Cell Line Authentication and Overexpression in Pancreatic Cancer

Panc1 (CRL-1469) and 293T (CRL-3216) cells were purchased from ATCC. mT3 and mT4 mouse PDAC cell lines were provided by Dr. Tuveson (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and authenticated by genotyping PCR to compare with Kras^+/LSL-G12D; Trp53^+/LSL-R172H; Pdx1-Cre (KPC) model^{87 (link)}. Established primary PDAC cell lines were authenticated by genotyping PCR to compare with the corresponding mice. Lentiviral plasmids pTRIPZ, TLCV2, and pCW57 were purchased from Addgene. Specific shRNA and sgRNA sequences were listed in Supplementary Table 4. Gateway Entry plasmids for SOX2 (HsCD00436328), SOX5 (HsCD00442638), TWIST2 (HsCD00330331), and NR2F2 (HsCD00005215) were purchased from DNASU (Tempe, AZ), and transferred into the pCW57 destination vector using Gateway LR Clonase II Plus (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Lentiviral production was performed as previously described^{17 (link)}. For pTRIPZ or TLCV2-infected PDAC cells, puromycin selection was used to eliminate uninfected cells. For pCW57-infected PDAC cells, FACS was used to purify PDAC cells carrying overexpressing vectors. Cells were grown in DMEM supplemented with 10% Fetal Bovine Serum and 1% Pen/Strip (Thermo Fisher Scientific). All cell lines were free of mycoplasma infection using PlasmoTest Mycoplasma Detection Kit (InvivoGen, San Diego, CA).