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Analytical Chemistry
Cl
Cl
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available p53-MDM2 inhibitor (100 µM Nutlin-3) as the
positive control incubation together with 5 µM each
probe at the same conditions. Then the fluorescence
imaging in living A549 and NCI-H1299 cell lines were
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S
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obtained on
a Zeiss Axio Observer A1 fluorescent
c
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O
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microscope. The backgrounds of all the images were
adjusted by Image J software. Objective lens: 63×.
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O
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NH2
n
N
N
O
NH
N
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n
N
O
N
H
N
O
H
O
Cl
Flow cytometry analysis. The flow cytometry test was
performed on A549 cell line. When the cells were in the
proliferation period, cells were collected and washed with
1×PBS for three times. Probes 9-11 was added at a final
concentration of 5 µM, respectively or together with the
positive control (100 µM Nutlin-3) into the flow tubes
with 300 μL PBS and 1×105 cells each tube. After
incubation for about 30 min at 37 °C in dark environment,
the samples were analyzed by Beckman Cell Counter.
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N
d
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9 11
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5-7
Scheme 2. Synthetic routes of fluorescent probes 9-11. (a)
120–130 °C, 6 h, 71.0%; (b) Triethylamine, r.t., 0.5 h;
°
dimethylamine hydrochloride, tetrahydrofuran, 1 h, 0 C,
43.7%; (c) Acetonitrile, 60 °C,
Triethylamine, DCM, r.t., 30 min, 79.6%.
6
h, 92.3%; (d)
Western Blot. The protein level of p53 and MDM2 in
A549 cancer cell line (wild-type p53) were detect by
western blot. When A549 cell reaching 60-70%
confluency, the cells were harvested and transferred into
6 wells plate (Corning), after cultured for 12h, probe 9-10
(10 µM, respectively) and Nutlin-3 (5 µM) were added in
different well. After 24 h drugs treatment, cells were
collected. Then, the cells were lysed by RIPA Lysis and
Extraction Buffer and the protein concentration was
quantitated by BCA Protein Assay Kit. Then, the protein
extract was denatured and run on 12% SDS
polyacrylamide gels. Gels were transferred to 0.45 μM
PVDF membranes. The membranes were blocked with 5%
milk buffer (5% nonfat dry milk in TBS/0.1% tween-20)
for 2 h at room temperature and washed three times by
TBST (each time for 5 min). The membranes were
incubated with the primary antibody specific for p53
(Abcam, ab7757; 1:800), MDM2 (Abcam, ab16895; 1:600),
Fluorescence spectroscopy test. The spectroscopic
properties of probes 9-11 were measured at a final
concentration of 5 μM diluted with PBS (pH = 7.4) by
BioTek multiscan spectrum, respectively. More details of
the spectroscopic properties of probe 9-11 can be found in
the Supporting Information.
MDM2 binding affinity assay. The fluorescence
polarization (FP) assay as described previously
reports,2,30,31 The FP values were read on BioTek
(Winooski, VT, USA) Synergy H4 with the filters (Ex: 485
nm and Em: 535 nm). More detail procedures are showed
in the Support Information.
In vitro anti-proliferative assay. We investigated the
cytotoxicity of probes 9-11 and the Nutlin-3 by the CCK-8
method. We select A549 (wild-type p53) and H1299 (p53
null) cell lines to test the cytotoxicity of probe 9-11. 5×103
cells per well were placed in 96-well plates with 100 μL
DMEM culture medium (include 10% fetal bovine serum)
and then cultured 37 °C in a humidified 5% CO2
atmosphere overnight. Subsequently, the probes or
Nultin-3 were added to the wells at different
concentrations (a series 2-fold dilutions among 3.125-100
μM), and then incubated for another 48 h. Then, follow
the protocol of CCK-8, the absorbance value of each well
was recorded by BMG microplate reader. The IC50
valuable of each probe was calculated by the GraphPad
Prism 5.0 software. More details can be found in the
supporting informations.
°
GAPDH (Abcam, ab181602; 1:10000) overnight at 4 C and
washed three times by TBST. Next, the PVDF membranes
were incubated with a secondary antibody (Abcam,
ab216777; 1:10000) carry out 2 h incubation at room
temperature. After washing by TBST, Each protein was
detected by BeyoECL Plus (Beyotime Biotechnology,
P0018S, Shanghai, China) with ChemiDoc XRS imaging
system (Bio-Rad, Hercules, CA), and the protein levels
were quantitated by the gray values of the bands by the
Image J software.
Immunohistochemical analysis of A549 and NCI-
H1299 tumor tissue slices. According to the protocol of
Immunohistochemistry, tissue slices were deparaffinized
in xylene, then rehydration in graded alcohols, after
antigen repaired by All-purpose Powerful Antigen
Retrieval Solution (Beyotime Biotechnology, P0088,
Fluorescence microscopy imaging. A549 (wild-type p53)
and H1299 (p53 null) cell lines were chosen for the
fluorescence imaging. When the cells were in the
proliferation period, these two types of cells were
transferred to the confocal dish and cultured overnight.
Then, the probes 9-11 were further diluted with DMEM
medium free fetal bovine serum at a final concentration
of 5 μM and co-staining with the nuclear dye Hoechst
33342 500 nM. Subsequently, probes were incubated with
A549 and NCI-H1299 cells at 37 °C for 15 min, respectively.
The A549 cell line were also performed by commercial
°
Shanghai, China) at 95-100 C for 20 min, the slices were
incubation with primary antibodies Anti-p53 antibody
°
[E26] ab32389 (1:100, abcam) overnight at 4 C. And then
were incubated with secondary antibody goat anti-mouse
Ig G and goat antibody rabbit Ig G polymer (Gene Tech,
GK600505, Shanghai, China) at room temperature for 15
min, followed by staining with DAB at room temperature
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