Additional file 1 of The N6-methyladenosine METTL3 regulates tumorigenesis and glycolysis by mediating m6A methylation of the tumor suppressor LATS1 in breast cancer
Creators: name: Xu, Youqin
name: Song, Mu
name: Hong, Ziyang
name: Chen, Wancheng
name: Zhang, Qianbing
name: Zhou, Jianlong
name: Yang, Chao
name: He, Zilong
name: Yu, Juanjuan
name: Peng, Xiaolin
name: Zhu, Qiuhong
name: Li, Shaotian
name: Ji, Kaiyuan
name: Liu, Minfeng
name: Zuo, Qiang
Publisher: name: figshare
Year: 2023
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Hematology
subject: Plant Biology

description: Additional file 1: Supplementary Figure 1. m6A modification levels in the Hippo pathway and metabolism in breast cancer. (a) Venn diagram (left panel) of common (the intersection area) and unique peaks (non-intersection region) of another pair of breast cancer tissue compared with adjacent normal tissue, |log2(fold change)|&gt;1 and q value&lt;0.05. The gene region (right panel) annotated by difference Peaks of breast cancer tissue compared with adjacent normal tissue, |log2(fold change)|&gt;1 and q value&lt;0.05. (b) Differential m6A and differential expression combined analysis diagram of another pair of breast cancer tissue compared with adjacent normal tissue. (c) GO analysis results of differential methylated genes. Differential methylated genes were obtained by TCGA clinical data of breast cancer and compared with MeRIP- seq data. (d) Enrichment analysis result of biological progress (BP) of differential methylated genes. (e) Location map of LATS1 in differential methylated KEGG pathway of Hippo. (f) Volcano map of differentially expressed genes based on transcriptome sequencing of two pairs of breast cancer tissue compared with adjacent normal tissue. (g) Differentially expressed genes of Hippo pathway in transcriptome sequencing of breast cancer tissues compared with adjacent normal tissues, n=2. (h) M6A peaks on LATS1 mRNA originated from MeRIP-seq. M6A peaks were visualized by IGV. The red peaks show the results of MeRIP and the blue peaks represent the input. Supplementary Figure 2. Expression of m6A proteins in breast cancer tissue and cells. (a) The correlation between METTL3 and the expression level of ER/ PR/ HER2 in breast adenocarcinomas (GSE70951, 195 breast adenocarcinomas and matched adjacent normal breast tissue samples). (b) The correlation between METTL3 protein expression and the prognosis of Luminal A (upper panel) and Luminal B (lower panel) breast adenocarcinomas. (c) Alter the mRNA expression level of METTL3 in MCF-7 and T47D cells, ** p&lt;0.01. (d) Alter the protein level of METTL3 in MCF-7 and T47D cells. Supplementary Figure 3. METTL3 promotes tumorigenesis of T47D cells. (a) The proliferation of T47D was determined with EdU cellular proliferation assay with flow detection after altering the expression of METTL3, n=3, * p &lt;0.05, ** p &lt;0.01. (b) The apoptotic cell number was counted with fluorescein assay when knocking out or overexpressing METTL3 in T47D, n=3, *** p &lt;0.001. (c) Migration ability of T47D was suppressed when knocking out the expression of METTL3 detected at 0h, 12h, 24h and 36h. (d) Cell invasion ability was inhibited after blocking the expression of METTL3 in cells detected by trans-well assay, n=3, ** p &lt;0.01. (e) The tumorigenesis of T47D in nude mice was significantly suppressed when knocking out the expression of METTL3, n=7, * p &lt;0.05, *** p &lt;0.001. Supplementary Figure 4. Large amounts of differently expressed metabolites were involved in the deletion of METTL3 in MCF-7. (a) Heat map of differentially expressed metabolites when knocking out the expression of METTL3 in MCF-7, n=6. (b) KEGG enrichment results of differential metabolites affected by METTL3 deletion in MCF-7. Supplementary Figure 5. METTL3 promotes glycolysis and proliferation of T47D cells. (a) The ECAR change between METTL3 KO group and control group of T47D cells in response to glucose, oligomycin and 2-DG, n=4, ** p &lt;0.01. (b) The level of glycolysis, glycolysis reserve and glycolysis capacity in T47D cells with METTL3 knock-out and control group, n=4, * p &lt;0.05, *** p &lt; 0.001. (c) The protein level (left panel) and mRNA level (right panel) of LATS1 after knocking out and overexpressing METTL3 in MCF-7 and T47D cells, n=3, * p &lt;0.05, ** p &lt; 0.01. (d) MeRIP-qPCR shown the m6A level of LATS1 mRNA after altered the expression of METTL3 in T47D, n=3, ** p &lt;0.01. (e) MeRIP-qPCR revealed the m6A level and METTL3 binding level of LATS1 mRNA when muted the m6A methylation sites on LATS1 mRNA in T47D, n=3, ** p &lt;0.01, *** p &lt; 0.001. (f) Rescued the expression of LATS1 with knocking out the expression of METTL3 and siRNA of LATS1 remarkably affected the proliferation of T47D cells. (g) The glycolysis was regained by rescuing the expression of LATS1 by METTL3 in T47D cells, n=4, *** p &lt; 0.001. Supplementary Figure 6. YTHDF2 helped promote the tumorigenesis of T47D breast cancer cells. (a) Knocking out YTHDF2 remarkably suppress the glycolysis progress in T47D cells, n=4, *** p &lt;0.001. (b) Deleting the expression of YTHDF2 significantly inhibited the tumor growth in vivo, n=7, ** p &lt;0.01, *** p &lt;0.001. (c)The colony formation ability of T47D cells was suppressed by the deletion of YTHDF2 and reversed by downregulated the expression of LATS1 at the same time. (d) YTHDF2 knock out led to the slower proliferation of T47D cells and such inhibition could be rescued by LATS1 knockdown, n=3, ** p &lt;0.01. (e) YTHDF2 deletion increased the apoptosis rate of T47D cells, n=3, ** p &lt;0.01, *** p &lt;0.001. (f) Trans-well assay revealed the invasion ability of T47D could be controlled by missing expression of YTHDF2, but also could be rescued by LATS1 siRNA, n=3, * p &lt;0.05, ** p &lt;0.01.