Pancreatic Duct

Pancreatic ducts were isolated from the bulk of the pancreas of mice older than 8 weeks by collagenase dissociation (Collagenase type XI 0.012% (w/v) (Sigma), dispase 0.012% (w/v) (Gibco), FBS (Gibco) 1% in DMEM media (Gibco)) at 37°C. Isolated ducts were mixed with Matrigel (BD Bioscience) and seeded and cultured as we described previously (Sato et al, 2009 (link); Barker et al, 2010 (link)). After Matrigel formed a gel, culture medium was added. Culture media was based on AdDMEM/F12 (Invitrogen) supplemented with B27 (Invitrogen), 1.25 mM N-Acetylcysteine (Sigma), 10 nM gastrin (Sigma) and the growth factors: 50 ng/ml EGF (Peprotech), 10% RSPO1-conditioned media (kindly provided by Calvin Kuo), 100 ng/ml Noggin (Peprotech) or 10% Noggin-conditioned media (in-house prepared), 100 ng/ml FGF10 (Peprotech) and 10 mM Nicotinamide (Sigma). One week after seeding, organoids were removed from the Matrigel, mechanically dissociated into small fragments, and transferred to fresh Matrigel. Passage was performed in a 1:4–1:8 split ratio once per week for at least 9 months. To prepare frozen stocks, organoid cultures were dissociated and mixed with Recovery cell culture freezing medium (Gibco) and froze following the standard procedures. When required, the cultures were thawed using standard thawing procedures, embedded in Matrigel and cultured as described above. For the first 3 days after thawing, the culture medium was supplemented with Y-27632 (10 μM, Sigma-Aldrich).

Pancreatic cancer cell lines (AsPC-1 and BxPC-3) were cultured in RPMI-1640 (Corning, NY, USA) with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. Two additional pancreatic cancer cell lines (PANC-1, MIA Paca-2) were cultured in DMEM (Dulbecco’ modified eagle medium) (Gibco, Grand Island, NY, USA) supplemented with 10% FBS and 1% penicillin–streptomycin. Human pancreatic ductal epithelium (hTERT-HPNE) cells were cultured in Medium D with mixtures of M3 and DMEM medium containing one volume of medium M3TM Base F culture media (InCell Corp., San Antonio, TX, USA), three volumes of glucose-free DMEM, 5% FBS, 5.5 mM glucose, 10 ng/ml EGF, and 50 µg/ml gentamycin [26 (link)]. All these cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. RNA was extracted from tissues using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and was reverse-transcribed into cDNA using the PrimeScript RT Master Mix (Takara, Otsu, Shiga, Japan). RT-qPCR analyses were quantified with PowerUp™ SYBR® Green Master Mix (Applied Biosystems, Austin, TX, USA), and expression levels were normalized to GAPDH levels. Proteins were extracted in RIPA buffer supplemented with a complete, EDTA-free protease and phosphatase inhibitor single-use cocktail (Thermo Scientific). Proteins were separated by SDS-PAGE and blotted onto a PVDF membrane. Anti-TSC22D2 (1:1000 dilution, #25,418–1-AP, Proteintech) was used as primary antibodies for immunoblotting. Reacted antibodies were detected using an enhanced chemiluminescence detection system.

Human normal renal tubular epithelial cells (HK-2), human GBM cell lines (U87 MG, T98G), human pancreatic duct epithelial cell line (hTERT-HPNE), and human PAAD cell line (PANC-1) were cultured in Dulbecco's modified Eagle's medium (Gibco, United States) containing 10% Fetal Bovine Serum (FBS, Gibco, United States) and 1% penicillin/streptomycin. Human KIRC cell lines (786-O, CaKi-1), human normal astrocytes (HA1800), and human PAAD cell line (ASPC-1) were cultured in RPMI-1640 medium (Gibco, United States) containing 10% and 1% penicillin/streptomycin. Cells were cultured in incubator containing 5% CO₂ at 37 °C.

Animal experiments were performed according to the rules and regulations of the Animal Care and Experimental Committees of Gunma University (permit number: 22-010; Maebashi, Japan). Only male mice and their tissues and cells were phenotypically characterized in this study. Leaden (C57J/L) mice with nonfunctional mutation of the gene encoding melanophilin (Mlph) (Matesic et al., 2001 (link)) were purchased from The Jackson Laboratory (Strain #:000668, RRID:IMSR_JAX:000668), and were backcrossed with C57BL/6N mice 10 times to generate MlphKO mice. Exo8KO mice in the genetic background of C57BL/6N mice were described previously (Fan et al., 2017 (link)). ME8DKO mice were obtained by mating Exo8KO mice with the MlphKO mice described above. GrphKO mice in the genetic background of C3H/He mice were described previously (Gomi et al., 2005 (link)). The male Exo8KO mice were mated with the female GrphKO mice. Because the granuphilin and exophilin-8 genes are on the mouse X and 9 chromosomes, respectively, the resultant F1 generation is either male (Grph^-/Y, Exo8^+/-) or female (Grph^+/-, Exo8^+/-). By crossing these F1 mice, GE8DKO mice, as well as the WT, GrphKO, and Exo8KO mice, were generated in the F2 generation and used for experiments. Although the resultant F2 mice have a mixture of C57BL/6N and C3H/He genomes, we expected that significant phenotypic changes due to the loss of Rab27 effectors may be preserved despite any influences due to randomly distributed differences in the genome. In fact, we found similar differences in exocytic profiles between WT and Exo8KO cells both in the C57BL/6N background (Figure 2) and in the mixture of the C57BL/6N and C3H/He backgrounds (Figure 6). Furthermore, GrphKO cells in the mixture of the C57BL/6N and C3H/He backgrounds showed changes in granule localization and exocytosis (Figure 6), consistent with the reported phenotypes of GrphKO cells in the C3H/He background (Gomi et al., 2005 (link)). Pancreatic islet isolation and dissociation into monolayer cells and insulin secretion assays were performed as described previously (Gomi et al., 2005 (link); Wang et al., 2020 (link)). Briefly, islets were isolated from cervically dislocated mice by pancreatic duct injection of collagenase solution, and size-matched five islets were cultured overnight in a 24-well plate. Monolayer islet cells were prepared by incubation with trypsin-EDTA solution and were cultured for further 2 days. Insulin released from isolated islets or monolayer cells was measured by an AlphaLISA insulin kit (PerkinElmer) or insulin high range and ultra-sensitive kits (Cisbio).