Panc 1

A total of six pancreatic cancer cell lines (BXPC-3, CFPAC-1, MIA PaCa-2, ASPC-1, PANC-1 and SW1990) were provided by Shanghai GeneChem Co., Ltd. and cultured in RPMI-1640 basic medium (Corning, Inc.). All cells were routinely subcultured at 37˚C in the presence of 5% CO₂ in an incubator with saturated humidity. Total RNA was extracted from the six cell lines using TRIzol reagent according to the manufacturer's instructions. RNA was reverse transcribed to complementary DNA using Promega M-MLV at 42˚C (Promega Corp.). The mRNA expression levels of the ITGB6 gene in different cell lines of interest were detected by quantitative PCR using a LightCycler 480 II (Roche Molecular Systems, Inc.). The composition of the reaction mixture was SYBR premix ex taq 6.0 µl, primer mix 0.3 µl, reverse transcription product 0.6 µl and RNase-free H₂O 5.1 µl. The reaction conditions were as follows pre-denaturation at 95˚C for 30 sec, followed by denaturation for 5 sec at 95˚C and annealing for 30 sec at 60˚Cfor a total of 40 cycles. The primer sequences were as follows: ITGB6 forward, 5'-TGATCTTCGCTGTAACCC-3' and reverse, 5'-CAGACCGCAGTTCTTCATA-3'; GAPDH forward, 5'-TGACTTCAACAGCGACACCCA-3' and reverse, 5'-CACCCTGTTGCTGTAGCCAAA-3'. The experimental results were analyzed by the 2^-∆∆Cq method (23 (link)) for relative quantitative analysis.

BXPC-3, PANC-1 and SW1990 (all from Shanghai Genechem Co., Ltd., Shanghai, China) pancreatic cancer cell lines were cultured at 37°C in an atmosphere containing 5% CO₂ in RMPI-1640 (Hyclone; Thermo Fisher Scientific, Inc.) or DMEM, supplemented with 10% FBS. Subsequently, CD44⁺CD24⁺ cells were sorted from the three cell lines using flow cytometry as previously described (19 (link),20 (link)).