Plvx puro vector

For rescue experiments, the human TDP-43 cDNA sequence was PCR-amplified from a previously described TDP-43–tdTomato expression vector (kindly provided by Dr. Zuoshang Xu via Addgene [Yang et al, 2010 (link)], see Table S3 for primer sequences). The PCR product was introduced into the pLVX-puro vector (Clontech) by Gibson assembly (NEB). For ALS-associated TDP-43 mutations, cDNAs containing the mutations of interest were synthesized (gBlocks; IDT) and introduced into the pLVX-puro vector by Gibson Assembly (these new plasmids will be made publicly available through Addgene). For lentivirus production, 293FT cells (Invitrogen) were co-transfected with pLVX-TDP-43 along with the lentiviral packaging vectors pCMV-VSV-G (#8454; Addgene) and pSPAX2 (#12260; Addgene) in equal amounts using FuGENE 6 transfection reagent. The lentivirus-containing media was collected 24 h posttransfection, filtered through a 0.45-μm filter, combined with 8 μg/ml polybrene and added to the TDP-43 KO cell line. Transduced cells were selected with 2 μg/ml puromycin to establish polyclonal stable lines.

Wildtype CTDNEP1 and mutant D67N, D69N, and L72H were cloned with Myc-tag into a pLVX-puro vector (Addgene). c-Myc and mutant S62E, D62A were amplified and then cloned into pcDNA3.1-3xFLAG-CMV. shRNAs against CTDNEP1 were designed at https://rnaidesigner.thermofisher.com/rnaiexpress/ (Supplementary Data 1) and then cloned into pGreen-puro vector. siRNA targeting CTDNEP1 or control siRNA was ordered from Sigma-Aldrich (www.sigmaaldrich.com, siRNA ID: SASI_Hs01_00188422 and SASI_Hs01_00188427). siRNA interfering CTDNEP1 was performed using Lipofectamine RNAiMAX Transfection Reagent (Qiagen) according to the manufacturer’s instructions. For the QPCR, 1 μg RNA was used to generate the 1st stand of the cDNA using iScript Reverse Transcription Supermix (BioRad, #1708 841). QPCR was performed using SYBR green PCR mix (BioRad) and the primers as listed (Supplementary Data 1).
To produce lentiviruses, HEK293T cells were co-transfected with shRNA or GFP vector packaging using Lipofectamine 3000 reagent (Life Sciences). Supernatants were collected and filtered at 48 and 72 hr following transfection. Viral supernatant was concentrated by centrifugation at 25,000 rpm for 2 hr at 4°C and used to infect cells (MOI = 5) overnight in the presence of 10 μg/mL polybrene. Cells were selected and maintained with puromycin (2 μg/ml). Gene expression was verified by western blot or real-time PCR.

The plasmid for shYTHDF1 was constructed based on the pLKO.1 vector (Addgene, #8453) with the shYTHDF1 sequence (5′-GGACATTGGTACTTGGGATAA-3′), while mouse YTHDF1 overexpression plasmid was prepared by inserting YTHDF1 (NM_173761.3) into pLVX- puro vector (Addgene, #125839). Human YTHDF1 and PKM2 overexpression vectors were constructed by inserting YTHDF1 (NM_017798.4) and PKM2 (NM_002654.6) into pCDNA3.1-3×FLAG (Addgene, #53556) plasmid respectively, of which the sequences were synthesized by Miaolingbio Ptd Ltd.
The as-prepared plasmids (1 μg) were transfected individually in HEK-293T together with 1 μg of packing constructs pMD2.G (Addgene, #12259) and pSPAX2 (Addgene, #12260). Then the cultured supernatant containing the lentivirus was collected and filtered with a sterile filter membrane with a diameter of 0.45 μm. 500 μL of the filtered lentivirus mixture was then diluted with culture medium to a volume of 1 mL and further transfected into the breast cancer with Lipo3000 (Thermo). Breast cancer cell lines with YTHDF1 knockdown or overexpression were screened with puromycin (1 mg/mL).

The optimized cDNA encoding the THD domain of EDA·A1 (residues 233–391) was cloned into the pMAL-c2x vector (Addgene) with an N-terminal MBP (maltose binding protein)-tag (primers: EDA-opt-233-391-F and EDA-opt-233-391-R, Supplementary Table 3). The sequence encoding the ectodomains of EDAR (EDAR_CRDS, residues 30–150) was cloned into the baculovirus transfer vector pFastBac (Invitrogen), in-frame with an N-terminal EDAR (residues 1–28) signal peptide for secretion, an MBP-tag for purification, an N-terminal 3 C cleavage site and a linker sequence (GGSGGSGGSGGS) (primers: EDAR-30-150-F and EDAR-30-150-R, Supplementary Table 3). Secreted EDA·A1 THD domain was cloned into the expression vector pcDNA3.1 (Addgene), containing an HA signal peptide, a Flag tag, a linker (GGSGGSGGSGGS), and amino acids 233–391 of EDA·A1 (primers: EDA-233-391-F and EDA-233-391-R, Supplementary Table 3). The cDNA of full-length EDAR was cloned into the pLVX-puro vector (Addgene) with a C-terminal Flag tag (primers: EDAR-1-448-F and EDAR-1-448-R, Supplementary Table 3). All mutants were generated by the site-directed mutagenesis kit (Agilent) with primers listed in Supplementary Table 3 and verified by DNA sequencing.