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: Mesenchymal to Epithelial Transition Induced by Reprogramming Factors Attenuates the Malignancy of Cancer Cells

doi: 10.1371/journal.pone.0156904

Figure Lengend Snippet: (A) Parental human SCC cells (left) of HOC313 and TSU show fibroblast-like morphology; however, the reprogramming factors introduced cells (RICs, right panels) exhibit polygonal, epithelial-like morphology. The RICs from OSC-19 cells show more dens cell colony than the parental cells. (Scale bar, 100μm) (B) Cellular morphology is quantified by length to width ratio of each cell from at least 20 cells each from parental SCC cells (open circles) and RICs (closed circles). The ratio of RICs approximates to one, indicating them as epithelial, polygonal shaped cells. ** P < 0.01 by Student t -test. (C) The introduced reprogramming factors were evaluated by western blot analysis. P, Parental cells. R, RICs. β-Actin (ACTB) was used as loading control. (D-F) Expression of TRA-1-60 and TRA-81 was studied by western blotting (D) and flowcytometry (E, F). The representative data and mean ± SD of three independent experiments of flowcytometry were shown in E and F, respectively. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was used as loading control in (D). Blue lines; the specific antibodies, red lines; isotype control mouse IgM in (E). NCCIT, a human teratoma cell line, was used as a control in (D-F).

Article Snippet: The OSC-19 cells and NCCIT cells were purchased from the Japanese Collection of Research Bioresources Cell Bank (Ibaraki, Japan).

Techniques: Western Blot, Control, Expressing