Additional file 1 of Programmable N6-methyladenosine modification of CDCP1 mRNA by RCas9-methyltransferase like 3 conjugates promotes bladder cancer development
Creators: name: Xiaoling Ying
name: Jiang, Xu
name: Haiqing Zhang
name: Bixia Liu
name: Yapeng Huang
name: Xiaowei Zhu
name: Defeng Qi
name: Yuan, Gang
name: Junhang Luo
name: Weidong Ji
Publisher: name: figshare
Year: 2020
Subjects: subject: Biophysics
subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Sociology
subject: FOS: Sociology
subject: FOS: Sociology
subject: 69999 Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Computational Biology

description: Additional file 1: Supplementary Figure 1. MeRIP and RT-qPCR of epidermal growth factor receptor (EGFR) targeted by RCas9-M3 editors. (a) Western blot analysis of EGFR expression in control and METTL3-depleted cells (HeLa-KO-M3). (b) m6A enrichment of the EGFR mRNA 3′ UTR in METTL3-depleted cells (HeLa-KO-M3) with the RCas9 system. All qRT-PCR data are presented as the mean ± SEM (n = 3). *p 2) are shown in red and counted in each volcano plot. Over 14,730 total genes were analyzed in each experiment. Statistical significance was calculated using a two-tailed Student’s t-test with a false discovery rate correction. Data are presented as mean ± SEM from three independent experiments. NS, no significant. Supplementary Figure 4. Targeted methylation using RCas9-METTL3 promotes translation. (a) A psiCHECK-2 luciferase reporter plasmid carrying a fragment of the human CDCP1 3′ UTR containing three putative m6A motifs (target sites for RCas9). The positions of the m6A sites (155, 173, and 212) were numbered relative to the first nucleotide of the 3′ UTR. The sgRNAs were designed to target the three putative m6A motifs. Relative luciferase activity reveals the translational effects of RCas9-M3 binding to mRNA. (b) Relative luciferase activity of psiCHECK-2-CDCP1–3′ UTR after transient transfection of the RCas9-M3 system in HEK293T cells targeting the CDCP1 mRNA compared to non-targeting control. (c) Relative luciferase activity of the psiCHECK-2-CDCP1 3′ UTR after stable transfection of the CDCP1 mRNA-targeting RCas9-M3 system in SV-HUC-1 cells compared to non-targeting control. (d) qRT-PCR analysis of CDCP1 mRNA levels in control and SV-HUC-1 cells expressing the RCas9 system. mRNA was normalized to β-Actin mRNA. The relative ratio (fold-change) obtained from SV-HUC-1 cells without RCas9-M3 was set to 1. (e) Western blot analysis of CDCP1 expression in control (SV-HUC-1 without RCas9 and RCas9 without fused METTL3) and stably transfected SV-HUC-1 cells expressing the RCas9 system. (f) Western blot analysis of EGFR expression in control (METTL3-depleted HeLa cells without RCas9-M3, RCas9 without fused METTL3CD, or EGFP alone) and METTL3-depleted HeLa cells transfected with the RCas9 system. (g) qRT-PCR analysis of EGFR mRNA levels. (h) Protein expression of CDCP1 in stable SV-HUC-1 cells expressing the RCas9-M3 system. All bar plot data are presented as the mean ± SEM of three independent experiments. *p