Journal: Clinical and Translational Medicine

Article Title: FTO up‐regulation induced by MYC suppresses tumour progression in Epstein‒Barr virus‐associated gastric cancer

doi: 10.1002/ctm2.1505

Figure Lengend Snippet: FTO restrains EBVaGC cell migration and invasion in vitro. (A) Wound healing assays of AGS B95.8 cells with FTO silencing and FTO overexpression were recorded and quantitatively analysed. Scale bar: 200 µm. (B and C) Images and quantification of cell migration (top) and invasion (bottom) assays of FTO‐knockdown and FTO‐overexpressing EBVaGC cells. Scale bar: 200 µm. (D) Western blotting analysis was used to detect EMT markers (β‐Catenin, ZEB1, Slug) in EBVaGC and EBVnGC cells. (E) The protein levels of EMT markers (β‐Catenin, ZEB1, Slug) in AGS B95.8 cells after FTO deficiency were measured using immunoblotting. (F) Western blotting assay of FTO in AGS B95.8 cells overexpressing wild‐type and catalytic mutant FTO. (G) RNA m6A dot blot assay of wild‐type and catalytic mutant FTO‐overexpressing AGS B95.8 cells. MB staining served as a loading control. (H) The migration ability of AGS B95.8 cells overexpressing wild‐type and catalytic mutant FTO was determined (left), and the cell migration assay results were quantitatively analysed (right). Scale bar: 200 µm. The data in (A–C and H) are presented as the means ± SDs. p ‐Values were determined by Student's t test. * p < 0.05; ** p < 0.01; *** p < 0.001. Vinculin was included as a loading control.

Article Snippet: Moreover, an N‐terminal Flag‐tagged FTO overexpression wild‐type (WT) plasmid, FTO double mutant (H231A/D233A) plasmid, FOS overexpression plasmid, Flag‐tagged expression vectors for MYC overexpression and luciferase reporter plasmids containing the FTO promoter WT or deletion mutant were constructed by OBiO Technology.

Techniques: Migration, In Vitro, Over Expression, Knockdown, Western Blot, Mutagenesis, Dot Blot, Staining, Control, Cell Migration Assay

Journal: Clinical and Translational Medicine

Article Title: FTO up‐regulation induced by MYC suppresses tumour progression in Epstein‒Barr virus‐associated gastric cancer

doi: 10.1002/ctm2.1505

Figure Lengend Snippet: FOS is a functionally critical downstream target of FTO in EBVaGC metastasis. (A) Venn diagram illustrating the shared peaks between EBV‐related hypo peaks and FTO‐related hypo peaks. A total of 1222 shared peaks corresponding to 982 specific genes were obtained. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the above 982 specific genes, and the enriched top five enriched pathways were the TNF signalling pathway, Adherens junction, Basal transcription factors, Fanconi anaemia pathway and Homologous recombination. (C and D) The RT‐qPCR assay was performed to assess mRNA expression of the shared genes in the TNF signalling pathway (CSF1, TNFRSF1A, FOS, MAPK3, TRADD, MAP2K4, CREB1, ITCH, TNFAIP3, MAPK14 and TRAF3) with FTO knockdown (C) and FTO overexpression (D) in AGS B95.8 cells. (E) Western blotting analysis of FOS and ITCH protein levels in FTO‐knockdown (left) and FTO‐overexpressing (right) AGS B95.8 cells. (F) Representative H&E and IHC images of FTO and FOS expression in FTO‐deficient and control AGS B95.8 cell‐induced xenograft tumours (top). Scale bar: 200 µm. Quantification of FOS expression (bottom) in the FTO knockdown and control groups ( n = 9 per group). (G) Scatterplot showing the correlation between FOS expression level and the enrichment scores of EMT pathway obtained from single sample gene set enrichment analysis (ssGSEA) using the RNA‐seq data of AGS, AGS B95.8, FTO‐overexpressing AGS B95.8 and control cells. (H) Images of the cell migration assay of FOS‐overexpressing (FOS) versus empty vector (EV) AGS B95.8 (left) and AGS AKATA (right) cells. Scale bar: 200 µm. (I) Western blotting assay of FTO, FOS, MMP9 and Vimentin protein levels in AGS B95.8 cells transfected with siNC, siFTO#1, siFOS#1 and siFTO#1 + siFOS#1. (J) Representative images (left) and quantification (right) of the transwell migration assay of AGS B95.8 cells with siNC, siFTO#1, siFOS#1 and siFTO#1 + siFOS#1. (K) Immunoblotting assays were conducted to detect FTO, FOS, MMP9 and Vimentin expression in AGS B95.8 cells with EV, FTO, FOS and FTO + FOS. (L) Images (left) and quantification (right) of the cell migration assay of AGS B95.8 cells with EV, FTO, FOS and FTO + FOS. Scale bar: 200 µm. The data in (C, D, F, J and L) are presented as the means ± SDs. p ‐Values were determined by Student's t test. * p < 0.05; ** p < 0.01; *** p < 0.001. Vinculin was included as a loading control.

Article Snippet: Moreover, an N‐terminal Flag‐tagged FTO overexpression wild‐type (WT) plasmid, FTO double mutant (H231A/D233A) plasmid, FOS overexpression plasmid, Flag‐tagged expression vectors for MYC overexpression and luciferase reporter plasmids containing the FTO promoter WT or deletion mutant were constructed by OBiO Technology.

