Post on 25-Dec-2015
Fig. S1. Modulation of chromatin structure of AS52 cells by increasing concentrations of resveratrol, probed by limited digestion with Dnase I: densitometric analysis of electrophoresis gels shown in Fig. 1A. Nuclei were isolated from cells treated for 24 hours with indicated concentrations of resveratrol. DNA fragment size analyses were performed after the agarose gel electrophoresis by the Image J software. Fluorescence intensity profiles of the lanes corresponding to samples treated with the indicated amount of DNase I are shown on the right. Mode values are indicated by arrows and position of undigested DNA by the dashed line. Molecular size markers (M) are shown for reference.
resveratrol (μM)
DNase I (units)
0 10 10
0 10 30 75 M0 10 30 75
0 10 30 75
resveratrol (μM)
M
5000 bp
500 bp
1500 bp
Fig. S2. Modulation of chromatin structure of AS52 cells by the HDAC inhibitors butyrate (BA) and trichostatin A (TSA), probed by limited digestion with DNase I: densitometric analysis of electrophoresis gels shown in Fig. 1D. Nuclei were isolated from cells treated for 24 hours with indicated concentrations of the substances. DNA fragment size analyses were performed after the agarose gel electrophoresis by the Image J software. Fluorescence intensity profiles of the lanes corresponding to samples treated with the indicated amount of DNase I are shown on the right. Mode values are indicated by arrows and position of undigested DNA by the dashed line. Molecular size markers (M) are shown for reference.
con
tro
l
BA
(0.
3 m
M)
BA
(2
mM
)
TS
A (
30 n
M)
con
tro
l
BA
(0.
3 m
M)
BA
(2
mM
)
TS
A (
30 n
M)
0 7 7
DNase I (units)
5000 bp
500 bp
1500 bp
con
tro
l
BA
(0.
3 m
M)
BA
(2
mM
)
TS
A (
30 n
M)
M M
resveratrol (μM)
0 10 30 75 M 0 10 30 75
0 10 30 75
0 5 5 15
DNase I (units)
resveratrol (μM)
M
5000 bp
500 bp
1500 bp
resveratrol (μM)
0 10 30 75
Fig. S3. Effect of increasing concentrations of resveratrol on the global chromatin structure of HeLa cells, probed by limited digestion with DNase I. Nuclei were isolated from cells treated for 24 hours with indicated concentrations of the substance. DNA fragment size analyses were performed after the agarose gel electrophoresis by the Image J software. Fluorescence intensity profiles of the lanes corresponding to samples treated with the indicated amounts of DNase I are shown on the right. Mode values are indicated by arrows and position of undigested DNA by the dashed line. Molecular size markers (M) are shown for reference.
Fig. S4. Effect of resveratrol (24-hour treatments) on the proliferation of HeLa cells.
cell
coun
t (lo
g 10)
0 24 48 72
time (hours)
6
7
5
8
resveratrol removed
solvent
10 µM
30 µM
75 µM
1 µM
100 µM
5 μM
Fig. S5. Effect of resveratrol (75 µM, 24 hours) on the total glutathione levels in AS52 and HeLa cells. The numbers of independent determinations are indicated above the columns. Columns indicate means ± S.D.
glut
athi
one
(nm
ol /
100
µg
pro
tein
)
(14)
(7)
(5)
(4)
0 10 30 75resveratrol (µM]
0
50
100
T4E
V s
ites
(% u
nrep
aire
d)
0
0.5
1.0
1.5
2.0
0 10 30 75resveratrol (µM]
T4E
V s
ites
/106
bp
not irradiated
2.9 J/m2 UV-B
0
50
100
0 4 8 12 16
repair time (hours)
T4E
V s
ites
(% u
nrep
aire
d)
solvent
75 µM resveratrol
*
Fig. S6. Influence of resveratrol on the generation and repair of UV-B induced damage in HeLa cells. CPDs in chromosomal DNA were quantified by alkaline elution as T4 endonuclease V sensitive (T4EV) sites. (A) Effect of a pretreatment with resveratrol (24 h) on the generation of CPDs (n≥3). (B) Effect of resveratrol on the CPD repair kinetics (n≥3). (C) Concentration-dependent effect of resveratrol on the fraction of unrepaired CPDs measured 8 hours after UV-B exposures (n≥3). Data indicate means ± S.D. Student´s t-test: * p < 0.05.
A B C
0
50
100
0 10 30 75
resveratrol (µM)
Fpg
site
s /1
06 b
p
no damage induction
Ro19-8022+light
0
50
100
0 1 2 3 4 5 6
repair time (hours)
Fpg
site
s (%
unr
epai
red)
solvent
75 µM resveratrol
0 10 30 75
resveratrol (µM)
0
50
100
Fpg
site
s (%
unr
epai
red)
Fig. S7. Influence of resveratrol on the generation and repair of DNA base modifications induced by photosensitization in HeLa cells. Oxidized purine species (predominantly 8-oxoG) in chromosomal DNA were quantified by alkaline elution as Fpg-sensitive sites. (A) Effect of a pretreatment with resveratrol (24 h) on the induction of the Fpg-sensitive base damage by irradiation with light in the presence of 100 nM Ro19-8022 (n≥3). (B) Effect of resveratrol on the repair kinetics of the Fpg sensitive DNA lesions (n≥3). (C) Concentration-dependent effect of resveratrol on the fraction of unrepaired Fpg sensitive DNA lesions 3 hours after the damage generation (n≥3). Data indicate means ± S.D.
A B C
Fig. S8. Influence of TSA on the repair of DNA single-strand breaks (SSB) induced in AS52 cells by H2O2. Cells were pretreated 24 hours with 30 nM trichostatin A (TSA) and exposed to H2O2 (150 µM) 15min at 37°C. DNA damage was quantified by alkaline elution. Data indicate means ± S.D.
0
50
100
0 10 20 30 40 50 60
repair time (min)
solvent
30 nM TSA
SS
B
(% u
nre
pai
red
)
Fig. S9. Effect of resveratrol (75 µM) and sirtuine inhibitor EX-527 (10 µM) on the global chromatin structure of AS52 cells, probed by its accessibility to DNase I. Nuclei were isolated from cells treated for 24 hours with the indicated substances. DNA fragment size analyses were performed after the agarose gel electrophoresis by the Image J software. Fluorescence intensity profiles of the lanes corresponding to samples treated with the indicated amounts of DNase I are shown on the right. Mode values are indicated by arrows (two arrows in graphs with bimodal distribution) and position of undigested DNA by the dashed line. Molecular size markers (M) are shown for reference.
5000 bp
500 bp
1500 bp
Resveratrol
- + - - + -
- + + - + + - + +
- + - EX 527 - + -
- + +
DNase I (units)
0 2 3 3
M M