Additional file 3 of m6A-dependent glycolysis enhances colorectal cancer progression
Creators: name: Chaoqin Shen
name: Baoqin Xuan
name: Tingting Yan
name: Yanru Ma
name: Pingping Xu
name: Xianglong Tian
name: Xinyu Zhang
name: Yingying Cao
name: Ma, Dan
name: Xiaoqiang Zhu
name: Youwei Zhang
name: Fang, Jing-Yuan
name: Haoyan Chen
name: Hong, Jie
Publisher: name: figshare
Year: 2020
Subjects: subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences

description: Additional file 3 Figure S1. METTL3 is closely correlated with glycolytic metabolism in CRC. (a) The relative expression of METTL3 was measured by real-time PCR in normal colonic epithelial cell line FHC and CRC cell lines, n = 3, nonparametric Mann–Whitney test. (b) Schematic diagram of METTL3 genomic sequence in HCT116 wild-type (WT) and METTL3-knockout (METTL3-KO) cells. Red label, target sgRNA sequence; Yellow label, A 161 bp DNA fragment was inserted in the cutting site; Red circle, a premature stop site was generated after insertion of 161 bp DNA fragment. (c) The knockout efficiency of METTL3 was confirmed by Western blot. (d) GSEA analysis was conducted to identify the differential gene profiles between HCT116 METTL3-KO and WT cells. (WT, wild type; METTL3-KO, METTL3-knockout). Figure S2. METTL3 drives glycolytic metabolism in CRC. (a-b) The knockdown efficiency of METTL3 siRNA1/2 was evaluated in HCT116 (a) and SW480 (b) cells, n = 3, nonparametric Mann–Whitney test. (c-e) Lactate production (c), glucose uptake (d) and ECAR (e) were measured after transfection of control siRNA and METTL3 siRNA1/2 in SW480 cells, n = 3, nonparametric Mann–Whitney test. (f) OCR was measured in HCT116 WT and METTL3-KO cells (left). OCR was measured in SW480 cells transfected with control siRNA and METTL3 siRNA1/2 (right). (g) Schematic domain structures of METTL3. METTL3 MTase (AA residues 369–580). (h-i) Real-time PCR (h) and Western blot assay (i) were performed to detect METTL3 expression after transfection with pcDNA3.1-METTL3 and pcDNA3.1-METTL3-mut in DLD1 cells. (WT, wild type; METTL3-KO, METTL3-knockout). Figure S3. METTL3 is an oncogenic gene in colorectal cancer. (a-b) GSEA analysis was conducted to identify the differential gene profiles between HCT116 METTL3-KO and WT cells. (c) The expression of CDK1, PCNA and CDCA7 were detected in HCT116 WT and METTL3-KO cells, n = 3, nonparametric Mann–Whitney test. (d-e) The expression of CDK1, PCNA and CDCA7 were detected after transfection with control siRNA and METTL3-siRNA1/2 in HCT116 (d) and SW480 cells (e), n = 3, nonparametric Mann–Whitney test. (f) Cell proliferation of HCT116 cells was measured by CCK8 assay after transfected with control siRNA or METTL3 siRNA, n = 6, nonparametric Mann–Whitney test. (g) Colony formation assay was performed in HCT116 cells after transfection with control siRNA or METTL3 siRNA1/2, n = 3, nonparametric Mann–Whitney test. (h) Cell proliferation assay was performed by CCK8 assay in SW480 cells with METTL3 knockdown, n = 6, nonparametric Mann–Whitney test. (i) Colony formation assay was performed after transfection with control siRNA or METTL3 siRNA1/2 in SW480 cells, n = 3, nonparametric Mann–Whitney test. (j) Representative images of tumors in nude mice bearing DLD1 cells treated with PBS, control adenovirus and shControl adenovirus, n = 8. (k-l) Tumor volumes and (k) tumor weights (l) were measured in mice bearing DLD1 cells treated with PBS, control adenovirus and shControl adenovirus, n = 8, nonparametric Mann–Whitney test. (WT, wild type; METTL3-KO, METTL3-knockout). Figure S4. Transcriptome-wide m6A-seq and RNA-seq assays identified potential targets of METTL3 in colorectal cancer. (a) The distribution of m6A IP signal across the mRNA transcripts. m6A IP signal was enriched around the stop codon of mRNAs. (b) Comparison of the abundance of m6A peaks between HCT116 WT and METTL3-KO cells. A total of 2632 (hypo-methylated) and 584 (hyper-methylated) m6A peaks showed a significant decrease and increase in abundance in HCT116 METTL3-KO cells compared with WT cells. (c) Gene ontology analysis of genes with a significant decrease in m6A modification as well as a significant decrease in mRNA expression level (Hypo-down, p