Journal: Genome biology

Article Title: ADAR1 is a new target of METTL3 and plays a pro-oncogenic role in glioblastoma by an editing-independent mechanism.

doi: 10.1186/s13059-021-02271-9

Figure Lengend Snippet: Fig. 3 YTHDF1 binds m6A on ADAR1 mRNA boosting its translation without affecting decay. a Box plot showing YTHDF1 mRNA levels in GBM (red box) versus normal brain (blue box) (GEPIA 2, * p ≤0.05). b qRT- PCR of YTHDF1 and ADAR1 in siYTHDF1 U87MG cells (24–48 h post transfection-pt). On the right, western blotting analysis (48 h pt) of ADAR1 in siYTHDF1 U87MG cells is shown. GAPDH was used as control. c Relative enrichment of ADAR1 mRNA in YTHDF1-RIP (using two different antibodies Ab1 and Ab2) over IgG in U87MG cells. HPRT expression was used as negative control (n = 3) Values are represented as means ± SD, * p ≤0.05. d Ribosomal immunoprecipitation was performed in siYTHDF1 and siscr U87MG cells transfected with an RPL22-FLAG construct. Left, is shown a control western blotting analysis, while on the right, a qRT-PCR is shown. HPRT was used as control (n = 3). Values are represented as means ± SD, ** p ≤0.01. e ADAR1 mRNA stability in control and siYTHDF1 U87MG cells was determined by qRT-PCR after actinomycin D (5 μg/ml) treatment in the time indicated. Values are represented as means ± SD. On the right, the qRT-PCR of YTHDF1 silencing. Values are represented as means ± SD, *** p ≤0.001, n = 2. f Western blotting analysis of siYTHDF1 and control U87MG cells treated with or without MG132 at different concentrations (indicated in the figure); ADAR2 and ubiquitin Ab (Ubi) were used as controls. On the right, siYTHDF1 and sictrl U87MG cells were treated with or without 1.25 μM MG132 for 24 h. GAPDH was used as loading control. Values are represented as means ± SD, * p ≤0.05

Article Snippet: The antibodies used were as follows: ADAR1 (Santa Cruz Biotechnology), ADAR1 (Bethyl), CDK2 (Santa Cruz Biotechnology), YTHDF1 (Abcam), METTL3 (Abcam), METTL14 (Bethyl), cyclinE (Santa Cruz Biotechnology), p57 (Santa Cruz Biotechnology), Skp2 (Santa Cruz Biotechnology), CDC14B (LifeSpan), ADAR2 (Santa Cruz Biotechnology), Ubiquitin (Thermo Fisher), β-actin (Santa Cruz Biotechnology), GAPDH (Cell Signaling), and the anti-rabbit and anti-mouse peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology).

Techniques: Quantitative RT-PCR, Transfection, Western Blot, Control, Expressing, Negative Control, Immunoprecipitation, Construct, Ubiquitin Proteomics

Journal: Genome biology

Article Title: ADAR1 is a new target of METTL3 and plays a pro-oncogenic role in glioblastoma by an editing-independent mechanism.

doi: 10.1186/s13059-021-02271-9

Figure Lengend Snippet: Fig. 7 Targeting ADAR1 in growing tumor mass blocks glioblastoma progression. a Quantitative analysis of tumor size (tumor volume) of U87MG cells subcutaneously injected into the flank of NOD-SCID mice (n = 16 mice). Tumors generated by control (n = 8) or shADAR1 (n = 8) inducible U87MG cells were treated with DOXY (in drinking water). Tissues were collected and analyzed at the end of treatment (60 days p.i.). *p ≤0.05. Right, a representative picture of tumors. Representative sections and relative quantification of Ki67 (b), ADAR1 (c), and CDK2 (d) staining are shown. e qRT-PCR of CDK2 using mRNA obtained from the same samples. Data were normalized to the mean of controls values set to 1. *p ≤0.05, **p ≤0.01. f Schematic representation of METTL3/ADAR1 modulation in glioblastoma. METTL3/METTL14 methylates ADAR1 mRNA allowing the reader YTHDF1 to boost ADAR1 translation. The high level of ADAR1 protein (with unaltered ADAR1 mRNA) correlates with GBM patient OS and promotes cell proliferation by stabilizing CDK2. Moreover, the ablation of ADAR1 in an METTL3 unaltered background is sufficient to inhibit glioblastoma in vivo

Article Snippet: The antibodies used were as follows: ADAR1 (Santa Cruz Biotechnology), ADAR1 (Bethyl), CDK2 (Santa Cruz Biotechnology), YTHDF1 (Abcam), METTL3 (Abcam), METTL14 (Bethyl), cyclinE (Santa Cruz Biotechnology), p57 (Santa Cruz Biotechnology), Skp2 (Santa Cruz Biotechnology), CDC14B (LifeSpan), ADAR2 (Santa Cruz Biotechnology), Ubiquitin (Thermo Fisher), β-actin (Santa Cruz Biotechnology), GAPDH (Cell Signaling), and the anti-rabbit and anti-mouse peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology).

