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Extremely low frequency magnetic field (ELF-MF) exposure sensitizes SH-SY5Y cells to the pro-Parkinson’s Disease toxin MPP+
Mol Neurobiol
Barbara Benassi1*, Giuseppe Filomeni2,3, Costanza Montagna 2,3, Caterina Merla1, Vanni Lopresto1, Rosanna Pinto1, Carmela Marino1 and Claudia Consales1*
1 Unit of Radiation Biology and Human Health, ENEA-Casaccia, Rome, Italy
2 Department of Biology, Tor Vergata University, Rome, Italy
3 Danish Cancer Society Research Center, Copenhagen, Denmark
*Corresponding authors: B. Benassi and C. Consales, Unit of Radiation Biology and Human Health, ENEA-Casaccia, Via Anguillarese 301, 00123 Rome, Italy.
Tel: +39-06-30483921/4031; Fax: +39-06-3046559. email: [email protected]; [email protected]
a
ESM_1
b
MPP+ (mM)
0
120
●
Staurosporine
1
25,7 %
33,5 %
27,6 %
51,4 %
74,2 %
73 %
Relative growth
 Trypan blue + (non-viable)
Ann V-FITC
100
Cell percentage
0,5
10,5 %
80
60
PI
40
20 %
Caspase 3/7
20
0
0
1
2
3
4
5
MPP+ (mM), 24h
PI
d
12
MPP+ (mM)
0
0,1
0,5
1
10
MPP+ (1 mM)
Doxo
8
3
Events
Relative gene expression
Untreated
MFI (H2-DCFDA)
Arbitrary units
c
6
4
2
1
0
0
2
H2-DCFDA fluorescence
0.1
0.5
1
MPP+ (mM)
0
p53
p21
puma
bax
e
MPP+
6
Control
MPP+ (1 mM)
MFI (DHE)
Arbitrary units
Control
MPP+ (0,5 mM)
Events
Control
MPP+ (0,1 mM)
4
2
0
DHE fluorescence
0
0.1
0.5
1
MPP+ (mM)
Supplementary Material_1 The pro-PD MPP+ toxin drives apoptosis in proliferating SH-SY5Y cells through caspase 3/7 activation, oxidative stress,
p53 and its down-stream targets activation. Proliferating SH-SY5Y cells have been treated with different doses of MPP+ (0.1-5 mM) for 6-24 hours,
collected and immediately evaluated in terms of (a) proliferation index and viability (trypan blue exclusion), (b) percentage of Annexin V+/PI- and
caspase-3/7 activating cells (early apoptotic cells), (c) p53/p21/puma/bax gene expression, and ROS generation by (d) H2-DCFDA (5 µM in HBSS, 30’,
37°C in the dark) and (e) DHE (5 µM in PBS, 20’, 37°C in the dark) viable staining and flow cytometric analysis. In panel b, staurosporine (1 µM, 24
hours) has been used as positive control for inducing apoptosis and defining the early apoptosis and caspase positive regions (highlighted by box in the
dot plot with relative percentage). In panel c, doxorubicin (2 µM, 24 hours) has been added to cells as positive control of p53 and its target genes
activation. Gene expression analysis has been performed by real time PCR. Primers sequences and PCR setup are available upon request. FACS plots are
representative images out of three different experiments leading to similar results.