SNU878 cells were purchased from the Korean Cell Bank, while Hep3B, PLC/PRF/5, and HUH7 cells were procured from ATCC. Hep3B, PLC/PRF/5, HUH7 cells, and SNU878 cells were maintained in RPMI 1640 medium (Thermo Fisher Scientific, 11875-093) supplemented with 10% fetal bovine serum. The cells were incubated in a humidified incubator at 37 °C with 5% CO2.
RNA extraction and real-time PCR
The cells were seeded in 60 mm dishes and allowed to adhere overnight at 37 °C in an atmosphere of 10% CO2. The total RNA was extracted using a RNeasy Mini Kit (Qiagen, Valencia, CA), and the complementary DNA (cDNA) was synthesized using a PrimeScript™ 1st strand cDNA Synthesis Kit (Takara, Japan). In order to quantify the transcripts of the genes of interest, real-time PCR was performed using a CFX96 Real-Time PCR system and iQ SYBR Green Supermix (Bio-Rad) for human FGF19 (Forward—5′-GGAGGAAGACTGTGCTTTCG-3′, Reverse—3′- GGCAGGAAATGAGAGAGTGG-5′), human FGFR4 (Forward—5′-CTGCAGAATCTCACCTTGAT-3′, Reverse—3′-TTCTCTACCAGGCAGGTGTA-5′), and human GAPDH (Forward—5′-AGGGCTGCTTTTAACTCTGGT-3′, Reverse—3′-CCCCACTTGATTTTGGAGGGA-5′). The relative mRNA level was calculated using the 2− ΔΔCT method.
siRNAs and transfection protocol
We purchased pre-designed FGFR4, FGF19, and Src siRNAs, along with the siRNAs in the negative control from Bioneer (Daejeon, South Korea). For transfecting the siRNAs, 3 × 105 cells were seeded in 60 mm culture plates and maintained in culture medium supplemented with 10% FBS at 37 °C in an atmosphere of 5% CO2 for 24 h. The cells were transfected with 100 nM siRNA using 40 µL lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) in 300 µL of opti-MEM (ThermoFisher Scientific). The cells were harvested for further analyses after 48 h of transfection.
Treatment with FGF19 and Src inhibitors
The cells were grown in 60 mm dishes to 80% confluency and maintained in RPMI 1640 medium supplemented with 1% BSA (Sigma-Aldrich, A2058). After 24 h, the cells were treated with 20 µM saracatinib (Selleckchem, Houston, TX, USA) for 24 h. The next day, the cells were treated with 100 ng/ml FGF19 (Rocky. Hill, NJ, USA) for 1 h.
Cell viability assay
For the cell viability assay, 5 × 103 cells were seeded in 96-well plates and allowed to adhere overnight. The triplicate wells were subsequently treated with dasatinib or BLU9931 (Selleckchem, Houston, TX, USA), serially diluted from 10 µM for 72 h. Cell viability was determined using the Cell Counting kit-8 assay (CCK-8; Dojindo, Japan).
For protein extraction, the cells directly harvested with 2X lysis buffer (Cell Signaling Technology, 9803), 100X phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, 78,830), 10X PhosSTOP, and cOmplete Protease Inhibitor cocktail (Roche). The cell lysates (30 µg protein per sample) were separated by electrophoresis, transferred to PVDF membranes (Millipore, IPVH00010), and were incubated with the antibodies. The anti-Src, anti-pSrc, anti-STAT3, anti-pSTAT3, and anti-EEA1 antibodies (1:1000) were purchased from Cell Signaling Technology, while the anti-FGFR4, anti-LaminB1 (1:200), and anti-beta-actin (1:1000) antibodies were purchased from Santa Cruz Biotechnology. The anti-FGF19 (1:500) antibody was procured from R&D Systems, while the anti-phosphotyrosine antibody, clone 4G10 (1:1000) was procured from Millipore. The secondary antibodies used (1:5000) were HRP-conjugated mouse anti-rabbit IgG-HRP, goat anti-mouse IgG-HRP, and bovine-anti goat IgG-HRP-linked antibodies (Santa Cruz Biotechnology). Detection was performed using an ECL™ Prime Western Blot System (GE Healthcare) and an Amersham Imager 600 (GE Healthcare). The bands were quantified with ImageJ, a Java-based image analysis package that is widely used for measuring density.
For detection of pFGFR4, cell lysates were incubated with 2 μg of the anti-FGFR4 antibody bound to protein G magnetic beads (Bio-Rad) for 1 h at room temperature. The bound proteins were detected with anti-phosphotyrosine antibody.
The cells were lysed with 2X lysis buffer, as previously described. Following centrifugation, the whole-cell lysates were incubated with 2 μg of the specified antibody (anti-Src or anti-FGFR4) bound to protein G magnetic beads (Bio-Rad) for 1 h at room temperature. The bound proteins were detected by western blotting.
The cells were fixed in 4% paraformaldehyde for 10 min. Following permeabilization with 0.3% Triton X-100, the cells were blocked with 5% normal goat serum for 30 min at room temperature. The samples were subsequently incubated with the primary antibodies (anti-pSrc, anti-pSTAT3, 1:250, Cell Signaling Technology, and anti-FGFR4, 1:50, Santa Cruz Biotechnology) for 24 h at 4 °C. After washing, the samples were incubated with the secondary antibodies (goat anti-mouse IgG H&L -Alexa Fluor® 488, goat anti-rabbit IgG H&L-Alexa Fluor®, 594, 1:250, Abcam) for 1 h at room temperature. After three final washes, the cells were mounted with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories).
The cells were washed twice with 1 × PBS, lysed in hypotonic buffer comprising 10 mM Tris–HCl, pH 8.0, 10 mM KCl, 1.5 mM MgCl2, 0.5% Nonidet P-40, 1 mM dithiothreitol, 1 mM PMSF, 10X PhosSTOP, and cOmplete Protease Inhibitor cocktail, and incubated on ice for 15 min. Following incubation, the lysates were centrifuged at 1500×g for 5 min. The supernatants were further centrifuged at 16,100×g for 20 min, and the pellets were resuspended in nuclear extraction buffer comprising 400 mM NaCl, 1 mM EDTA, 20 mM Tris–Cl, pH 8.0, 1 mM dithiothreitol, 1.5 mM MgCl2, 25% glycerol, 1 mM PMSF, 10X PhosSTOP, and cOmplete Protease Inhibitor cocktail. The resuspended nuclear fractions were centrifuged at 16,100×g for 30 min.
All experiments were repeated at least triplicate. GraphPad Prism 5 was used for data analyses. Statistical significance was measured by two-way ANOVA. IC50 values were calculated from a log([drug]) versus normalized response curve fit.