Culturing Pancreatic Cancer Cell Lines

HEK293T, MIA-PaCa-2, BxPC-3, and PANC-1 cell lines were obtained from the American Type Culture Collection (ATCC). They were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (#S11150; FBS, Atlanta Biologicals), antibiotics (#P4458; Gibco) and L-glutamine (#17921004; Corning). The murine pancreatic cancer cell line KPC1 was originally described in our recent publication (Parajuli et al., 2018 (link)). The cell line was established from a KP53 mouse, which harbored KrasG12D and one conditional allele of Trp53 (LSL-KrasG12D;LSL-Trp53fl/+;Pdx1-Cre). Freshly isolated specimen from the KP53 mouse with terminal PDAC was gently dissected, minced with scissors, and digested with Dispase II at 2.4 U/ml (#4942078001; Sigma-Aldrich) and Collagenase D at 0.5 mg/ml (#11088858001; Sigma-Aldrich) for 1 h at 37°C in an atmosphere of 5% CO2. Then, cells were washed three times with PBS, suspended in RPMI 1540 containing 20% FCS, and seeded on fibronectin-coated plates. Cell colonies were subsequently passaged by trypsinization, pooled, and propagated in DMEM supplemented with 10% FBS, antibiotics, and L-glutamine. To generate the PANC-1-SMAD4KO and PANC-1-SMAD2/3KO cell lines, cells were transduced with the corresponding lentiCRISPRV2-gRNA lentiviruses, selected with puromycin (for SMAD4) or hygromycin (for SMAD2/3), and all resistant clones were pooled and expanded as a single population. Lentiviruses were produced by transfecting HEK293T cells with lentiviral constructs and the One-Step Lentivirus Packaging System as described by the manufacturer (#631275; Takara). Lentiviral particles in the conditioned media were harvested after a period of 48–72 h. The conditioned media were then cleaned of cell debris by centrifugation at 5,000×g for 15 min, filtered through a 0.45-μm filter, and used immediately for cell transduction.