M3 base

As described in previous studies [19 (link),20 ], the human ductal cell line (hTERT-HPNE) and pancreatic cancer cell lines, MIA PaCa-2, AsPC-1, PANC-1, T3M4, and BxPC-3 were bought from ATCC. Pancreatic cancer line PaTu8988 was provided from PharmLab (PharmLab, Beijing, China). MIA PaCa-2, PANC-1 (DMEM, Gibco, Cat. no. C11995500), AsPC-1, BxPC-3, and T3M4 (RPMI 1640, Gibco, Cat. no. C11875500) were cultured in cell culture dishes (NEST Biotechnology, Wuxi, China) in a humidified incubator at 37 °C with 5% CO₂. The hTERT-HPNE was cultured in 75% DMEM without glucose and 25% M3 Base (Incell, Cat. no. M300F-500) supplemented with 10 ng/mL human recombinant EGF (CST, Cat. no. 72528), 5.5 mM D-glucose (1 g/L), 5% FBS, and 0.75 mg/mL puromycin (MCE, Cat. no. 58-58-2). All cell lines were authentic by short tandem repeats profile. Cells were maintained in a cell incubator (ThermoFisher, Waltham, MA, USA) with 5% CO₂ and 20% O₂ for a normoxic condition and incubator chamber (Billups-rothenberg, San Diego, CA, USA) flushed with 5% CO₂ and 95% N₂ for hypoxic condition.

HPNE cells were obtained from Drs Der and Campbell (University of North Carolina), and were originally isolated from the ductal structure of a normal human pancreas and were immortalized with the catalytic subunit of telomerase (h-Tert), as was described previously [50 (link)]. HPNE cells were maintained in Medium D that is comprised of one volume of M3 base (InCell Corp., San Antonio, TX, USA), three volumes of glucose-free DMEM, and 5% FBS, 5.5mM glucose, 10ng/ml EGF, and 50μg/ml gentamycin, at 37°C in humidified atmosphere containing 5% CO2. HA-wt-Aurora A was created by PCR using pcDNA3-Aurora A as template, as previously described [9 (link)] and Flag-tagged JAK2 was purchased from GeneCopoeia (Rockville, MD). Transfections were performed using 0.3 ugs of DNA constructs in Lipofectamine 2000 (Invitrogen).

Human pancreatic cancer cell lines, including AsPC-1, BxPC-3, MIA PaCa-2, PANC-1, T3M4 and normal pancreatic ductal cell (hTERT-HPNE) were purchased from the American Type Culture Collection (ATCC). Human pancreatic cancer cell line PaTu8988 was from DSMZ. Human umbilical vein endothelial cell (HUVEC) was purchased from the Pharmlab in china. The HEK-293T was generously provided by professor Mao in the department of Biochemistry and Molecular Biology in Peking university. All cell lines were authenticated by short tandem repeats (STR).
The hTERT-HPNE was cultured in in 75% DMEM without glucose (Gibco, USA) and 25% M3 Base (Incell, USA) supplemented with 10 ng/ml human recombinant EGF (CST, USA), 5.5 mM D-glucose (1 g/L), 5% fetal bovine serum (Gibco, USA), 0.75 mg/ml puromycin (sigma, USA). AsPC-1, BxPC-3, T3M4 were cultured in RPMI 1640 (Gibco, USA). MIA PaCa-2, PANC-1, PaTu8988, HEK-293T were cultured in DMEM. HUVEC was cultured in Endothelial Cell Medium (ScienCell, USA) supplemented with 5% fetal bovine serum, endothelial cell growth supplement, 1% penicillin-streptomycin solution (KeyGEN, china). Cells were maintained in cell incubator (ThermoFisher, USA) with 5% CO2 and 20% O2 for normoxic condition and incubator chamber (Billups-rothenberg, USA) flushed with 5% CO2 and 95% N2 for hypoxic condition.

Panc-1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS). Miapaca-2 and C2C12 were maintained in DMEM supplemented with 10% FBS, 1 mM sodium pyruvate and 4 mM L-glutamine. PaTu8988T were cultured in DMEM supplemented with 5% FBS, 5% horse serum, and 2 mM L-glutamine. BxPC-3 were maintained in RPMI1640 supplemented with 10% FBS, 1 mM sodium pyruvate, 10 mM HEPES, and 2.5 g/L of glucose. HPNE required a medium composed of 75% DMEM, 25% M3 Base (Incell Corp, San Antonio, TX, USA), 2.5% FBS, 0.01% epidermal growth factor, 2 mM L-glutamine, and 1 g/L of glucose. The cells were cultured in a humidified 5% CO2 incubator at 37 °C and were used between passage 1 and passage 10. The cells were monthly tested for mycoplasma.