Cell culture and maintenance
MDA-MB-231 was maintained in Dulbecco’s modified Eagle’s medium (DMEM), and SUM-159 was maintained in DMEM with F12 at 370C in a 5% humidified CO2 incubator. All cells were supplemented with 10% (vol/vol) fetal bovine serum and 1% (vol/vol) antibiotic- antimycotic solution and routinely checked to be free of mycoplasma contamination.
Reagents and antibodies
Antibodies against β-ACTIN, YAP, TEAD, LATS1, p21, p27, and Caspase 3 were purchased from Santa Cruz Biotechnology (USA), antibodies against TAZ, CTGF, CYR61, p-YAP (Ser127), MYC, Cyclin D1, PARP and cleaved Caspase 3 were purchased from Cell Signaling Technology (Danvers, MA), antibody against Ki-67 were purchased from Abcam (UK). Anti-mouse, anti-rabbit secondary antibodies and Chemiluminescence detection luminol reagents (SuperSignalTM West Pico Plus Chemiluminescent Substrate- 3458) were obtained from Thermo Scientific, USA. Alexa Flour 488 conjugated anti-mouse secondary antibody were purchased from Cell Signaling Technology (Danvers, MA). Benzoporphyrin derivate verteporfin (VP) was purchased Sigma Aldrich, USA. Small interfering RNA (siRNA) targeting YAP (siYAP) and control siRNA (siCN) was purchased from Santa Cruz Biotechnology.
Drug treatment and siRNA transfection
For VP treatment, cells at 70–80% confluence were used and incubated 24 hours prior to all functional assays. Cells at 60% confluence were taken for the siRNA transfection according to the instructions provided by the manufacturer. Cells were subjected for all further experiments after 48 hours of transfection.
Western blot assay
Whole cell lysate was prepared in RIPA lysis buffer and 1% protease inhibitor and phosphatase inhibitor supplemented. The total protein was quantified using BCA reagent kit (Thermo Scientific, USA). 50–80µg of proteins was used; equal concentrations of protein loading were made sure at each time of immunoblotting. Polyvinylidene fluoride membrane (Bio-Rad, USA) was used for the protein transferring. Blots were probed with corresponding antibodies against protein of interests and β-actin was used as internal control. HRP conjugated secondary antibodies were used and the chemiluminescent signal was detected using ECL .
Quantitative real-time PCR
Total RNA from cells was extracted using Trizol (Ambion) isolation method. Quality and quantity of the RNA isolated was confirmed using Epoch microplate spectrophotometer (BioTek, USA). RNA then subjected for cDNA synthesis using PrimeScript™ 1st strand cDNA synthesis kit (TAKARA, Japan). Real time PCR was done using Takyon™ Rox SYBR® MasterMix (Eurogentec), in Real-Time PCR machine (StepOne, Applied Biosystems, USA). The gene expressions were calculated by comparative the 2−ΔΔCT method.
Immunofluorescence Assay
Cells were grown in coverslips and fixed with 4% paraformaldehyde. Cells were washed with 1xTBS for 10 minutes, and permeabilized with 0.1% TritonX for 10 minutes. Followed by 1% Glycine wash, and blocked using 3% BSA for 30 minutes. Primary antibody against YAP were added to the cells and incubated for 2 hours at 370C. After 1xTBS wash for 10 minutes, cells were incubated with secondary antibody conjugated with Alexa Flour 488 for 1 hour at 370C. 1xTBS wash given, counter stained with Hoechst 33342, and images were captured using EVOS imaging system fluorescence microscope (Invitrogen).
MTT assay
The MTT colorimetric assay was used for determining the viability of the cells. Cells were seeded in 96 well plates and after 24 hours of incubation different concentrations of VP added. Cells were incubated for 24, 48 and 72 hours at 370C with 5% CO2 incubator. 1mg/ml MTT were added and incubated at dark for 2 hours. After the incubation the insoluble formazan crystals formed were solubilized using DMSO-isopropanol (1:1) for 15 minutes. The spectrophotometric readings were recorded and viability was calculated relative to the non-treated control cells.
Colony formation assay
Cells were treated with VP for 24 hours and seeded at 1000–2000 cells/ well density in a 6 well plate. After 7–10 days of incubation with alterative days of media change, cells were fixed using ice cold methanol and stained using 1% crystal violet solution for 15 minutes. Excess stain was washed out using water gently and carefully, and plates were air dried. The colonies were counted under microscope and images were recorded (Axiovert 25-Zeiss, Germany). The colony is defined to consist of at least 50 cells.
Cell cycle assay
Cells were fixed in 70% cold ethanol and incubated at 40C for 1 hour, followed by wash with 1xPBS. Resuspended cells in 1xPBS to obtain 5x105 cells/0.5ml to which 50ug/ml RNAse A was added and incubated for 30 min at room temperature. Subsequently 100ug/ml propidium iodide was added to the cells and incubated in dark for 15 min. The cells were analyzed by flow cytometry (FACS Jazz, BD).
Annexin V-PI apoptosis analysis
Cells were trypsinized and subjected to apoptosis assay using the Annexin V-FITC assay kit according to the manufacturer’s instructions (BD Pharmingen). Samples were analyzed by Flow cytometry (FACS JAZZ, BD).
Statistical analysis
For statistical analysis and graphical representations GraphPad Prism (version 5) was used. All experiments mentioned were repeated for minimum of 3 times and representative images are exemplified. All data were statistically evaluated by one way or two-way ANOVA, and p values > 0.05 were only considered significantly accepted.