Transcript Fig 1A - Mie University

Research Seminar 111031 Yasuhito Shimada
A High-throughput Quantitative ATP
Assay using Zebrafish.
• We applied the conventional cell viability assays
to zebrafish to create the high-throughput whole
animal screening systems.
• ATP-based zebrafish assay is more sensitive to
phenotypic changes and reveal latent toxicity in
vivo.
• This screen technology will be applicable to check
the toxicity of chemical compounds such as
anticancer drug in vivo.
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Supplementary Figure
A
MTT assay of zebrafish (4 dpf)
0.07
B
Resazurin assay of zebrafish (4 dpf)
40
Intact
Fluorescent (560 / 590 nm)
0.06
OD 450nm
0.05
0.04
0.03
0.02
30
25
20
15
10
0.01
5
0
0
0
1
2
Intact
35
3
EggWater
0
Res (-)
1
2
3
blank
fish without
resazurin
blank
fish without
resazurin
Fish numbers
Fish numbers
0.4
12
Homogenize
Fluorescent (560 / 590 nm)
0.35
OD 450nm
0.3
0.25
0.2
0.15
0.1
0.05
Homogenize
10
8
6
4
2
0
0
0
0
1
2
3
Fish numbers
1
2
3
EggWater without Res
Fish numbers
2
C
Calcein-AM assay of zebrafish (4 dpf, 1/50 dilution)
Fluorescent (485 / 535 nm)
7000
1/500 dilution
Intact
6000
5000
4000
1/50 dilution
3000
2000
1000
0
0
1
2
3
Fish numbers
Fluorescent (485 / 535 nm)
40000
Homogenize
35000
1/20 dilution
30000
25000
20000
15000
10000
5000
0
0
1
2
Fish numbers
3
3
Fig 1A
Fish number-dependent ATP increase in 96-well plate-based zebrafish assay
200000
180000
Counts per second
160000
140000
120000
100000
80000
60000
40000
20000
0
0
1
2
3
4
Fish numbers / well
Intracellular ATP levels were correlated with the numbers of zebrafish
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Fig 1B-G
2,4-Dinitrophenol (DNP) exposure to zebrafish (4 dpf) for 30 min
B
Control
C
E
5 μM
F
0.5 μM
15 μM
D
1.5 μM
G
50 μM
DNP-exposed zebrafish represented phenotypic change over the concentration of
15 μM.
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Fig 1H-M
DNP exposure to zebrafish (4 dpf) for 150 min
H
Control
I
K
5 μM
L
0.5 μM
15 μM
J
1.5 μM
M
50 μM
DNP-exposed zebrafish represented phenotypic change over the concentration of
1.5 μM with cardiac arrest. 15 and 50 μM samples lost the transparency, suggesting
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protein degradation.
Fig 2A
DNP exposure to zebrafish (4 dpf) decreased ATP contents in dose dependent manner.
60000
30 min
Counts per second
50000
150 min
40000
*
30000
*
20000
*
10000
*
*
*
0
0
0.5
1.5
5
15
50
n = 8, * P < 0.01
Concentration (μM)
5 μM of DNP decreases ATP contents significantly in 30 min treatment (blue line)
without phenotypic change. In 150 min treatment (red line), 0.5 μM DNP also decreseas
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ATP contents without phenotypic change. See previous figures.
Table 1
IC50 of ATP synthase inhibitors in zebrafish (30 min treatment)
Chemical
Category
Zebrafish
(MK002)
Antimycin A
Cytochrome c reductase inhibitor
75 nM
CCCP
Uncoupling agent
150 nM
FCCP
Uncoupling agent
15 nM
DNP
Uncoupling agent
7.5 μM
Oligomycin
Proton channel inhibitor
3.6 μM
Rotenone
Complex I inhibitor
1.2 μM
Interestingly, theses ATP synthase inhibitors could not decrease intracellular
ATP in Hela cells in 30 min exposure, similar to described in other articles.
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Fig. 3A
Anti-cancer drug (10 μM) treatment during 4 to 7 dpf (3 days)
Control
Dasatinib
Celecoxib
Imatinib
Cisplatin
MTX
Celecoxib and Dasatinib induced phenotypic change of pericardial and whole
body edema.
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Fig. 3B
Phenotypic analysis of anti-cancer drug (10 μM) exposure (7 dpf, MK001)
100
90
80
Percentage
70
60
50
40
Survival
30
20
Anomalies
10
0
Celecoxib and Dasatinib increased dead phenotype (decrease of transparency)
and phenotypic change
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Fig. 3C
ATP assay of anti-cancer drug (10 μM) exposure (7 dpf, MK001)
2.5
Mean
Median
ATP normalized to control
2
*
Outlier
1.5
*
1
*
0.5
n = 5 – 8, *P < 0.05
0
Control
Celecoxib Cisplatin Dasatinib Imatinib
MTX
Celecoxib decreased ATP contents while Dasatinib increased. MTX, a cytotoxic
anticancer drug decrease ATP contents without phrnotypic change.
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Supplementary Figure
Anti-cancer drug treatment over night (Day 1, 5 dpf, MK001)
Control
Dasatinib
Celecoxib
Imatinib
Cisplatin
MTX
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Supplementary Figure
ATP assay of anti-cancer drug exposure (Day 1, 5 dpf, MK001)
1.4
n. s.
1.2
Ratio to control
1
0.8
0.6
0.4
0.2
0
Control
Celecoxib
Cisplatin
Dasatinib
Imatinib
MTX
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