EGF and Alexa Fluor 647-EGF were purchased from Sigma-Aldrich (St. Louis, MO, USA). Human PKR siRNA (SR303767) and control siRNA (SR30004) were purchased from OriGene (Rockville, MD, USA). The siRNAs against HIP1 were purchased from Ambion (Foster City, CA, USA). Antibodies against clathrin and EGFR were purchased from Abcam (Cambridge, UK). Tetramethylrhodamine isothiocyanate-labeled affinity purified goat anti-mouse IgG and goat anti-rabbit IgG (H+L) were purchased from KPL (Guildford, UK). Electrochemiluminescence kit was bought for Boshide (Wuhan, China). All other chemicals were purchased from Sigma-Aldrich.

Human hepatocarcinoma cells lines 7402, 7703, 7721, Hep3B, HepG2, H460, SPCA1, SKOV-3, HeLa, and MCF-7 and glioma cells U87, U251, C33a, PC-3, NCI-H1299, NCI-H446, and K562 were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 1X antibiotic-antimycotic solution (15240-096; Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified atmosphere with 5% CO₂.

The sequences of siRNAs targeting human HIP1 were as follows: siRNA1, taattgagcgactatacagag; siRNA2, acagcgatatagcaagctaaa; siRNA3, accgcttcatggag cagttta. These siRNAs were designed using siRNA Target Finder developed by Ambion, Inc. Cells were transfected with siRNA using Lipofectamine 2000 (11668-019; Invitrogen) according to the manufacturer's protocol. Transfection was confirmed by an immunoblot analysis.

Cells were collected, pelleted, and lysed in ice-cold lysis buffer (25 mmol/l Tris-HCl, 1 mmol/l edetic acid, 150 mmol/l NaCl, 50 mmol/l NaF, 1% Triton-100, 1 mmol/l PMSF, 1 mg/l leupeptin, 1 µmol/l aprotinin, pH 7.6). Cell lysates were centrifuged at 4°C and 12,000 g/min, rotated with polyclonal rabbit anti-human antibody for 2 h, followed by precipitation with Protein A Sepharose at 4°C overnight. Beads were washed five times with cold wash buffer (20 mmol/l Tris, pH 7.8, 150 mmol/l NaCl, 1 mmol/l EDTA, 0.1% Triton X-100, 100 µM PMSF and 1 mmol/l Na3VO4) and the bound protein was eluted with Laemmli sample buffer and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After SDS-PAGE, the protein was transferred to a nitrocellulose membrane using a semidry transfer apparatus (Bio-Rad, Hercules, CA, USA), blocked with 3% bovine serum albumin in TBST [10 mmol/l Tris (pH 8.0), 150 mmol/l NaCl, 0.1% Tween-20], incubated at room temperature for 1 h with rabbit anti-human HIP1, EGFR, and GAPDH antibodies, rotated for 1 h at room temperature of 25°C, and washed with TBST three times. The protein on the nitrocellulose membrane was then detected by electrochemiluminescence using a kit with a goat anti-rabbit antibody according to the manufacturer's instructions. Based on the western blotting results, HeLa cells exhibited high coexpression of HIP1 and EGFR and accordingly were used in subsequent experiments.

HeLa cells were plated in 6-well dishes and grown to ~50% confluency. After cells were serum-starved for 2 h at 37°C, they were incubated with serum-free DMEM containing Alexa Fluor 647-EGF at a final concentration of 1.5 ng/ml or 100 ng/ml for 1 h at 4°C. Cells were then incubated at 37°C for 1 h to allow internalization. EGFR internalization was stopped by placing the cells at 4°C. The cells were then washed three times for 10 min with PBS, fixed, and permeabilized with 4% paraformaldehyde and 0.1% Triton X-100 in PBS for 10 min. They were then washed with PBS again at room temperature and visualized under a Zeiss LSM confocal microscope (Oberkochen, Germany).

EGFR internalization experiments were carried out as described previously. The cells were blocked with 5% normal goat serum in PBS for 30 min, washed three times for 5 min each with PBS, and then incubated with mouse anti-clathrin monoclonal antibody (Abcam) diluted 1:50 in PBS for 3 h at room temperature. After another three washes for 10 min each with PBS, tetramethylrhodamine isothiocyanate-labeled affinity-purified goat anti-mouse IgG and goat anti-rabbit IgG (H+L; KPL) diluted 1:50 in PBS were added and incubated for 30 min. Following a final rinse (3×10 min) with PBS, the cells were visualized under a Zeiss LSM confocal microscope.

Images were collected using a Zeiss LSM510-Meta laser scanning confocal microscope with a 63× water immersion objective. Colocalization was calculated using ImageJ (NIH, Bethesda, MD, USA) with the JACoP plug-in to estimate Manders coefficients with automated thresholding. Statistical analyses were implemented in GraphPad Prism (GraphPad, La Jolla, CA, USA).

Based on the image analysis data, differences among treatment groups were examined by analysis of variance (ANOVA). Data are represented as means ± SEM of three experiments. P<0.05 was considered statistically significant. When significant differences were detected, specific post-hoc comparisons between treatment groups were performed using Student-Newman-Keuls tests.

Using 17 cell lines, western blotting was performed to identify cells with high expression levels of both HIP1 and EGFR. As shown in Fig. 1, the 7703, 7721, SKOV-3, U251, and HeLa cell lines exhibited higher expression levels of HIP1 than those of other cell lines. As shown in Fig. 2, the Hep3B, HeLa, PC-3, NCI-H1299, and NCI-H466 cell lines had higher expression levels of EGFR than those of other cell lines. Combining these results, the HeLa cell line had high expression levels of both HIP1 and EGFR.

Based on western blot assays, in HeLa cells, the expression of HIP1 was significantly blocked by siRNA1 (taattgagcgactatacagag) and siRNA2 (acagcgatatagcaagctaaa), both of which were more efficient compared with siRNA3 (accgcttcatggagcagttta) (Fig. 3). Neither siRNA1 nor siRNA2 influenced the expression levels of EGFR and GAPDH in HeLa cells (Fig. 4).

NCI-H1299 cells exhibited high expression of EGFR and low expression of HIP1 (Figs. 1 and 2). Accordingly, NCI-H1299 was chosen as a control to examine the effects of HIP1 siRNA. As shown in Fig. 5, the expression of HIP1 was significantly blocked by siRNA1, siRNA2 and siRNA3. The three siRNAs had no effect on the expression levels of EGFR and GAPDH in NCI-H1299 cells (Fig. 6).

After stimulation with 1.5 ng/ml EGF, endocytosis of EGF-bound EGFR was significantly accelerated after the expression of HIP1 was blocked (Fig. 7). After simulation with 100 ng/ml EGF, endocytosis of EGF-bound EGFR was also significantly accelerated after the expression of HIP1 was blocked (Fig. 8). There was no obvious difference between 1.5 and 100 ng/ml EGF with respect to EGFR endocytosis (Figs. 7 and 8).

Clathrin-mediated endocytosis was also accelerated after the expression of HIP1 was blocked, and exhibited a positive correlation with the internalization of EGF-EGFR for both 1.5 ng/ml (Fig. 7) and 100 ng/ml EGF (Fig. 8). There was no obvious difference between 1.5 and 100 ng/ml EGF with respect to clathrin endocytosis (Figs. 7 and 8). EGFR and clathrin were colocalized in the cytoplasm.