Pcdh mcs t2a puro vector

Human epithelial colorectal adenocarcinoma cell line Caco-2 and human breast cancer cell line MDA-MB-231 were purchased from American Type Culture Collection (ATCC). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen) in a 37 °C incubator containing 5% CO₂. MNPs used in this study were purchased from Beijing Tong Ren Tang Chinese Medicine Co Ltd. SPIO nanodots were synthesized according to our previously reported procedures [11 (link)]. Caco-2 with overexpression of ASBT was generated through lentiviral transduction and selected by puromycin. Lentiviral construct was established by subcloning ASBT cDNA (also called SLC10A2, R&D Systems) into pCDH-MCS-T2A-Puro vector (System Biosciences). Artificial lentiviruses were produced using a second generation of packing systems, including pMD2.G and psPAX2 (Addgene) according to our previous reports [12 (link), 13 (link)]. Other reagents, if not specified, were purchased from Sigma-Aldrich.

cDNA for mouse Sptlc2 was obtained from TransOMIC (pCS6 BC003227) and subsequently subcloned into pCDH-MCS-T2A-Puro vector (System Biosciences). Sptlc2^R507A was generated through overlap extension PCR. Codon optimized CD300lf-FLAG, described previously ^{5 (link)}, was subcloned into pCDH-MCS-T2A-Puro or synthesized into pCMV-CD300lf-Flag-P2A-Blast (VectorBuilder). For the pCMV-CD300lf-Flag-P2A-Blast, we routinely see lower expression compared to the pCDH-CD300lf-T2A-Puromycin. All plasmid constructs were sequenced verified.