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Gene & Protein in Disease Verteporfin therapy for triple-negative breast cancer

isolation method. The quality and quantity of the RNA 2.9. Cell cycle assay
isolated were confirmed using an Epoch microplate Flow cytometric analysis of cell cycle assay was performed
spectrophotometer (BioTek, USA). RNA was then to analyze the effect of YAP inhibition on cell proliferation.
subjected to cDNA synthesis using PrimeScript 1 strand For the assay, cells were fixed in 70% cold ethanol
st
™
cDNA synthesis kit (TAKARA, Japan). The specific primer and incubated at 4°C for 1 h, followed by a wash with
sequences used are listed in Table S1. Real-time PCR was 1× phosphate-buffered saline (PBS). Resuspended cells in
done using Takyon Rox SYBR MasterMix (Eurogentec) in 1× PBS to obtain 5 × 10 cells/0.5 mL, to which 50 µg/mL
™
®
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the real-time PCR machine (StepOne, Applied Biosystems, RNase A was added and incubated for 30 min at room
USA). The gene expressions were calculated using the temperature. Subsequently, 100 µg/mL propidium iodide
comparative 2 −ΔΔCT method. was added to the cells and incubated in the dark for 15 min.
2.6. Immunofluorescence assay The cells were analyzed by flow cytometry (FACS Jazz, BD).
To analyze the cellular localization of the protein, an 2.10. Annexin V-PI apoptosis analysis
immunofluorescence assay was carried out. Cells were Cells were trypsinized and subjected to an apoptosis assay
grown in coverslips and fixed with 4% paraformaldehyde using the Annexin V-FITC assay kit according to the
for the assay. Cells were washed with 1× tris-buffered manufacturer’s instructions (BD Pharmingen). Briefly, after
saline (TBS) for 10 min and permeabilized with 0.1% treatment with either VP or siRNA, cells were trypsinized
Triton X-100 for 10 min, followed by washing with 1% and counted. Cells were resuspended in 1× binding buffer
glycine and blocked using 3% bovine serum albumin for to obtain 1 × 10 cells/mL, from which 100 µL (1×10 cells)
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6
30 min. Primary antibodies against YAP were added to the of the cell suspension was transferred to 5 mL flow
cells and incubated for 2 h at 37°C. After 1× TBS wash for cytometry-compatible tubes. 5 µL of Annexin V-FITC and
10 min, cells were incubated with a secondary antibody propidium iodide were added to the cells, mixed enough,
conjugated with Alexa Flour 488 for 1 h at 37°C. After that, and incubated in the dark for 15 min. After incubation,
the cells were washed with 1× TBS, then counterstained 400 µL of 1× binding buffer was added to the cells. Samples
with Hoechst 33342, and images were captured using EVOS were analyzed by flow cytometry (FACS JAZZ, BD).
imaging system fluorescence microscope (Invitrogen).
2.11. Statistical analysis
2.7. MTT assay
For statistical analysis and graphical representations,
The MTT colorimetric assay was used to determine the GraphPad Prism (version 5) was used. All experiments
viability of the cells. Cells were seeded in 96-well plates, mentioned were repeated a minimum of 3 times, and
and after 24 h of incubation, different concentrations of VP representative images are exemplified. All data were
were added. Cells were incubated for 24, 48, and 72 h at statistically evaluated by one-way or two-way ANOVA,
37°C with a 5% CO incubator. 1 mg/mL MTT was added and P < 0.05 were considered significant.
2
and incubated in the dark for 2 h. After the incubation,
the insoluble formazan crystals formed were solubilized 3. Results
using DMSO-isopropanol (1:1) mixture for 15 min. The
spectrophotometric readings were recorded, and viability 3.1. YAP and its downstream targets upregulated in
was calculated relative to the non-treated control cells. TNBC cell lines
We initiated the study by evaluating a comparative
2.8. Colony formation assay analysis of the activity of Hippo transducers in TNBC and
To analyze the proliferation and survival ability of the cells non-TNBC cells. MDA-MB-231 and SUM159 were used
with VP treatment and transient silencing of YAP, a colony as TNBC cell lines, and MCF-7 and T47D were used as
formation assay was carried out. Cells treated with VP for non-TNBC cell lines. Apart from YAP and TAZ, we also
24 h or siRNA-treated were seeded at a 1000 – 2000 cells/well assessed the basal level protein expressions of the upstream
density in a 6-well plate. Cells were incubated for 7 – 10 days, YAP/TAZ regulator LATS, the major binding partner
with the medium changed every other day. Cells were fixed TEF1, and the downstream targets CYR61, CTGF, and
using ice-cold methanol and stained using 1% crystal violet cMYC. From the immunoblot analysis, we consistently
solution for 15 min. Excess stain was washed out using observed a relatively higher basal level protein expression
water gently and carefully, and plates were air dried. The of YAP/TAZ and downstream targets in TNBC cells
colonies were counted under a microscope, and images compared to non-TNBC cell lines (Figure 1). We also
were recorded (Axiovert 25-Zeiss, Germany). The colony is noted a relatively increased expression of TEF1 and a lower
defined as a group of at least 50 cells. expression of LATS1 in TNBC cells.

Volume 2 Issue 2 (2023) 3 https://doi.org/10.36922/gpd.0658