E1751

For METTL3-promoter activity assay, the upstream −2000 bp of a promoter of METTL3 was constructed into a pGL3-basic vector (Promega # E1751). CRC cells were transfected with pcDNA3.1(+)/myc-His C vector (Invitrogen # V80020) control or pcDNA3.1(+)/myc-His C-FOXD3 plasmid, and co-transfected with the reporter plasmids (pGL3-basic-METTL3 (Promoter) vector and TK-renilla vector). For CTGF-promoter activity assay, CRC cells were transfected with the reporter plasmids (pGL4-basic-CTGF^{27 (link)} and TK-renilla vector). The transfected cells were treated with or without F. nucleatum (MOI of 100:1) for 2 h. Luciferase assays were measured at 24 h after transfection, using Dual-Luciferase Reporter System (Promega, Fitchburg, WI, USA) according to the manufacturer’s protocol. The transfection efficiency data were presented by normalizing the Firefly luciferase activities to that of Renilla luciferase signals. Each assay was performed independently in triplicate.

Overexpression plasmids were constructed using the pCDH puromycin resistant or PLX304 blasticidin resistant vectors. Overexpression sequences of YTHDC1, SPRY1, TUG1, and SMAD3 were cloned by PCR from cDNA of MDA-MB-231 cells. The W377A and W428A mutants of YTHDC1 were constructed using the Q5® Site-Directed Mutagenesis Kit (New England Biolabs). For rescue experiments using WT or mutant YTHDC1, the coding sequence was altered to carry synonymous mutations within the sgA site to prevent targeting by sgA. A full list of overexpression vectors is available in Table S1. sgRNA sequences were cloned into the lentiGuide-Puro vector. The sgRNA sequences used were YTHDC1 sgA: 5'-AGATGAGCCGACTTAGTGAA-3' and YTHDC1 sgB: 5'-ACGGAGGATCTCCTATACAC-3'. shRNA targeting YTHDC1 were obtained from Sigma-Aldrich (sh1: TRCN0000243989, sh2: TRCN0000243987). Plasmids used for dual luciferase assay were obtained from Promega. The first 800bp of WT SMAD3 3'UTR was cloned from DNA isolated from MDA-MB-231 cells and fused to a firefly luciferase gene (E1751, Promega). All the 18 RRACH motif sites within the first 800 bp of SMAD3 3'UTR were mutated to RRTCH by site-directed PCR mutagenesis and fused to a firefly luciferase gene to generate mutant SMAD3 3'UTR. As a control, pRL-TK (E2241, Promega) expressing renilla luciferase was used.

Full-length esr2b (XM_020611557.1) was cloned into pcDNA3.0 using EcoRI and SalI to generate pCMV-esr2b. Three deletion fragments of the atg13 (XM_020600753.1) promoter were amplified from genomic DNA, using KpnI and XhoI to generate the pGL3-basic vector (E1751, Promega, Madison, WI, USA). Full-length rad23ba (XM_020600944.1) was cloned into pSico-Cherry-FLAG using EcoRI and XhoI to generate Cherry-FLAG-rad23ba. Full-length Rad23b (NM_009011.4) was cloned into pSico-Cherry-FLAG using EcoRI and XhoI to generate Cherry-FLAG- Rad23b. Full-length Psmb2 (NM_011970.4) and psmb2 (XM_020597689.1) were cloned into pEGFP-N1 using XhoI and EcoRI to generate GFP-Psmb2 and GFP-psmb2. The primers and PCR conditions are described in Table S4. Site-directed mutagenesis for the Esr2b binding sites was performed using the primers described in Table S4. All constructs were confirmed by sequencing (TSINGKE, Beijing, China).