Hupt4

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.

The human PC cell lines, SW1990, HUPT4, PATU8988, PANC1, and ASPC1, were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). All the cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco). All mediums were supplemented with 10% fetal bovine serum (FBS) and maintained in a 37°C and 5% CO₂ atmosphere.
ATF6, EMC6, and APAF1 expression and functions were investigated by Western blotting, qRT-PCR, CCK8 assay, and Transwell assay. Si-ATF6, Si-EMC6, and Si-APAF1 were, respectively, used to inhibit the expression of ATF6, EMC6, and APAF1. The constructs OE-ATF6, OE-EMC6, and OE-APAF1 were used to, respectively, overexpress ATF6, EMC6, and APAF1. The negative control (NC) and the vector were designed and synthesized by RiboBio Co., Ltd. (Guangzhou, China). PC cells were separately seeded in 24-well plates at a density of 5 × 10⁵ cells and transfected with Si-ATF6, Si-EMC6, Si-APAF1, OE-ATF6, OE-EMC6, OE-APAF1, NC, and the vector using riboFECT mRNA Transfection Reagent (RiboBio Co., Ltd. Guangzhou, China) according to the recommendations of the manufacturer. After 48 h of transfection and incubation in a 37°C and 5% CO₂ atmosphere, the cells were harvested for subsequent experiments.

The human pancreatic cancer cell lines AsPC-1, MIA PaCa-2, PANC-1, HuP-T4, HuP-T3, PSN1, and CFPAC-1 were purchased from American Type Culture Collection (ATCC) and provided by Suzhou Truway Biotechnology Inc. All cell genetic information was analyzed and downloaded from the Cancer Cell Line Encyclopedia (CCLE) or Catalogu of Somatic Mutations in Cancer (COSMIC) database. Cells were cultured in RPMI 1640, McCoy's 5a, MEM or DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin under the recommended conditions. The ATCC has performed morphological, cytogenetic and DNA profile analyses for characterization of these cell lines. The cell passages were limited to 15 generations for all experiments in this study. Mycoplasma contamination was excluded using the antibiotic mycoplasmincin (InvivoGen) and was periodically examined using a MycoFluor Mycoplasma Detection Kit (Invitrogen, #M7006).