Techniques: Injection, Generated, Control, Quantitative Proteomics, Staining, Quantitative RT-PCR, In Vivo

Journal: BMC Cancer

Article Title: N6-methyladenosine RNA modification (m6A) is of prognostic value in HPV-dependent vulvar squamous cell carcinoma

doi: 10.1186/s12885-022-10010-x

Figure Lengend Snippet: Summary of the analyzed m6A proteins as indicated and their correlation with overall survival (indicated as %alive) for the entire study cohort, HPV-independent, and HPV-dependent VSCC. The HPV-status was not available for 24 patients. Samples were grouped according to high and low expression based on the staining intensities. p -values for the group comparisons are based on log-rank tests (significance threshold p < 0.5). q -values are based on multiple hypotheses testing using the method of Benjamini and Hochberg with a significance threshold of q < 0.1

Article Snippet: Immunostaining of METTL3, METTL4, METTL14, WTAP, KIAA1429, FTO, ALKBH5, HNRNPA2B1, HNRNPC, YTHDC1, YTHDF1,YTHDF2, and YTHDF3 was performed on the TMAs using an automated staining system (BenchMark ULTRA; Ventana Medical Systems) which performed deparaffinization, pretreatment with cell conditioning buffer (CC1 buffer, pH8), and incubation with primary antibodies (FTO (1:50; Atlas Antibodies #HPA041086), ALKBH5 (1:200; Novus #NBP1-82,188), METTL3 (1:1000; Biorbyt #orb374082), METTL4 (1:40; Atlas Antibodies #HPA040061), METTL14 (1:100; Atlas Antibodies #HPA038002), WTAP (1:100; Atlas Antibodies #HPA010550), KIAA1429 (1:25; Atlas Antibodies #HPA031530), HNRNPC (1:25; Atlas Antibodies #HPA051075), HNRNPA2B1 (1:100; Atlas Antibodies #HPA001666), YTHDC1 (1:25; Atlas Antibodies #HPA036462), YTHDF1 (1:10; Biorbyt #orb179018), YTHDF2 (1:200; Biorbyt #orb39199), YTHDF3 (1:200; Biorbyt #orb374095) at 4 °C overnight.

Techniques: Expressing, Staining

Journal: Frontiers in Genetics

Article Title: Identification of m6a-related signature genes in esophageal squamous cell carcinoma by machine learning method

doi: 10.3389/fgene.2023.1079795

Figure Lengend Snippet: Identify ESCC-related m6A regulators, build risk models, and validate model predictive capabilities (A) Venn diagrams determined YTHDF1 and HNRNPC as ESCC risk factors after overlapping m6A regulators with higher meanDecreaseGini score in both RNA-seq data and TCGA-ESCA database. (B) Nomogram was constructed using YTHDF1 and HNRNPC to visualize the predictive ability of ESCC-related m6A regulators for Normal group and ESCC group. (C) Calibration curve with nomogram predicted probabilities on the x-axis and actual probabilities on the y-axis. The dashed line represents the true value, and the solid line is bias-corrected by Bootstrapping (1000 repetitions). (D) ROC curve was applied to assess the predictive efficiency of the model. (E) Clinical decision curve. The red line represents the outcome predicted by the model, the grey line represents “all positive diagnosis (ESCC)” and the black line represents “all negative diagnosis (Normal)”. The horizontal axis is Threshold Probability, and the vertical axis is the net profit rate after subtracting the disadvantages. (F) Clinical Impact Curve (CIC). The red curve (Number of high risk) represents the number of people classified as ESCC by the model at each threshold probability; the blue curve (Number high risk with outcome) represents the number of true ESCC at each threshold probability. AUC, Area Under Curve; CIC, Clinical Impact Curves; ESCC/ESCA, Esophageal quamous cell carcinoma; HNRNPC, RNA-binding protein, is a member of the heterogeneous ribonucleoproteins C; m6A, N6-methyladenosine; TCGA, The Cancer Genome Atlas; YTHDF1, YTH N6-methyladenosine RNA-binding protein 1.

Article Snippet: Antigen retrieval was performed by heating sections in 10 mM citrate (pH 6.0) boiling buffer for 15 min. ESCC tissue sections were incubated overnight at 4°C with rabbit monoclonal anti-YTHDF1 antibody (1:1,000, A18126, Abclonal, China) and anti-HNRNPC (1:2000, A19137, Abclonal, China).

