Cell lines, culture conditions and reagents
Pancreatic cancer cell lines (AsPC-1, BxPC-3, PANC-1, CFPAC-1 and SW1990) were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and maintained in Dulbecco’s modified Eagle media (DMEM) supplemented with 10% fetal bovine serum (Gibco, NY, USA). The cells were incubated at 37℃ in a humidified atmosphere containing 5% CO2. Antibodies against YAP (#14074), ATG5 (#2630) were purchased from Cell Signaling Technology. Anti-active YAP (ab205270) antibody was from Abcam. Antibodies against p62 (sc-28359) and GAPDH (sc-32233) were purchased from Santa Cruz Biotechnology. Anti-LC3 (NB100–2220) antibody was obtained from NOVUS. Anti-Flag antibody (F3165), cycloheximide (CHX, 01810), verteporfin (SML0534) and chloroquine (CQ, C6628) were obtained from Sigma-Aldrich.
RNA isolation and qRT-PCR
Total RNA was isolated using Total RNA Kit I (OMEGA) according to the manufacturer’s instruction. For qRT-PCR, RNA was reverse transcribed to cDNA by using a PrimeScript™ RT reagent kit (Takara, Dalian, China). The cDNA was amplified on a 7500 Real-Time PCR System (Applied Biosystems) using SYBR Green Master Mix (Roche). All samples were normalized to GAPDH. The primer sequences were listed in supplementary Table 1.
Cell proliferation assays
Cell viability was detected by CCK8 and EdU assays. Cells were seeded in 96-well plates (4,000 cells/well) and incubated overnight for attachment, and were then treated with indicated agents for different times. The medium was replaced with CCK8 at 37℃ for 2 hours and absorbance at 450 nm was measured in a microplate reader (BIO-RAD xMark). EdU assays were performed using the EdU Cell Proliferation Assay Kit (Ruibo, China) according to the manufacturer’s instructions. All experiments were performed in triplicate.
Wound-healing assay
AsPC-1 and BxPC-3 cells were seeded in 12-well plates and grown to 90% confluence. Then, scratch wounds were generated by using a plastic pipette tip, which was recorded as 0 h. Then, the scratch was imaged at 24 h or 48 h. Cell migration was assessed by measuring the movement of cells into the scratch wounds.
Transwell invasion assay
Matrigel-coated invasion assay was performed using a 24-well Transwell chamber system (Corning, USA) according to our previous work [31]. Briefly, 5×104 cells in 400 μL serum-free culture medium were placed into the upper chamber, which was coated with Matrigel (BD, New Jersey, USA). A total of 600 μL medium supplemented with 20% FBS was added into the lower chamber. After incubation for 24 h, cells were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet (Sigma-Aldrich, USA) for 30 min. The stained cells were analyzed.
ChIP analysis
ChIP assays were carried out using the EZ-ChIP kit (Millipore) according to the manufacturer’s instructions, using the antibody against YAP. Briefly, cells were lysed and then sonicated to obtain DNA fragments (500-800 bp). Next, samples were immunoprecipitated overnight at 4°C with YAP antibody, supplemented with magna ChIP™ protein A/G beads. After washing, elution and de-cross-linking, the amount of immunoprecipitated DNA was analyzed by qRT-PCR using the indicated primers listed in supplementary Table 2.
Luciferase reporter assay
293T cells were seeded in 24-well plates and transfected with the pGL3-Atg5 (wild type or mutated TEAD1-binding site) promoter reporter plasmid, pcDNA3.1/YAP or an empty vector, and a Renilla luciferase vector for normalization. Relative luciferase activity was measured with the Dual-luciferase Reporter Assay System (Promega).
Plasmid constructs and transfection
Plasmid encoding the human YAP was 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. siRNAs against human YAP, Atg5 and scramble control RNA oligos were produced by GenePharma (Suzhou, China) and transfected using Lipofectamine RNAiMAX Transfection Reagent (Invitrogene). For stable YAP knockdown, the following Addgene plasmids were used, pLKO1-shYAP#1 (27368) and pLKO1-shYAP#2 (27369). Stable cell lines with YAP knockdown established as previously described [31].
Tissue microarray slides and immunohistochemistry (IHC)
In vivo active-YAP expression was detected by IHC using tissue microarrays (PA2081a, AlenaBio, Xi’an, China). The tissues were incubated with primary anti-active-YAP antibody (1:100, ab205270, Abcam) and biotin conjugated secondary antibody. Hematoxylin was used as the counterstain. Immunostaining degree of each sample was evaluated independently by two pathologists,based on nuclear staining intensity (0, negative; 1, weak; 2, moderate; 3, strong) and percentage of positive cells (0, <5% positive cancer cells; 1, 6-25% positive cancer cells; 2, 26-50% positive cancer cells; 3, 51-75% positive cancer cells; 4, ≥76% positive cancer cells). The final immunoreactivity score is the product of the intensity score and the extent score.
Cycloheximide (CHX) chase assay
AsPC-1 cells were incubated at 90% confluency in complete growth media containing the protein synthesis inhibitor CHX (50 μM) or vehicle. Cells were collected in RIPA buffer containing proteinase inhibitors at 0, 4, 8, 16 and 24 h after incubation. The lysate was sonicated, centrifuged for 10 min at 12,000 × g and the resulting supernatants analyzed by immunoblotting.
Immunofluorescence analysis
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 (BSA) in PBST. Cells were then exposed to primary antibodies (anti-YAP 1:200) diluted in PBST containing 5% BSA overnight at 4℃. After washing three times with PBS, secondary antibody (Alexa Fluor 488 goat anti-rabbit 1:200) diluted in PBST was added and incubated for 1h 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).
Autophagy analysis
Autophagy was measured by quantitation of GFP-LC3 puncta using fluorescence microscopy according to our previous work [26]. Cells were infected with appropriate amounts of lentivirus carrying GFP-LC3 to express the close-to-endogenous level of GFP-LC3. After treatment, cells were fixed with 4% paraformaldehyde for 20 min and rinsed with PBS twice. Total number of cells on images was determined by nuclei staining with 4,6-diamidino-2-phenylindole. Cells were mounted and visualized under a confocal microscope (Olympus FV-1000).
Xenograft tumor-formation assay and therapeutic treatment
Female BALB/C nude mice at 4-5 weeks of age were obtained from Beijing Vital River Laboratory Animal Technology Co.,China. 5×106 AsPC1 cells were subcutaneously inoculated into the right flank of mice to establish pancreatic cancer xenografts. Approximately 8 days after subcutaneous implantation, the mice were randomly divided into four groups and delivered verteporfin (50 mg/kg; intraperitoneally), CQ (60 mg/kg; intraperitoneally), verteporfin plus CQ, or PBS as a control every 2 days. During the treatment, tumour volume was measured every 4 days and calculated using the formula: length × width2 × π/6. Mice with tumor implants were euthanized 32 days after drug treatment, and the tumor xenografts were excised and weighed.
Statistical analysis
GraphPad Prism software (Version 5.0) was used for experimental data analysis. All experiments were independently repeated at least three times with triplicate samples. The Student’s t test was used to detect significance between groups and statistical significance was determined when p < 0.05 (two-tailed). Values are expressed as the mean ± SEM.