Animals
Five-week-old female mice (C57BL6×DBA2 F1-hybrid) were used. All animal experiments were approved by the Institutional Animal Care and Use Committee of Seoul National University (SNU-IACUC; Approval number: SNU-180403-4). All protocols involving animals were conducted in accordance with the Animal Protection Act, Laboratory Animal Act, and Guide for the Care and Use of Laboratory Animals of the SNU-IACUC. Furthermore, the protocol complied with the ARRIVE guidelines.
Primary isolation and culture of salivary gland epithelial cells (SG-Epis)
Single cells were obtained from mouse submandibular glands. Isolated cells were suspended in Dulbecco’s modified Eagle’s medium (DMEM)/F12 culture medium 1:1 (v/v; Thermo Fisher, Waltham, MA, USA) supplemented with 10% FBS (Hyclone Laboratories, Logan, UT, USA), 1% penicillin-streptomycin (Thermo Fisher), and 10 μM Y-27632 (Selleckchem, Houston, TX, USA) and plated on 0.1% gelatin-coated culture dishes. The next day, the media was replaced with serum-free DMEM/F12 culture medium 1:1 (v/v) containing 20 ng/mL epithelial growth factor (Peprotech, Rocky Hill, NJ, USA), 1% N-2 supplement (Thermo Fisher), 1% insulin (Cell application, San Diego, CA, USA), 1 μM dexamethasone (Sigma, Louis, MO, USA), 1% penicillin-streptomycin, and 10 μM Y-27632. Trypsin (0.05%) (Thermo Fisher) was used for 2 min to remove mesenchymal cells. Then, cells were passaged by incubation with 0.05% trypsin for 6 min.
Viability assay
A water-soluble tetrazolium salt 1 (WST-1) based EZ-Cytox kit (Daeil Lab Service, Seoul, Korea) was used for determining cell viability. SG-Epis were cultured for three days with 10 μM Y-27632 in 96-well plates. WST-1 reagent was added to each well, and plates were incubated at 37°C for 30 min. An Emax Plus Microplate reader measured the absorbance of each well at 450 nm. (Molecular Devices, CA, USA). All small molecules were purchased from Selleckchem.
RNA extraction, cDNA synthesis, and qRT-PCR
Total RNA was extracted using a PureLinkTM RNA Mini Kit (Invitrogen, Camarillo, CA, USA). cDNA synthesis used M-MLV reverse transcriptase following the manufacturer’s protocol (M.biotech, Hanam, Korea). Real-time PCR was performed using SYBR Pre-mix Ex Taq™ Ⅱ (Takara, Tokyo, Japan) and a StepOnePlus Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). Cycling was performed for 30 s at 95°C, followed by 40 amplification cycles of 5 s at 95°C and 30 s at 60°C. Gapdh was used for normalization.
Western blot analysis
Total protein was extracted using Cell Culture Lysis 5X Reagent (Promega) on ice. The protein concentration was measured using a BCA protein Assay Kit (Thermo Fisher). Twenty μg of proteins from each group were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 10% skim milk or 5% BSA for 1 h at room temperature and incubated with primary antibodies overnight at 4°C. Membranes were washed three times with TBS-T (Tris-buffered saline, 0.1% Tween 20) buffer and incubated with secondary antibodies. The proteins were visualized with an EZ-Western Lumi La (Daeil Lab Service).
Characterization of SG-Epis by flow cytometry
SG-Epis of passages 7, 12, and 17 were harvested and washed with ice-cold PBS containing 5% FBS. Cells were then incubated on ice for 30 min with monoclonal antibodies against CD44, CD49f, and CD117. Flow cytometry analysis was performed using FACSVerse (BD bioscience, Erembodegem, Belgium).
Immunocytochemistry
SG-Epis were fixed with 4% paraformaldehyde for 20 min then permeabilized for 10 min with 0.2% Triton X-100 (Sigma) at room temperature (RT). Non-specific antibody binding was prevented by incubating cells in a blocking solution (3% BSA in PBS) for 15 min at RT. Cells were then treated with primary antibodies and incubated overnight at 4°C. Samples were then incubated with Alexa Fluor 488- or cyanine 3 (Cy3)-conjugated secondary antibodies, as well as Alexa Fluor 647-conjugated phalloidin (Cayman, Ann Arbor, MI, USA) for F-actin staining. ProLong Glass Antifade Mountant with NucBlue (Thermo Fisher) was applied as a mounting and nuclear counterstaining solution.
Colony-forming assays
SG-Epis were seeded at a density of 2 × 103 cells/well on 6-well culture plates and incubated for seven days. Colonies were stained using 0.2% crystal violet solution (Sigma) for 20 min, and the total number of colonies was counted.
RNA interference
Rock2 siRNA was purchased from Thermo Fisher. A scrambled siRNA was used as a control, and Hprt siRNA was used as a positive control, and these siRNAs were purchased from Integrated DNA Technologies (Coralville, IA, USA). SG-Epis were transfected with siRNAs at a final concentration of 25 nM using TranIT-X2 transfection reagent (Mirus Bio, Madison, WI, USA) following the manufacturer’s instructions. Cells were harvested for further analysis after 24 h.
Statistical analysis
All data were presented as mean ± SD and Student’s t-test, as well as one-way and two-way ANOVA followed by Tukey’s and Bonferroni post-hoc tests, were performed to determine the significance of observed differences. Statistical analyses were performed using GraphPad Prism 5.0. All experiments were independently repeated at least three times. *p < 0.05 was considered statistically significant.