Collagen 1 coated dishes

The establishment of the human CSCs used in this study (GS-Y03, PANC-1 CSLC, and A2780 CSC) has been described [17 (link), 34 (link), 35 ]. These cell lines were maintained under the monolayer stem cell culture condition reported previously [17 (link), 34 (link), 35 ]. In brief, cells were cultured on collagen-I-coated dishes (IWAKI, Tokyo, Japan) in the stem cell culture medium (DMEM/F12 medium supplemented with 1% B27 [Gibco-BRL, Carlsbad, CA, USA], 20 ng/mL EGF and FGF2 [Peprotech, Inc., Rocky Hill, NJ, USA], D-(+)-glucose [final concentration, 26.2 mM or 5 mM where indicated as such], L-glutamine [final concentration, 4.5 mM], 100 units/mL penicillin and 100 μg/mL streptomycin). In principle, the stem cell culture medium was changed every 3 days, and EGF and FGF2 were added to the culture medium every day. Throughout the study, the cell number was determined using a hemocytometer, and the cell viability was examined by the dye exclusion method (0.2% trypan blue). Cell viability (%) was defined as 100 x ‘the number of viable cells’/(‘the number of viable cells’ + ‘the number of dead cells’).

The mouse embryonic fibroblast lines, MEF-1 (LRP1-wild-type (WT)) and PEA-13 (LRP1-knockout (KO)), obtained from American Type Culture Collection, and human embryonic kidney (HEK) 293 cells were cultured in DMEM supplemented with 10% fetal bovine serum. HEK293 cells were cultured on collagen I-coated dishes or plates (Iwaki). Primary neuronal cultures were prepared from cerebral cortices of rat embryos at embryonic day 17, as described previously (Araki et al., 2001; Motoki et al., 2012 (link)). Cells were plated on poly-L-lysine–coated dishes or plates and maintained in Neurobasal medium containing B27 supplements (Invitrogen).

Five-week-old mice were anesthetized with an intraperitoneal injection of a mixture of medetomidine hydrochloride (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol tartrate (5 mg/kg), and the parotid glands were extracted. Acinar cells were isolated from the parotid glands as previously described [21 (link)]. Isolated acinar cells were diluted with Waymouth's medium containing 10% rat serum, 1% ITS-X supplement, 1% penicillin–streptomycin, 1 µm hydrocortisone, and 10 nM cystatin, and cultured in collagen I-coated dishes (Iwaki, Tokyo, Japan) at 37°C in a 5% CO₂ incubator. One day after cellular isolation, the cells were then divided into groups and cultured as follows: the first group had the same Waymouth's medium concentration at the initial culture medium (10% rat serum), the second had Waymouth's medium concentration adjusted to 2% rat serum, and the third had Waymouth medium supplemented with 100 ng/mL recombinant human BMP2 (Chicago, Humanzyme, IL, USA) to 2% rat serum. The cells were seeded at a concentration of 0.02–0.06 mg/mL cells per well in a 96-well plate for the cell proliferation assay.