The following inhibitors or reagents were used in this study: LY294002 (Enzo Life Sciences, USA), MK-2206 (Invitrogen, USA), and pertussis toxin (PTX; Invitrogen, USA). YAP, phospho-YAP (Ser127), CTGF, ANG-2, and GAPDH antibodies were from Santa Cruz Biotechnology (Santa Cruz, USA). Alexafluor secondary antibodies were from Life Technologies, USA.
Cell lines and culture
The MDA-MB-231 cells and human umbilical vein endothelial cells (HUVECs) were obtained from China Center for Type Culture Collection (CCTCC, China). All cell lines were maintained in a humidified atmosphere at 37 oC with 5% CO2. MDA-MB-231 cells and HUVECs were cultured in DMEM/F12 (GIBCO, USA) with 15% FBS (GIBCO, USA) and 100 µg/mL penicillin/streptomycin (P/S). For serum starvation, cells were incubated in growth medium without FBS or antibiotics.
Plasmid and RNA transfection
To established stable cells with different YAP expression, 6-well plates were seeded with 2 × 105 cell/well in 2 mL media 24 hr before transfection; cells were 80–90% confluent. Cells were transfected with YAP CRISPR/Cas9 KO plasmid and YAP CRISPR activation plasmids using UltraCruz® Transfection Reagent (Santa Cruz, USA) according to manufacturer’s instruction. After 48 hr of transfection, stable cells were selected with puromycin. For lentiviral particles transduction, Polybrene® (Santa Cruz, USA) reagent was used to introduce retroviral vectors into HUVECs according to manufacturer’s instruction. All CRISPR plasmids and shRNA lentiviral particles were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).
Conditioned medium preparation
MDA-MB-231 cells with different YAP expression were cultured to reach at 80–85% confluent. The original medium was removed and cells were washed three times by PBS. Then MDA-MB-231 cells were cultured with FBS and P/S free DMEM. After 24 hr, the supernatant was collected and labeled as YAP over-expressed medium (CM-YAP+), YAP knock down medium (CM-YAP-) and control medium (CM-Ctrl) for future studies.
Western blot analyses were conducted using standard procedures and proteins were detected using primary antibodies and fluorescent secondary antibodies (IRDye800CW-conjugated or IRDye680-conjugated anti-species IgG (Li-Cor Biosciences, USA). The fluorescent signals were captured on an Odyssey Infrared Imaging System (Li-Cor Biosciences, USA) with both 700- and 800-nm channels. Boxes were manually placed around each band of interest, and the software returned near-infrared fluorescent values of raw intensity with background subtraction(Odyssey 3.0 analytical software, Li-Cor Biosciences, USA).
Tube formation assay
60-µl matrigel matrix (Corning, USA) was transferred to a 96-well plate and then incubated in 37 °C incubator for 30 min. The HUVECs (2 × 104 cells/well) were seeded on the matrigel matrix with different conditioned medium (CM), and incubated at 37 °C for 12 hr. The tube-like structures were photographed under Qimage Retiga 2000R camera (Surrey, Canada) at 100-fold magnification, and the total tube lenght and total branching lenght from six representative fields of each group were analyzed by Image J software.
Plate cloning formation experiment
HUVECs were digested and 300 cells of each group were seeded into 6-well plates. After 24 hr, the adherent cells were cultured with conditioned medium and the medium was changed every day. Cell culture was performed for 2–3 weeks when macroscopic apophyses were found in plates. Cells were washed and fixed with 20% methanol for 15 min. Then, fixed cells were stained with Giemsa solution for 40 min. Clones were photographed and counted using Image J software. Cloning formation rate (%) = (number of clones/number of inoculated cells) × 100%.
Quantitative real-time PCR
After transfection for 48 hr, cells were washed with cold PBS and collected in the Qiagen RLT lysis buffer (Qiagen, USA). RNA was extracted with a RNeasy mini kit (Qiagen, USA) and reverse transcribed by M-MLV reverse transcriptase. Quantitative real-time PCR was performed on a Light Cycler 480 (Roche, USA) with a SYBR Green I Master Mix (Roche, USA). mRNA abundance was normalized to GAPDH. Negative controls contained no transcript or reverse transcriptase. RNA from three separate cell pellets pertreatment was analyzed. Relative gene expression was calculated using themethod given in Applied Biosystems User Bulletin No. 2. (P/N 4303859B), with non-targeting RNA-treated cells acting as the control in each data set. Primer pairs used in this study were: GAPDH: F, 5’-GAAGGTGAAGGTCGGAGT-3’/R, 5’-GAAGATGGTGATGGGATTTC-3’; CTGF: F, 5’-CTAAGACCTGTGGAATGGGC-3’/R, 5’-CTCAAAGATGTCATTGTCCCC-3’; ANG-2: F, 5’-ATCTTCCTCCAGCCCCTACAT-3’/R, 5’-GCTTCCACATCAGTCAGTTTCC-3’.
HUVECs were seeded in chamber slides. After treatment, cells were fixed with 4% paraformaldehyde-PBS for 15 min. Following blocking in 5% goat serum with 0.3% Triton X-100 in PBS for 60 min, cells were incubated with YAP primary antibody (1:100 dilution) overnight at 4 oC. After three washes with PBS, cells were incubated with Alexa Fluor 488- or 555-conjugated secondary antibodies (1:500 dilution, Invitrogen, USA) for 2 hr at room temperature. Slides were then washed three times and mounted. Immunofluorescence was detected using a Qimage Retiga 2000R camera (Surrey, Canada) at 40 × magnification.
Cell migration Assays
Migration assays were conducted using Transwell plates with 8 µm pore size membranes (Corning Inc., USA). After incubation for 12 hr, cells remaining in the upper side of the filter were removed with cotton swabs. The cells attached on the lower surface were fixed and stained using crystal violet and washed with water. Cells were counted with five high power fields per membrane and results were presented as the mean number of cells migrated per field per membrane. All experiments were conducted in triplicate.
Tumor xenograft experiments.
All in vivo experiments were approved by the Institutional Research Committee of Xi’an Jiaotong University (Xi'an, China). All mice received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. MDA-MB-231 cells (2.5 × 106) were mixed in a 1:1 (v:v) ratio with Growth Factor Reduced Matrigel (BD Biosciences, USA), and the mixture was injected subcutaneously into the left (control cancer cells)and right (YAP overexpression cancer cells) flanks of 6- to 7-week-old BALB/c nu/nu nude mice. Mice were sacrificed by cervical dislocation on day 40. Implanted tumors were extracted and the volume (measured in mm3) was determined using calipers and calculated using the modified ellipse formula: Volume = length × width2/2.
Immunohistochemistry (IHC) analysis
The formalin-fixed paraffin-embedded sections (5 µm thick) of the tumor tissues were analyzed by IHC using the primary YAP or CD34 antibody (1:100) and a biotin-conjugated secondary antibody. Four randomly selected areas were photographed at 40 × magnification using a Qimage Retiga 2000R camera (Surrey, Canada). The images were analyzed using the Image-Pro Plus image analysis software (Media Cybernetics, USA).
The Student's t-test was utilized to assess the statistical significance of the difference between two treatments. Tumor volume and CD34 positive cells between two groups were analyzed by paired t-test. A P value of less than 0.05 was considered significant.