Tissues and cells
Surgically resected liver tissue specimens were obtained from the Department of Pathology, National Taiwan University Hospital and Keelung Hospital, Ministry of Health and Welfare. These specimens were used in accordance with appropriate regulations and with approval by the Institutional Review Board of the Ethics Committees of the NTUH (200701095R) and KLH (TYGH100038). The HCC cell lines used were PLC-PRF-5 (CRL-8024) cells, which were purchased from ATCC (VA, USA), and HuH-7 cells (JCRB-0403) (19), which were from JCRB (Osaka, Japan). HEK293 (CRL-1573) and HEK293T cells were obtained from ATCC (CRL-3216). Prior to use, reauthentication of these cells was performed by Short Tandem Repeat (STR) DNA profiling analysis. NIH3T3 cells (BCRC-60008) were purchased from BCRC in 2003 (Hsinchu, Taiwan); NIH3T3 is a nonhuman cell line, and the morphology and growth speed of these cells have not changed since we obtained them (data not shown). HEK293T and NIH3T3 cells were used for transient transfection and stable clone selection. HeLa cells (BCRC-60005), which were authenticated through STR DNA profiling analysis, were purchased from BCRC in 2014. They were used for immunofluorescence staining within 6 months of cell resuscitation. Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. G-418 (Promega, Fitchburg, WI, USA) was used for the selection of stable clones.
Plasmids and constructs
The wild-type GPC3 expression vector GPC3, convertase-resistant mutant RR→AA (11), proline residue mutant P26-30A, 70–453 a.a. deleted GPC3 expression vector (∆GPC3), Grb10 and neural precursor cell-expressed developmentally downregulated 4 (Nedd4) have all been previously described (6). Activator protein-1 (AP-1) luciferase reporter was purchased from Promega (USA).
Antibodies and immune assays
The antibodies used in the present study included anti-GPC3 (1G12, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-IGF1Rβ (#3027, Cell Signaling Technology, Danvers, MA, USA) (6), anti-Grb10 (Santa Cruz Biotechnology), anti-Flag (Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan) and anti-HA (New England Biolabs, Ipswich, MA, USA). Paraffin-embedded tissue sections were deparaffinized, and immunochemical staining was performed after antigen retrieval. Endogenous peroxidase activity was blocked with EnVision FLEX Peroxidase-Blocking Reagent (Dako, Agilent). For Western blot analyses, tissues and cells were extracted with HNTG buffer (20 mM HEPES buffer, pH 7.5; 150 mM NaCl; 0.1% Triton X-100; and 10% glycerol). Immunoprecipitation was performed by treating 0.5–1 mg of the cell lysate with HNTG buffer containing antibodies, and precipitated proteins were subsequently subjected to Western blotting.
After transfection, cells were cultured in a serum-free medium, stained with IGF-1R (CD221, ThermoFisher, USA) antibody and stimulated with 50 ng/ml of IGF-1 for 10 min. They were then treated with 0.5% Triton X, fixed in 4% paraformaldehyde and stained with anti-Grb10 (Santa Cruz Biotechnology). The secondary antibodies were conjugated with Alexa 488 (green) for IGF-1R and Alexa 594 (red) for Grb10, and images were captured using a confocal laser scanning microscope.
Cell culture in 3D collagen I gels
We added the desired amount of collagen I (Corning, 354249) to obtain a final collagen I concentration of 2.3 mg/ml in a solution containing 10% 10 × minimal essential medium, 1% fetal calf serum (Sigma-Aldrich), and distilled water. The pH was adjusted to 7.4 with 1 N NaOH. Cells (2.5 × 105 cells/ml) were added to the aforementioned solution before it solidified. The mixture was then immediately transferred to a 96-well plate and allowed to solidify at 37 °C. After 30 min, 100 µl of normal complete culture medium was added on top of the cells and the collagen I gel mixture. Cells were cultured in the 3D collagen I gel for 3 d.
Luciferase assays for AP-1 reporter activities
Cells were transiently transfected with a combination of plasmids, as described in the legend of Fig. 4. After overnight transfection, they were treated with serum-free media for 48 h. Firefly luciferase (Promega) and Renilla luciferase (Applied Biosystems) assays were performed according to manufacturer instructions. For each experiment, transfections were performed in triplicate. Values represent means ± SD of at least three independent experiments. All values were normalized for transfection efficiency (Renilla luciferase activity).
PLC-PRF-5 cells were cultured in serum-free medium for 3 d. The conditioned media with 8 µg of protein–after spin concentration–was mixed with nonreducing sodium dodecyl sulfate gel sample buffer and applied without boiling to a 10% polyacrylamide gel containing 0.1% sodium dodecyl sulfate and 1 mg/ml gelatin solution. After electrophoresis, the gels were washed with 50 mmol/l Tris-HCl (pH 7.5) containing 0.15 mol/l NaCl, 5 mmol/l CaC12, 5 µmol/l ZnCl, 0.02% NaN3 and 0.25% Triton X-100 (three changes) at room temperature and then incubated in the same buffer without Triton X-100 (two changes) at 37 °C for 20 h. Proteins were stained with Coomassie Brilliant Blue R-250.