Techniques: RNA Sequencing, Construct, Biomarker Discovery, RNA Binding Assay

Journal: Frontiers in Genetics

Article Title: Identification of m6a-related signature genes in esophageal squamous cell carcinoma by machine learning method

doi: 10.3389/fgene.2023.1079795

Figure Lengend Snippet: Clinical significance of risk grouping in TCGA-ESCA database (A) The heatmap shows the clinicopathological features were compared between the high- and low-risk groups. (B) Univariate Cox analysis of the risk score and clinicopathological features. (C) Multivariate Cox analysis of the risk score and clinicopathological features to identify the independent prognostic predictors in TCGA-ESCA database. (D) Kaplan-Meier analysis for patients of TCGA-ESCA database in high- and low-risk group. Data are shown as the means ± SD. * p < 0.05. ESCA, esophageal squamous cell carcinoma; TCGA, The Cancer Genome Atlas; HNRNPC, RNA-binding protein, is a member of the heterogeneous ribonucleoproteins C; YTHDF1, YTH N6-methyladenosine RNA-binding protein 1.

Article Snippet: Antigen retrieval was performed by heating sections in 10 mM citrate (pH 6.0) boiling buffer for 15 min. ESCC tissue sections were incubated overnight at 4°C with rabbit monoclonal anti-YTHDF1 antibody (1:1,000, A18126, Abclonal, China) and anti-HNRNPC (1:2000, A19137, Abclonal, China).

Techniques: RNA Binding Assay

Journal: Frontiers in Genetics

Article Title: Identification of m6a-related signature genes in esophageal squamous cell carcinoma by machine learning method

doi: 10.3389/fgene.2023.1079795

Figure Lengend Snippet: Experimental validation of YTHDF1 and HNRNPC with Western blot, immunohistochemical staining and PPI network analysis. (A) Western blot analysis of YTHDF1 and HNRNPC expression in 5 pairs of fresh frozen ESCC specimens with related adjacent normal tissues. (B) Representative immunohistochemical images of YTHDF1 and HNRNPC expression in ESCC tissues (scale bar: 50 μm). (C) Kaplan-Meier analysis for patients with high or low expression levels of YTHDF1 and HNRNPC from ESCC tissue sections. (D) PPI network showed the interactions between HNRNPC and other m6A RNA methylation regulators. Data are shown as the means ± SD. * p < 0.05. ESCA, esophageal squamous cell carcinoma; TCGA, The Cancer Genome Atlas; HNRNPC, RNA-binding protein, is a member of the heterogeneous ribonucleoproteins C; YTHDF1, YTH N6-methyladenosine RNA-binding protein 1; METTL3, Methyltransferase-like 3.

Article Snippet: Antigen retrieval was performed by heating sections in 10 mM citrate (pH 6.0) boiling buffer for 15 min. ESCC tissue sections were incubated overnight at 4°C with rabbit monoclonal anti-YTHDF1 antibody (1:1,000, A18126, Abclonal, China) and anti-HNRNPC (1:2000, A19137, Abclonal, China).

Techniques: Biomarker Discovery, Western Blot, Immunohistochemical staining, Staining, Expressing, Methylation, RNA Binding Assay

Journal: Frontiers in Genetics

Article Title: Identification of m6a-related signature genes in esophageal squamous cell carcinoma by machine learning method

doi: 10.3389/fgene.2023.1079795

Figure Lengend Snippet: Clinicopathological characteristics of YTHDF1 expression on ESCC tissue sections.

Article Snippet: Antigen retrieval was performed by heating sections in 10 mM citrate (pH 6.0) boiling buffer for 15 min. ESCC tissue sections were incubated overnight at 4°C with rabbit monoclonal anti-YTHDF1 antibody (1:1,000, A18126, Abclonal, China) and anti-HNRNPC (1:2000, A19137, Abclonal, China).

Techniques: Expressing

Journal: Frontiers in Genetics

Article Title: Identification of m6a-related signature genes in esophageal squamous cell carcinoma by machine learning method

doi: 10.3389/fgene.2023.1079795

Figure Lengend Snippet: Univariate and multivariate Cox regression analyses of clinical factors associated with 5-year overall survival on ESCC tissue sections.

Article Snippet: Antigen retrieval was performed by heating sections in 10 mM citrate (pH 6.0) boiling buffer for 15 min. ESCC tissue sections were incubated overnight at 4°C with rabbit monoclonal anti-YTHDF1 antibody (1:1,000, A18126, Abclonal, China) and anti-HNRNPC (1:2000, A19137, Abclonal, China).

Techniques: Expressing