Pa tu 8988t

Human PDAC cell lines, PaTu8988T, MiaPaCa2, and HupT4, were purchased from the American Type Culture Collection. Stable cell lines for GRN suppression were established by transfecting GRN shRNA into PaTu8988T and MiaPaCa2. Scramble shRNA was included as negative control (nc) for transfection. Sequences of shRNA and nc are as follows,
sh_GRN1: AGGCCCTGATAGTCAGTTCGAATtgacaggaagATTCGAACTGACTATCAGGGC
sh_GRN2: AGGAAGGACACTTCTGCCATGATtgacaggaagATCATGGCAGAAGTGTCCTTC
sh_GRN3: AGGTGACCTGATCCAGAGTAAGTtgacaggaagACTTACTCTGGATCAGGTCAC
nc: AGGGAATCTCATTCGATGCATACtgacaggaagGTATGCATCGAATGAGATTCC
All transfectants were maintained in 10% FBS-supplemented Dulbecco’s Modified Eagle Medium (DMEM) with 2 mg/mL of G418 (Life Technologies, Thermo Fisher Scientific, MA). Exogenous PGRN stimulation was performed by incubating HupT4 with or without 0.4 ng/mL rPGRN for 1 day.
For GP82, it was established from the tumor of an FKPC2GP mouse no. 82. After enzymatic digestion of the tumor, the desegregated cells were grown and maintained in 10% FBS-supplemented DMEM. After 3rd passage, the cell expression of epithelial marker EpCAM and fibroblast marker α-SMA was assessed by immunofluorescence staining and flow cytometry, and confirmed enrichment of epithelial cells with <1% contamination of fibroblasts.

HEK293, HEK293T and PDAC cell lines L3.6, BxPC3, Panc1, PaTu8988T, and HPNE (an HTERT-immortalized normal pancreatic epithelial cell line) were obtained from American Type Culture Collection (ATCC). They were maintained under recommended culture conditions. Cells were detached from the plates using 0.25% Trypsin/EDTA (Gibco) and transferred to a new 6 well dishes 24 h before transfection. Plasmid and Lipofectamine 2000 (Invitrogen) were mixed in Opti-MEM (Gibco) medium and incubated for 10 min before transfection. The medium was changed 4–6 h after transfection.

Human pancreatic cancer cell lines PANC-1, BxPC-3, PaTu8988T were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). BxPC-3 cells were maintained in RPMI 1640 medium supplemented with 2.5 g/L glucose, 1 mM sodium pyruvate, and 10% fetal bovine serum. PANC-1 and PaTu8988T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 2 mM L-glutamine. Cells were cultured in a 37 °C, 5% CO₂ incubator with routine mycoplasma check once every month.
Plasmids for lentivirus packaging were purchased from Shanghai Genechem Inc (Shanghai, China). Plasmids were transfected in HEK-293 T cells by using Lipofectamine 3.0 (Invitrogen) following the manufacturer's instructions. The lentivirus-containing supernatant was collected 48 and 72 h after transfection, and cells were transfected with lentivirus. The pancreatic cancer cell lines with stable gene expression were selected in culture medium supplemented with puromycin (1 μg/ml; Sigma-Aldrich, St. Louis, MO, USA).

Eight primary PDAC cell cultures (PDAC1, PDAC-2, PDAC-3, PDAC-4, PDAC-5, PDAC-6, PDAC-7 and PDAC-8) were isolated from patients at the University Hospital of Pisa (Pisa, Italy), as described previously [21 (link)], while the human pancreatic duct epithelial-like cells hTERT-HPNE, and the PDAC cell lines Pa-Tu-8988T (PaTu-T) and Pa-Tu-8988S (PaTu-S) were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany), respectively. The primary cells were cultured in RPMI-1640, supplemented with 10% heat-inactivated (HI) FBS and 1% streptomycin/penicillin at 37°C, in a 5% CO₂ humidified atmosphere and harvested with trypsin-EDTA in their exponentially growing phase. The cell lines hTERT-HPNE, Pa-Tu-8988S (PaTu-S) and Pa-Tu-8988T (PaTu-T), PaTu-T cells lentiviral transduced with Gal-4 (PaTu-T/Gal-4) or lentiviral mock-transduced (PaTu-T/Mock) were cultured in DMEM high glucose (GIBCO, Invitrogen), with 10% HI-FBS and 1:100 streptomycin/penicillin.

Pancreatic cancer cell lines (AsPC1, CaPan1, Colo357, Hs766t, IMIM-PC1, MiaPaca2, Panc1, Panc89, PaTu 8988t) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured under standard conditions (37 °C, 5% CO₂) in RPMI 1640 Medium (Sigma, Taufkirchen, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS Superior, Biochrom, Berlin, Germany), unless stated otherwise.