Stellate cell medium

Human PDAC cell lines MIA PaCa-2 and Capan-1 were purchased from ATCC (Manassas, VA, USA). Normal fibroblasts (NFs, id = 6 and 11) were obtained from patients undergoing surgery for PDAC as described previously [9 (link)]. Three pancreatic fibroblasts (PFs, id = 10295, 14289 and 14358) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) (Catalog #3830).
Capan-1 and MIA PaCa-2 were cultured in a Dulbecco’s modified Eagle medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Palo Alto, CA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in 5% CO₂. PFs were maintained in a Stellate Cell Medium (ScienCell Research Laboratories, Carlsbad, CA, USA) at 37 °C in 5% CO², while NFs were maintained in an MF-medium (Toyobo, Tokyo, Japan).

Human hepatic stellate cells (HSCs, Sciencell) were cultured in the Stellate Cell Medium (Sciencell) with 1% Stellate Cell Growth Supplement, 2% fetal bovine serum (FBS), and 1% penicillin/streptomycin solution (Sciencell). Cells were kept in the incubator (37 °C, 5% CO₂). At the beginning of the experiment, cells were seeded in the 96-well plates at a density of 5 × 10³ cells per well and incubated in the incubator for 24 h. After the incubation period, medium was removed and HSCs were exposed to 100 µL of the compound solutions over a range of concentrations (10–0.1 µM) or nothing but culture medium (blank control). Cells were further incubated for 24 h. At the end, medium was removed, cells were washed with PBS, and 50 µL of the MTT solution (0.75 mg/mL) was added to the cells. Plates were kept in the dark for 2 h at 37 °C. Finally, the MTT solution was removed and 100 µL of DMSO was added to each well. Plates with DMSO were kept at room temperature for 10 min. After this time, 5 µL of Sorensen Buffer was added to each well. Plates were swayed and the absorbance was measured at a wavelength of 570 nm by a microplate reader (Synergy H1, BioTek). The cell viability was expressed as a percentage of the control values (blank control) [45 ,46 (link),47 (link)].

HPaSteCs were purchased from ScienCell Research Laboratories (San Diego, CA, USA) and cultured in stellate cell medium (ScienCell Research Laboratories) supplemented with 2% fetal bovine serum (ScienCell Research Laboratories), 1% stellate cell growth supplement (ScienCell Research Laboratories), and 1% penicillin/streptomycin (ScienCell Research Laboratories) in a humidified atmosphere at 37 °C with 5% CO₂.
MIA PaCa-2 and PANC-1 cells were acquired from American Tissue Cell Culture (ATCC; Manassas, VA, USA), and OCUP-A2 is a PC cell line established by our group from a patient with malignant pancreatic neoplasm and liver metastasis [78 (link)]. These cell lines were grown in Dulbecco’s Modified Eagle’s Medium (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 2.5% horse serum (ATCC; for MIA PaCa-2 cells only). BxPC-3 cells were acquired from the Japanese Collection of Research Bioresources Cell Bank (JCRB, Osaka, Japan) and cultured in RPMI 1640 medium with 10% heat-inactivated FBS (Gibco) in a humidified atmosphere at 37 °C with 5% CO₂. Details of cell-based assays are provided in the Supplemental Materials.

The human pancreatic cancer cell lines BxPC3 and SW1990 were obtained from ATCC and cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% foetal bovine serum (FBS). The human primary pancreatic stellate cell line HPanSteC was purchased from ScienCell Research Laboratories (California, USA) cultured with Stellate Cell Medium (ScienCell, Cat #5301, 500 mL of basal medium supplemented with 10 mL of FBS, 5 mL of Stellate Cell Growth Supplement and 5 mL of penicillin/streptomycin solution) at a cell density of 5 × 10³/cm² as the supplier recommended. Cell culture medium was changed every three days until the culture reached approximately 90%, then the culture was passaged. The human primary pancreatic stellate cell line HPanSteC was used within five to six passages from initiation. All cell lines were grown at 37°C in a 5% CO₂ atmosphere.

Human HSC (TWNT-4) cells [11 (link)] were cultured in the stellate cell medium purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) at 37 °C in 5 % CO₂. Cells were seeded at 4 × 10⁴/well in a 6-well plate, grown until 70 % confluence, and treated with 20 μM SB431542 for 72 h. The levels of L⁵⁹ LAP-DPs in the culture media were measured by the ELISA developed in this study, and the mRNA levels in each cell lysate were quantitated by real-time reverse transcription-polymerase chain reaction (RT-PCR).

Human primary HSCs were purchased from ScienCell Research Laboratories (San Diego, CA, USA) and were grown at 37°C, 5% CO₂ in recommended Stellate Cell Medium provided by ScienCell according to the methods reported previously [14 (link), 25 (link), 26 (link)]. Consistent with these previous studies using Human primary HSCs, the cells showed phenotypic features of fully activated HSCs between passages 3 and 7 and thus the cells at passage 7 were used in the experiments of SSO transfection.

Human primary hepatic stellate cells (hHSCs) were obtained from ScienCell Research Laboratories and were cultured according to the manufacturer's instructions. Briefly, the cells were seeded into poly‐l‐lysine‐coated T‐25 flasks in Stellate Cell Medium (ScienCell Research Laboratories) containing 2% fetal calf serum (FCS) and stellate cell growth supplement (ScienCell Research Laboratories).