Capan 2

Six PDA cell lines (Capan-2, MIA PaCa-2, PANC-1, SW1990, BxPC-3, and HPAF-II) and the immortalized pancreatic cell line hTERT-HPNE were purchased from the Cell Bank of the Chinese Academy of Sciences. Mycoplasma contamination tests were negative, and short tandem repeat assays were performed to authenticate the cell lines. MIA PaCa-2, PANC-1, and hTERT-HPNE cells were seeded in high-glucose Dulbecco’s modified Eagle medium (DMEM; Gibco, USA). HPAF-II cells were plated in minimal essential medium (Boster, USA). SW1990, Capan-2, and BxPC-3 cells were grown in RPMI-1640 (Gibco, USA). The media were supplemented with 10% fetal bovine serum (FBS, Gibco, USA), and cells were cultured at 37 °C under 5% CO₂.
The METTL16 overexpression (OE) vector was purchased from Genechem (Shanghai, China). Short hairpin RNAs (shRNAs) against METTL16 were constructed by Genechem (Shanghai, China). For stable transfection, 2 × 10⁵ cells per well were plated in six-well plates 24 h in advance, then transfected with a 2E+6TU overexpression or shRNA plasmid using polybrene (Solarbio, China), followed by selection with puromycin (Solarbio, China). The shRNA sequences are listed in Supplementary Table S1.

Human pancreatic cell line HPDE6-C7 and pancreatic cancer cell lines PANC-1, Capan-2, SW1990, BxPC3 and AsPC-1 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Subsequently, these cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco, Australia), 100 U/ml penicillin, and 50 μg/ml streptomycin in incubators with humidified atmosphere of 5% CO₂ and 95% air at 37°C.

Human normal pancreatic epithelial cells HPDE6-C7 and PC cell lines PANC-1, SW1990, BxPC-3, Capan-2, AsPC-1 were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM medium (Hyclone) supplemented with 10% fetal bovine serum (GIBCO) at 37°C in a humidified atmosphere containing 5% CO₂. Stable PANC-1-luc-NC and PANC-1-luc-KD cell lines were established using lentivirus pGC-FU-LUC-IRES-Puromycin carrying control or RPL34-shRNA sequences. Cells were selected by culture in puromycin (2.5 μg/ml) and were continually passaged at low density to allow for election of subclones with acquired puromycin resistance.

Human pancreatic cancer cell lines, Capan-2, PANC-1, AsPC-1, BxPC-3, SW-1990, Patu-8988 and CFPAC-1, were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cells were maintained in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen). Cells were incubated at 37°C in a humidified atmosphere of 5% CO₂ and used for the assays during the exponential phase of growth. The study was approved by the Ethics Committee of Ruijin hospital, Shanghai Jiaotong University, Shanghai, China.