m 6 a-circrna epitranscriptomic microarray slide  (Arraystar inc)

Journal: Experimental Hematology & Oncology

Article Title: METTL3 modulates m6A modification of CDC25B and promotes head and neck squamous cell carcinoma malignant progression

doi: 10.1186/s40164-022-00256-3

Figure Lengend Snippet: METTL3 high expression is associated with poor prognosis of HNSCC patients. A Dot blot assay was conducted with mRNA extracted from HNSCC tissues and paired paracancerous normal tissues using an anti-m6A antibody, and MB (methylene blue) staining served as the loading control (representative images in left panel). The relative m6A contents on mRNA in HNSCC tissues and paired normal normal tissues were calculated (right panel, n = 7). B TCGA data showed that METTL3 expression was significantly upregulated in HNSCC (n = 520) than normal tissue (n = 44). C Disease-free survival (RFS) of HNSCC patients based on METTL3 expression obtained from GEPIA website ( http://gepia.cancer-pku.cn/ ). D The levels of METTL3 expression in HNSCC and paired normal tissues were measured by qRT-PCR (n = 10). E METTL3 protein levels were measured in HNSCC tissues and paired normal tissues by western blotting (n = 7). F METTL3 expression was significantly upregulated in HNSCC compared with the paired paracancerous normal tissue by IHC staining (n = 5, scale bars = 100 μm). G Kaplan–Meier OS analysis of HNSCC patients based on METTL3 expression measured by IHC of tissue microarray (n = 100). H Univariate Cox regression analysis was conducted in HNSCC patients (n = 100). All bars correspond to 95% confidence intervals. I Multivariate Cox regression analysis was conducted in HNSCC patients (n = 100). J The time-dependent receiver operating characteristic (ROC) analysis for the clinical risk score (TNM stage), the METTL3 risk score, and the combined METTL3 and clinical risk scores in HNSCC cohort

Article Snippet: After hybridizing these cRNAs onto a Arraystar Epitranscriptomic Microarray slide (8 × 60 K, Arraystar) and washing it, an Agilent Scanner G2505C was used to scan the array.

Techniques: Expressing, Dot Blot, Staining, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Microarray

Journal: Experimental Hematology & Oncology

Article Title: METTL3 modulates m6A modification of CDC25B and promotes head and neck squamous cell carcinoma malignant progression

doi: 10.1186/s40164-022-00256-3

Figure Lengend Snippet: METTL3 mediates the m6A modification on CDC25B mRNA in HNSCC. A m6A-mRNA epitranscriptomic microarray showed signal pathways in which most differentially expressed gene enriched in METTL3 knockdown cells (SAS), DE represents differentially expressed. B m6A-mRNA epitranscriptomic microarray showed signal pathways in which most differentially methylated genes enriched in METTL3 knockdown cells (SAS), DM represents differentially methylated. C m6A-mRNA epitranscriptomic microarray showed an overlap of the total differentially expressed gene, total differentially methylated gene, and differentially expressed and methylated gene enriched in the cell cycle pathway. D Genes selected from the overlap were used for qRT-PCR in METTL3 knockdown and their corresponding control cells, and CDC25B was the most significantly downregulated gene upon knockdown of METTL3. E , F CDC25B mRNA expression was confirmed by qRT-PCR in METTL3 knockdown (FaDu) and METTL3 overexpression (Hep2) cells. G CDC25B protein level was measured by western blot assay in METTL3 knockdown SAS cells. H CDC25B protein level was measured by western blot assay in METTL3 knockdown FaDu cells. I CDC25B protein level was measured by western blot assay in METTL3 overexpressed Hep2 cells. J MeRIP-qPCR was conducted to detect the m6A level of CDC25B mRNA in METTL3 knockdown (SAS) cells. K MeRIP-qPCR was conducted to detect the m6A level of CDC25B mRNA in METTL3 knockdown (FaDu) cells. L , M The levels of CDC25B expression in METTL3 knockdown and their corresponding control cells treated with actinomycin D (5 μg/mL) at the indicated time points were detected by qRT-PCR. The data are the means ± SD of three independent experiments. */# p < 0.05; **/## p < 0.01; ***/### p < 0.001; ****/#### p < 0.0001

Article Snippet: After hybridizing these cRNAs onto a Arraystar Epitranscriptomic Microarray slide (8 × 60 K, Arraystar) and washing it, an Agilent Scanner G2505C was used to scan the array.

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