Cell culture and reagents
Huh-7, HepG2 and LO2 cells were obtained from the American Type Culture Collection. These cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM; GIBCO BRL) supplemented with 10% (v/v) FBS, 100 U/mL penicillin and 100 U/mL streptomycin. Cultures were maintained at 37℃ in a humidified at mosphere with 5% CO2. Sorafenib and Verteporfin were purchased from Selleck Chemicals. Antibody to PARP, YAP, p-YAP(S127), survivin, Bcl-xl and Histone H3 were purchased from Cell Signaling Technology, Inc.; antibody to GAPDH was from Santa Cruz Biotechnology, Inc.; antibody to Flag was from Sigma.
Cell viability assay
Cells were seeded in 96-well plates (4,000 cells/well) and incubated overnight for attachment, and were then treated with indicated agents in 10% FBS-supplemented medium for 72 hours. The medium was replaced with CCK-8 at 37℃ for 2 hours and absorbance at 450 nm was measured.
For immunofluorescence analysis, cells were plated in chamber slides then fixed in methanol for 10 min at room temperature, permeabilized with 5% bovine serum albumin in PBST. Cells were then exposed to primary antibodies (anti-YAP 1:200 ) diluted in PBST containing 5% bovine serum albumin overnight at 4℃. After washing three times with PBS for 10 min, secondary antibody (Alexa Fluor 488- goat anti-rabbit 1:200) diluted in PBST was added and incubated for 1 h at room temperature. Cells were then washed in PBS and mounted using 4,6-diamidino-2-phenylindole (DAPI) to counterstain DNA. Images were collected using a confocal microscope (Olympus FV-1000).
Colony formation assay
For Colony formation, the cells were seeded into 6-well plates (500 cells per well), and then treated with sorafenib and verteporfin, alone or in combination. The medium was replaced with fresh medium containing the reagent every three days. After 10 days treatment, the medium was removed and cell colonies were fixed with 4% paraformaldehyde for 20 minutes and stained with crystal violet (0.1% in 20% methanol) for 30 minutes. Then they were washed slowly with running water and air dried naturally. Pictures were taken using a digital camera to record the result, and the number of cell clones with more than 50 cells was counted under the microscope.
Plasmids and transfection
Plasmids encoding the human YAP and survivin were cloned into pcDNA3.1 vector with the Flag-tag. For transient transfection, plasmids were pretransfected with lipofetamine 2000 (Invitrogene) for 24 hours and then processed with the indicated treatment as described.
Target siRNA was produced by GenePharma (Suzhou, China) and transfected using Lipofectamine RNAiMAX Transfection Reagent (Invitrogene) according to the manufacturer’s protocol. A non-targeting siRNA was used as a negative control. Target sequence as follows: survivin (5'-AAGGAGAUCAACAUUUUCA-3′).
For stable YAP knockdown, the following Addgene plasmids were used, pLKO1-shYAP#1(27368) and pLKO1-shYAP#2 (27369).
Quantitative real-time polymerase chain reaction (RT-PCR)
Total RNA was extracted with TRIZOL Reagent (Invitrogen), and then reverse transcribed to cDNA with M-MLVReverse Transcriptase (Promega). Real-time PCR was carried out by FastStart Universal SYBR Green Master (Roche) and cDNA amplification was detected by the StepOne RT-PCR System (Applied Biosystems). The primers used were as follows:
Flow cytometric analysis of apoptosis
Apoptotic rate was detected by flow cytometry with the Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Biosciences). Briefly, cells were collected after different treatment and the assay were performed according to the manufacturer's instruction. Samples were analyzed immediately using a Cytomics FC500 flow cytometer (Beckman Coulter).
Xenograft tumor growth
For the subcutaneous xenograft tumor model, each nude mice (nu/nu, 5-week-old females) were injected subcutaneously in the dorsal flank with 5×106 HepG2 cells suspended in 0.1 mL of serum-free medium. When tumors reached 100 to 200 mm3, mice were randomly divided into four groups, and received vehicle, sorafenib (50 mg/kg) orally once daily, verteporfin (100 mg/kg) intraperitoneally every other day or the combination of sorafenib and verteporfin respectively. Tumor volume was measured every 4 days using calipers and their volumes calculated using the following formula (tumor volume =π/6 (L×W2)). Mice were sacrificed on day 32, and the tumors were dissected and analyzed.
Xenograft tumors were fixed in 4% PFA, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Immunohistochemical staining of paraffin-embedded tumor tissues was performed using survivin (CST, 1:100 dilution) and Ki-67 (Abcam, 1:100 dilution) primary antibodies and the ABC Elite immunoperoxidase kit according to the manufacturer’s instructions.
The data were analyzed using SPSS 16.0 software. The Student’s t test was calculated to compare the mean of different groups and p < 0.05 were considered significant.