Epidermal growth factor (egf)

Single-cell suspension was prepared in a 6-well ultra-low attachment plate (1 × 10³ cells/well) (Corning, Cat# 3471). Cells were cultured in serum-free DMEM medium supplemented with 1× B-27™ Supplement (Gibco, Cat# 17504044), 20 ng/mL epidermal growth factor (SinoBiological, Cat# 10605), 20 ng/mL basic fibroblast growth factor (Novoprotein, Cat# C046), 5 μg/mL insulin (Yeasen, Cat# 40112ES25), 1 μg/mL hydrocortisone (Yeasen, Cat# 40109ES08), and 100 U/mL penicillin–streptomycin. After 7 days of culture, the number of cell spheres was counted under a microscope (Zeiss) and sphere diameters were measured by the AxioVision software (Zeiss)^{67 (link)}.

Human PDAC cell lines (Mia PaCa-2 and SUIT-2) and a nonmalignant pancreatic epithelial (hTERT-HPNE) cell line were obtained from Drs. Wun-Shaing Wayne Chang and Li-Tzong Chen, National Institute of Cancer Research (National Health Research Institutes, Miaoli, Taiwan). Mia PaCa-2 cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Corning), 2.5% horse serum, and 1% penicillin-streptomycin (TOKU-E, Bellingham, WA, USA). SUIT-2 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. hTERT-HPNE cells were cultured in low-glucose DMEM (Hyclone, Thermo Scientific) supplemented with 5% FBS, 1% penicillin-streptomycin, and 10 ng/mL epidermal growth factor (Sino Biological, Wayne, PA, USA). These cells were free of mycoplasma contamination, and their identities were confirmed by short tandem repeat (STR) profiling at the BCRC and Center for Genomic Medicine, National Cheng Kung University (NCKU; Tainan, Taiwan). All cells were maintained at 37 °C in a 5% CO₂ atmosphere. For drug treatment, cells were pre-cultured to 60–70% confluence and supplemented with medium containing BA (Sigma-Aldrich, St. Louis, MO, USA) for 24 or 48 h at indicated doses.