SUPPLEMENTARY MATERIAL
Inhibition of BRCT(BRCA1)-phosphoprotein interaction enhances the cytotoxic effect of Olaparib in breast
cancer cells: A proof of concept study for synthetic lethal therapeutic option.
Ziyan Yuan Pessetto · Ying Yan · Tadayoshi Bessho · Amarnath Natarajan1,2
Corresponding author
Amarnath Natarajan: anatarajan@unmc.edu
Eppley Institute for Cancer Research, Department of Pharmaceutical Sciences and Genetics Cell Biology and
Anatomy, University of Nebraska Medical Center Omaha NE 68198
Telephone: (402) 559 3793
Fax number: (402) 559 8270
Ziyan Yuan Pessetto: zyuan@kumc.edu
Eppley Institute for Cancer Research, University of Nebraska Medical Center Omaha NE 68198
Ying Yan: (402) 559-3036
Eppley Institute for Cancer Research, University of Nebraska Medical Center Omaha NE 68198
Tadayoshi Bessho: (402) 559-7018
Eppley Institute for Cancer Research, University of Nebraska Medical Center Omaha NE 68198
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Treatment of cells and preparation of cell lysates
Cell lysates were prepared by scraping cells (150 cm 2, 80-90% confluent) into
500 µl of lysis buffer A (10 mM HEPES, pH 7.8, 10 mM KCl, 0.1 mM ethylenediamine
tetraacetic acid (EDTA), 2 mM dithiothreitol (PMSF), 1×Halt protease inhibitor cocktail
(Thermo scientific), and 1×Halt phosphatase inhibitor cocktail (Thermo scientific)). The
cells were allowed to swell on ice (15 min), after which 32 µl 10% Nonidet P-40 (Sigma)
were added, and the tube were vortexed vigorously and the homogenates were
centrifuged. The nuclear pellet was resuspended in buffer B (20 mM HEPES, pH 7.8,
0.42 M NaCl, 5 mM EDTA, 5 mM dithiothreitol, 1 mM PMSF, 10% (v/v) glycerol, 1×Halt
protease inhibitor cocktail, and 1×Halt phosphatase inhibitor cocktail) and the tubes
were rocked gently at 4 ˚C. The supernatant were transferred to fresh tubes and protein
contents were determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Inc.)
with various concentrations of bovine serum albumin as standards. Aliquots of the cell
lysates were stored at - 80˚C.
Western blotting and immunoprecipitation
Equal amounts of protein were subjected to electrophoresis (Criterion TM precast
gels, 4-15% tris-HCl, 1.0 mm, Bio-Rad Laboratories, Inc.) in a 1×Tris-Glycine-SDS
running buffer (Bio-Rad Laboratories, Inc.) and transferred to poly (vinylidene difluoride)
(PVDF) membranes (Immun-Blot TM PVDF membrane for protein blotting; Bio-Rad
Laboratories, Inc.) for 12 h at 15V. Membranes were blocked in TBS containing 0.05%
Tween 20 (TBST) and 5% nonfat milk for 2 h at room temperature. Appropriately diluted
primary antibodies were incubated in TBS / 5% nonfat milk for 2 h at room temperature
or overnight at 4 C. Membranes were washed in TBST three times for 15 min each.
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Appropriately diluted secondary antibodies were incubated in TBS / 5% nonfat milk for 1
h at room temperature. Membranes were washed in TBST three times for 15 min.
Proteins were detected by AmershamTM ECL plus western blotting detection system
(GE healthcare). The following antibodies were used: anti-Abraxas (A302-181A, Bethyl);
anti-BRCA1 (c-20) (SC-642, Santa Cruz); anti-BRCA1 (D-9) (sc-6954, Santa Cruz);
anti-GAPDH (ab9484, abcam); anti-Lamin B1 (ab16048, abcam); secondary antibodies
(goat anti-mouse and goat anti-rabbit) were purchased from SouthernBiotech. Primary
antibodies were diluted 1:1000 and secondary antibodies at 1:10,000 dilutions.
Modified RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% sodium
deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1×Halt protease inhibitor
cocktail, and 1×Halt phosphatase inhibitor cocktail) was used to perform the
immunoprecipitation. Equal amounts of protein lysates were precleared for 30 min with
30 µl of Pierce® Protein A/G Agarose (Thermo scientific) and fractions were incubated
with appropriate primary antibody for 4 h at 4 ˚C. 30 µl of Pierce ® Protein A/G Agarose
were then added and incubated overnight. The beads were washed three times with
modified RIPA buffer and boiled in SDS-loading buffer. Western blotting assays were
performed as described above.
Immunofluorescence confocal imaging
Hela cells (4000 cells per well) or HCC1937 cells (3000 cells per well) were
seeded in 96-well optical plates (NUNC, New York) containing media (200 µl). After
allowing the cells to adhere for 24 h, cells were treated with or without peptide 2 (10
µM). The cells were incubated for 1 h and subjected to different doses of IR (0, 5, 10,
15, and 20 Gy). After an additional 30 min incubation the cells were fixed by replacing
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the growth medium with 100 µl of 100% ice-cold methanol for 10 min. The methanol
was removed and the cells were washed 3 times with 200 µl PBS. PBS was removed
and 100 µl BSA (5%) was added and incubated for 1 h at room temperature. BSA
solution was removed and the cells were washed 3 times with 200 µl PBS. The cells
were subjected to 50 µl of primary antibody (Anti-phospho-H2AX (S139), Active Motif)
diluted 1:250 in BSA/PBS. The plates were then sealed, covered, and incubated
overnight at 4 ˚C. The primary antibody was removed and the cells were washed 3
times with 200 µl PBS for 5 minutes each. The PBS was removed and 100 µl of
secondary antibody (ChromeoTM
488
Goat anti-Rabbit IgG, Active Motif) (1:1000) was
added to each well. Plates were incubated 1 h at room temperature. The secondary
antibody was removed and cells were washed with 200 µl of PBS for 5 min each. Cells
were analyzed on a Zeiss LSM 410 dual beam laser confocal scanning microscope.
Images were captured at 10× or 63× by using objective lenses. Pictures were analyzed
by using LSM image browser version 4.2.0.121 (Carl Zeiss Microimaging GmbH).
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