cropped images were used for Western blot, and uncropped images are presented in .

Article Snippet: NCCIT cells (Pasteur Institute, Iran, Tehran) were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin.

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Western Blot

Journal: Frontiers in Genetics

Article Title: Transcript Isoforms of SLC7A11-AS1 Are Associated With Varicocele-Related Male Infertility

doi: 10.3389/fgene.2020.01015

Figure Lengend Snippet: Assessment of oxidative stress markers after overexpression of SLC-AS6. (A) Transfecting SLC-AS6 diminished the glutathione (GSH) levels significantly relative to mock-transfected cells in NT2 and NCCIT cell lines. (B) Cytosolic reactive oxygen species (ROS) levels were elevated significantly after overexpression of SLC-AS6 in NT2 and NCCIT cells. (C) Transfecting SLC-AS6 in NT2 cell line leads to an increase in lipid peroxidation detected by C11-BODIPY staining. Error bars show standard error of mean (SEM) of triplicated experiments (* P < 0.05 and ** P < 0.01 using independent sample t -test).

Article Snippet: NCCIT cells (Pasteur Institute, Iran, Tehran) were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin.

Techniques: Over Expression, Transfection, Staining

Journal: Frontiers in Genetics

Article Title: Transcript Isoforms of SLC7A11-AS1 Are Associated With Varicocele-Related Male Infertility

doi: 10.3389/fgene.2020.01015

Figure Lengend Snippet: Effect of SLC-AS6 overexpression on cell viability. (A) Percentages of apoptotic (Annexin-positive, PI-negative) and necrotic cells (Annexin- and PI-positive) were elevated significantly in NT2 and NCCIT cell lines transfected with SLC-AS6. (B) MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium] assay results showed that overexpression of SLC-AS6 significantly decreased survival rate of NT2 and NCCIT cells lines. Error bars show standard error of mean (SEM) of triplicated experiments (* P < 0.05 using independent sample t -test).

Article Snippet: NCCIT cells (Pasteur Institute, Iran, Tehran) were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin.

Techniques: Over Expression, Transfection