Pen strip

ARPE-19 cells were seeded in a 6-well plate (VWR, Mississauga, ON, Canada. cat # 82050-842) at 600,000 cells/well (60,000 cells/cm²) and reached confluency at ~120,000 cells/cm². Cultures were maintained for up to 70 days in 2 mL of ARPE-19 culture media that consisted of: DMEM/F-12, HEPES (Thermo Fisher Scientific, Mississauga, ON, Canada. cat # 11330057), 10% FBS (VWR. cat # 97068-085), and 1% Pen/strip (Thermo Fisher cat # 15140122). Media was changed every 48 h by replacing the entire old media volume with 2mL of fresh media. For all experiments, E-RPE cells were cultured in an identical manner to ARPE-19 cells, using ES-RPE culture media that consisted of: 70% DMEM; 30% F12; 2% B-27 supplement and 1% Pen/strip (Thermo Fisher. cat # 11965, 11765, 17504, and 15140122).
To assess the oxygenation status of the cells under the outlined culture conditions, oxygen delivery was calculated using our previously published method [66 (link)]. A RPE oxygen consumption rate of 42 amol∙cell⁻¹∙s⁻¹ was used for the calculation [67 (link)]. We determined that cultured RPE cells should be receiving an adequate amount of oxygen, with a local oxygen concentration of 1.42 × 10⁻⁴ mol/L at the cells and a maximum oxygen delivery rate of 191 amol∙cell⁻¹∙s⁻¹.

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).