Bxpc 3

The human pancreatic cancer cell lines MIA PaCa-2, AsPC-1, PANC-1 and BxPC-3 were purchased from American Type Culture Collection (CRL-1420TM, CRL-1682, CRL-1469 and CRL-1687, respectively, ATCC; Rockville, MD, USA) and banked at Centre Paul Strauss. Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂ and 95% air. PANC-1 was cultured in Dulbecco’s modified Eagle’s medium (DMEM; PAN Biotech GmbH, Aidenbach, Germany) supplemented with 10% fetal bovine serum (FBS, PAN Biotech GmbH) and 1% of a solution of penicillin (10000 IU/mL) and streptomycin (10 mg/mL) (PAN Biotech GmbH). MIA PaCa-2 was cultured in the same conditions with 1% of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 1 mM, PAN Biotech GmbH), 1% of sodium pyruvate (10 mM, PAN Biotech GmbH) and 1% of non-essential amino acids (NEAA, PAN Biotech GmbH). AsPC-1 and BxPC-3 were cultured in Roswell Park Memorial Institute medium (RPMI; PAN Biotech GmbH) supplemented with 10% FBS, and 1% of a solution of penicillin (10000 IU/mL) and streptomycin (10 mg/mL). Subconfluent cell monolayers were trypsinized once a week using 0.5% trypsin containing 2% EDTA (PAN Biotech GmbH) and plated at passage ratios between 0.25:10 to 1:10, according to the cell line or used directly for study after enumeration determined with a Countess^® Cell Counter (Countess, Invitrogen, Carlsbad, CA, USA).

Human pancreatic cancer cell lines ASPC-1 and COLO357 were purchased from the Shanghai Chinese Academy of Sciences Cell Bank (Shanghai, China). Other cell lines, PANC-1, BXPC-3 and HPDE6-C7 were donated by Dr. Tan Peng from Southwest Medical University. PANC-1, BXPC-3, COLO357 and HPDE6-C7 were cultured in high glucose DMEM containing 10% fetal bovine serum (Pan Biotech, Germany). ASPC-1 was cultured in 1640 medium containing 10% fetal bovine serum. All cells were cultured under 5% CO2, 37°C and humidified conditions [40 (link)].
Lentiviruses targeting TNFSF9 (sh-TNFSF9-1, 5′-CGCCACAGTCTTGGGACTCTT-3′ and sh-TNFSF9-2, 5′-TGGAATACGCCTCTGACGCTT-3′) and negative control viruses (sh-NC, 5′-TTCTCCGAACGTGTCACGT-3′) were purchased from GeneChem (Shanghai, China). BXPC-3 and PANC-1 cells were transfected according to the manufacturer's instructions, and the knockdown efficiency was evaluated by western blot.

HUH7 were obtained from JCRB, BxPC3 and Panc03.27 were obtained from ATCC. STR profiling and routine testing for mycoplasma contamination were performed. HUH7 were grown in DMEM, 10% FCS, BxPC3 were grown in RPMI-1640, 10% FCS and Panc03.27 were grown in RPMI-1640, 15% FCS, 10 Units/ml human recombinant insulin (PAN-Biotech GmbH, Aidenbach, Germany & Sigma-Aldrich). All cells were cultured under constant humidity at 37 °C, 5% CO₂. For HUH7, all plastic ware was pre-coated with 0.001% collagen G (PBS). Archazolid A was provided by Rolf Müller, Torin1, CCCP, BPTES, UK5099 and Etomoxir were purchased from Sigma-Aldrich, and dissolved in DMSO (Sigma-Aldrich).