1. Cell lines and culture
MIA PaCa-2 and PANC-1 are p53-mutated pancreatic cancer cell lines that are derived from human pancreatic carcinoma . hTERT-HPNE is a human pancreatic normal ductal cell line. These pancreatic cancer cell lines were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (GIBCO Invitrogen Inc., USA) containing 4.5 mg/L glucose, 100 mg/L streptomycin, and 2 mM l-glutamine supplemented with 10% fetal bovine serum (FBS) (GIBCO Invitrogen Inc.). hTERT-HPNE (ATCC® CRL-4023™, USA) cells were grown in DMEM containing 1.0 mg/L glucose (low glucose), 100 mg/L streptomycin, and 2 mM l-glutamine supplemented with 10% FBS and 10 ng/mL human epidermal growth factor (hEGF) (MERCK, USA). The cells were sub-cultured following enzymatic digestion using trypsin-EDTA solution (GIBCO Invitrogen Inc.), and were maintained under a humidified atmosphere at 37 °C in a 5% CO2 incubator (Sanyo, Japan).
2. Flow cytometry analysis
MIA PaCa-2, PANC-1, and hTERT-HPNE cells were seeded in six-well cell culture plates at a density of 1 × 105 cells/well. After overnight culture, the cells were treated with various concentrations (0–400 μM) of silibinin for 24 h; then, the cells were harvested using 0.005 M trypsin-EDTA, and washed thrice with PBS. The cells were then stained with Annexin V Apoptosis Detection Kit I (51-66211E; BD Pharmingen™, USA). The stained cells were acquired and analyzed using Canto II (BD Pharmingen™) and FlowJo software (Tree Star Inc., USA).
Cells were stained with Ghost dye (BV510; #59863, CST, USA) and washed thrice with PBS. For intracellular staining, cells were stained using a FoxP3/transcription factor staining buffer set (00-5523; eBioscience, USA) and APC-conjugated anti-human Ki-67 (350514; Biolegend, USA).
3. Western blot analysis
Proteins were extracted from silibinin (200 µM/mL)-treated pancreatic cells (the cells were cultured to 70% confluence in 60 mm dishes) using a radioimmunoprecipitation assay buffer (Sigma R0278, Sigma-Aldrich Co. LLC, USA) containing a protease inhibitor (#p8340, Sigma-Aldrich) and a phosphatase inhibitor (#p2850, Sigma-Aldrich). The proteins were separated using 10% SDS–polyacrylamide gel electrophoresis, and blotted onto PVDF membranes (Millipore Corporation, Billerica, MA, USA). These membranes were blocked with 5% (w/v) skim milk in TBS-T (20 mM Tris, pH 7.6, 136 mM NaCl containing 0.1% (v/v) Tween-20) at 25℃ for 1 h. After washing thrice with TBS-T, the membranes were incubated overnight with diluted primary antibodies (all antibodies were from Cell Signaling Technology Inc., USA; antibody:TBS-T = 1:1,000) at 4 °C. After washing thrice with TBS-T for 10 min each, the membranes were incubated with either anti-rabbit or anti-mouse horseradish peroxidase–conjugated secondary antibodies. ECL SuperSignal chemiluminescent substrate (Millipore Corporation, Billerica, MA, USA) was used to develop the membrane. Protein bands were then visualized using a LAS 3000 Imaging System (FujiFilm, R&D Systems, Minneapolis, MN, USA).
4. Spheroid formation assay
Cells were seeded in six-well plates at a density of 1 × 103 cells/well, and cultured in F-12 DMEM (GIBCO Invitrogen Inc.) containing 10 ng/mL human recombinant basic fibroblast growth factor (bFGF) (R&D Systems) and 10 ng/mL hEGF (R&D Systems) with 1× N2 supplement (GIBCO Invitrogen Inc.). The cells were incubated at 37 °C under a humidified atmosphere containing 5% CO2. Spheroids were confirmed after 14 days.
5. Invasion assay
Cell invasion was carried out overnight using a transwell filter chamber (8.0-μm pores) coated with 1% gelatin/DMEM, followed by drying at RT. Pancreatic cancer cells were harvested, washed once with the growth culture medium, and seeded on the upper chamber at 2 × 105 cells in 120 μL 0.2% BSA medium. Then, 400 μL 0.2% BSA medium containing 20 μg/mL human plasma fibronectin (Calbiochem, La Jolla, CA, USA) was loaded into the lower chamber. The transwell apparatus was incubated at 37 °C for 24 h. Cells that invaded the bottom surface of the upper chamber were fixed with 70% ethanol and stained with Diff-Quik solution (Sysmex, Kobe, Japan), according to the manufacturer’s protocol. Non-invasive cells on the top surface were wiped off with cotton balls and stained. Cells on the bottom surface were counted in five selected fields (each 0.5 mm2), using a hematocytometer under a light microscope at ×400 magnification. The results were expressed as means ± SE of the number of cells per field from three individual experiments.
6. RNA isolation
Total RNA was extracted using TRIzol (Takara, Japan). Briefly, 1 mL TRIzol solution was added into each well, and the suspensions were transferred to 1.5 mL tubes. After adding 200 μL chloroform (Sigma-Aldrich) and vortex mixing for 15 s, the mixtures were centrifuged at 4℃ and 6,000 × g (grams) for 20 min. The supernatants were then collected, mixed with equal amounts of isopropyl alcohol (MERCK), and centrifuged at 4℃ and 6,000 × g for 20 min. The pellets were washed with 1 mL 70% ethyl alcohol (MERCK) and centrifuged at 4℃ and 6,000 × g for 5 min. After removing the remaining ethyl alcohol, the RNA pellets were air-dried at RT. They were then resuspended in 50 µL diethyl pyrocarbonate water.
7. Real-time polymerase chain reaction (PCR)
Total RNAs were converted to cDNAs using a reverse transcription system (Promega Corporation, WI 53711–5399, USA). Real-time PCR was performed with an Applied Biosystems StepOnePlus™ (Thermo Fisher Scientific, MA, USA) Real-Time PCR system, according to the manufacturer’s protocol. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH: HS02786624-g1) was used as a control, and the del/del threshold cycles ( 2-ΔΔCT CTs) values were calculated for stem markers (TWIST1: HS 01675818_s1, c-Jun: HS01103582_s1, and Snail: HS00195591_m1). The probes used were Thermo Fisher Taqman® Assay probes (Thermo Fisher Scientific).
8. Wild-type p53 gene transfection
Cells were seeded in six-well plates and incubated at 37℃ overnight. The cells were then transfected with pcDNA3/wild-type p53 (plasmid #69003, Addgene, MA 02472, USA) expression plasmid vector, using FuGENE®6 (Cat.#E2693, Promega Corporation) reagent, according to the manufacturer’s protocol.
9. Statistical analysis
The two-tailed paired t-test was used to assess statistical significance appropriately. The statistical significance of differences between data sets was determined using the paired t-test and one-way ANOVA test. All the reported p values are two-sided; p ≤ 0.05 was considered statistically significant. The Statistical Package for the IBM SPSS Statistics 23 software (SPSS Statistics Inc., Chicago, IL, USA) was used for all statistical analyses.