Techniques: Homologous Recombination, Quantitative RT-PCR, Expressing, Knockdown, Over Expression, Western Blot, Control, RNA Sequencing, Cell Migration Assay, Plasmid Preparation, Transfection, Transwell Migration Assay

Journal: Clinical and Translational Medicine

Article Title: FTO up‐regulation induced by MYC suppresses tumour progression in Epstein‒Barr virus‐associated gastric cancer

doi: 10.1002/ctm2.1505

Figure Lengend Snippet: FOS is regulated by FTO‐dependent m6A demethylation. (A) MeRIP‐seq analysis showing canonical RRACH m6A motifs of FOS transcripts in EBVaGC cells and EBVnGC cells. (B) The predicted m6A regions in FOS mRNA. (C) MeRIP‐qPCR analysis of m6A enrichment in regions 1−5 of FOS transcripts in AGS B95.8 cells. (D) MeRIP‐qPCR analysis of m6A enrichment in regions 1−5 of FOS mRNA in FTO‐knockdown and control AGS B95.8 cells. (E) The decay rate (left) and qPCR detection (right) of FOS mRNA at the indicated time point after actinomycin D (ActD) treatment in FTO‐knockdown versus control groups. (F and G) RT‐qPCR analysis (left) and immunoblotting (right) of FOS expression in EBVaGC cells with empty vector (EV), wild‐type FTO overexpression (FTO) and H231A/D233A‐mutant FTO overexpression (FTO H231A/D233A ). (H) MeRIP‐qPCR analysis was conducted to detect the relative m6A level of FOS mRNA regions in AGS B95.8 cells transfected with vectors expressing wild‐type or catalytic‐mutant FTO. (I) Relative FOS mRNA expression in EBVaGC cells treated with DMSO, FB23‐2 (5 µM), FB23‐2 (10 µM) and FB23‐2 (15 µM). (J) The decay rate (left) and qPCR assay (right) of FOS mRNA at the indicated times after ActD treatment in FB23‐2 (15 µM) versus DMSO‐treated AGS B95.8 cells. (K) The decay rate of FOS transcripts in AGS B95.8 cells transfected with EV, FTO and FTO H231A/D233A plasmids was analysed by nonlinear regression (left), and the relative FOS mRNA expression of different groups was detected by qPCR analysis at each time point (right). The data in (C–F and H–K) are presented as the means ± SDs. p ‐Values were determined by Student's t test (C, D, F, H, I and relative FOS level in E, J and K) and two‐way ANOVA (%RNA remaining in E, J and K). * p < 0.05; ** p < 0.01; *** p < 0.001. IgG was used as the negative control and the relative m6A level was normalized by the input in (C, D and H). Vinculin was included as a loading control.

Article Snippet: Moreover, an N‐terminal Flag‐tagged FTO overexpression wild‐type (WT) plasmid, FTO double mutant (H231A/D233A) plasmid, FOS overexpression plasmid, Flag‐tagged expression vectors for MYC overexpression and luciferase reporter plasmids containing the FTO promoter WT or deletion mutant were constructed by OBiO Technology.

Techniques: Knockdown, Control, Quantitative RT-PCR, Western Blot, Expressing, Plasmid Preparation, Over Expression, Mutagenesis, Transfection, Negative Control

Journal: Clinical and Translational Medicine

Article Title: FTO up‐regulation induced by MYC suppresses tumour progression in Epstein‒Barr virus‐associated gastric cancer

doi: 10.1002/ctm2.1505

Figure Lengend Snippet: EBV induces FTO expression by the transcription factor MYC in GC. (A) Immunoblotting of MYC protein levels in EBVaGC cells and EBVnGC cells. (B) Correlation of EBNA1 (left) and FTO (right) with MYC mRNA expression in 62 EBVaGC samples from SYSUCC. (C and D) RT‐qPCR analysis of FTO mRNA levels in EBVaGC cells upon MYC overexpression (C) and MYC knockdown (D). (E and F) Western blotting was performed to assess FTO and MYC protein expression levels in EBVaGC cells with MYC up‐regulation (E) and MYC down‐regulation (F). (G and H) Luciferase reporter assay of FTO transcriptional activity in MYC‐overexpressing (G) and MYC‐knockdown (H) EBVaGC cells. (I) Schematic illustration showing the FTO promoter containing the predicted MYC‐binding sites (−1365 to −1354). The strategy for mutating the FTO promoter is to delete the binding sequences. (J) Agarose gel electrophoresis (top) and qPCR analysis (bottom) of the ChIP assay indicated the enrichment of MYC on the FTO promoter at the predicted region of −1365 to −1354 in EBVaGC cells. (K and L) Luciferase reporter assay showing the transcriptional activity of FTO in AGS B95.8 (K) and AGS AKATA (L) cells overexpressing the FTO wild type and truncation mutant. The data in (C, D, G, H, K and L) are presented as the means ± SDs. p ‐Values were determined by Student's t test. * p < 0.05; ** p < 0.01; *** p < 0.001. Vinculin was included as a loading control.

Article Snippet: Moreover, an N‐terminal Flag‐tagged FTO overexpression wild‐type (WT) plasmid, FTO double mutant (H231A/D233A) plasmid, FOS overexpression plasmid, Flag‐tagged expression vectors for MYC overexpression and luciferase reporter plasmids containing the FTO promoter WT or deletion mutant were constructed by OBiO Technology.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Over Expression, Knockdown, Luciferase, Reporter Assay, Activity Assay, Binding Assay, Agarose Gel Electrophoresis, Mutagenesis, Control