9
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 M 0 10 30 75 0 10 30 75 resveratrol (μM) M 5000 bp 500 bp 1500 bp

resveratrol ( μ M)

  • Upload
    oistin

  • View
    90

  • Download
    1

Embed Size (px)

DESCRIPTION

resveratrol ( μ M). resveratrol ( μ M). M. 0 10 30 75. 0 10 30 75. M. 0 10 30 75. 5000 bp. 1500 bp. 500 bp. 0 10 10. DNase I (units). - PowerPoint PPT Presentation

Citation preview

Page 1: resveratrol ( μ M)

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

Page 2: resveratrol ( μ M)

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

Page 3: resveratrol ( μ 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.

Page 4: resveratrol ( μ M)

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

Page 5: resveratrol ( μ M)

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)

Page 6: resveratrol ( μ M)

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

Page 7: resveratrol ( μ M)

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

Page 8: resveratrol ( μ M)

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

)

Page 9: resveratrol ( μ M)

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