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