Stolpmann et al. 2012 Supplements
Supplementary Figures
fold-transcriptional i nduction
Sup 1
HepG2
NHEK
100
75
50
25
0
solvent
1
2
3.5
4
10
β-NF [µM]
fold-tr anscr iptional inducti on
Sup 2
HaCaT
5
NHEK
4
3
2
1
0
solvent
4
10
24
3.5 µM BP [h]
1
Sup 3
CD95
NHEK
HaCaT AhR e.V.
800
HaCaT AhR s.d.
600
400
200
0
-
+
+
BP [3.5 µM]
β-NF [10 µM]
mean fluorescence intensity
mean fluorescence intens ity
HaCaT
KBM
1000
KBM-
800
600
400
200
0
+
-
BP [3.5 µM]
TRAIL-R1
800
NHEK
HaCaT AhR e.V.
HaCaT AhR k.d.
600
400
200
0
-
+
+
BP [3.5 µM]
β-NF [10 µM]
mean fluorescence intensity
mean fluorescence intensity
HaCaT
KBM
1000
KBM-
800
600
400
200
0
+
-
BP [3.5 µM]
TRAIL-R2
800
NHEK
HaCaT AhR e.V.
HaCaT AhR k.d.
600
400
200
0
-
+
- BP [3.5 µM]
+ β-NF [10 µM]
2
mean fluorescence intensity
mean fluorescence inte nsity
HaCaT
KBM
1000
KBM-
800
600
400
200
0
-
+
BP [3.5 µM]
Sup 4
solvent
10 µM alpha NF
160
160
solvent
BP
120
viability [%]
viability [%]
120
solvent
BP
80
80
40
40
0
0
-
+
-
+
-
+
-
+
CD95L [20 ng/ml]
CD95L [100 ng/ml]
TRAIL [20 ng/ml]
TRAIL [100 ng/ml]
-
+
-
+
-
+
-
Sup 5
A
B
KRT1
keratin 1
keratin 5
1,8
Expression
(a.u.)
Rel.Rel.
expression
(, a.u.)
Expression
(a.u.)
Rel.Rel.
expression
(, a.u.)
35
35
35
30
30
30
25
25
25
20
20
20
15
15
15
10
10
10
555
KBM+
1,2
1
0,8
0,6
0,4
0,2
C
KBM+
-KBM-
KBM
KBM-
KBM-
KBM
D
KRT10
keratin 10
2,5
2,5
2.5
Rel.
Rel.expression
Expression(,(a.u.)
a.u.)
Rel. Expression (a.u.)
1,6
1,4
0
0
0
40
35
35
KRT5
30
25
20
15
10
5
KBM-
IVL
involucrin
2
2
1,5
1,5
1.5
1
1
0,5
0.5
0,5
0
0
KBM
KBM+
KBM+
KBM-
KBM-
KBM
3
KBMKBMKBM-
+
4
Sup 7
A
-
+ MG-132
160
160
solv ent
BP
120
viability [%]
viability [%]
120
80
40
80
40
0
0
-
+
-
B
+
-
+
-
+
CD95L [20 ng/ml]
CD95L [100 ng/ml]
TRAIL [20 ng/ml]
TRAIL [100 ng/ml]
-
-
+
-
+
-
+
-
+
+ MG-132
160
160
solve nt
BP
solvent
BP
120
viability [%]
120
viability [%]
solvent
BP
80
80
40
40
0
0
-
+
-
+
-
+
-
+
CD95L [20 ng/ml]
CD95L [100 ng/ml]
TRAIL [20 ng/ml]
TRAIL [100 ng/ml]
5
-
+
-
+
-
+
-
+
Captions and legends to supplementary figures
Supplementary Figure 1: Induction of CYP1A1 by β-NF in primary human
keratinocytes or HepG2 cells
Transriptional induction of the CYP1A1 gene by β-NF: Transcripts of CYP1A1 were
measured by real-time PCR in primary human keratinocytes (NHEK) and HepG2 cells after
exposure to β-NF for 6 h. Control cells were treated with solvent (0.1% DMSO) only. Total
RNA was isolated and a real-time PCR analysis was performed as described in the section
Materials and Methods.
Supplementary Figure 2: Levels of CD95 are not affected by BP treatment in primary
human keratinocytes or HaCaT cells
Transcriptional induction of the CD95 gene by BP: Transcripts of CD95 were measured by
real-time PCR in primary human keratinocytes (NHEK) and HaCaT cells after exposure to
3.5 µM BP as indicated. Control cells were treated with solvent (0.1% DMSO) only. Total
RNA was isolated and a real-time PCR analysis was performed as described in the section
Materials and Methods.
Supplementary Figure 3: Expression of CD95, TRAIL-R1 and TRAIL-R2 in HaCaT cells
and NHEK
HaCaT cells [AhR e.v.: parental (empty) control vector (black bars) and AhR k.d.: shRNA
AhR knock-down (white bars)] were treated with BP (3.5 µM), β-NF (10 µM) or 0.1% DMSO
(solvent) for 48 h as indicated (left side). NHEK [undifferentiated, KBM (black bars) and
differentiating, KBM- (white bars)] were treated with BP (3.5 µM) or 0.1% DMSO (solvent) for
48 h. FACS analysis for receptor expression was carried out using labelled anti-CD95 [top],
anti-TRAILR1 (DR4) [middle] or anti-TRAILR2 (DR5) [bottom] reagents obtained from R & D
Systems GmbH. The mean specific fluorescence intensities were calculated by subtracting
6
the mean fluorescence of specific isotype controls. Values represent means±S.D. of three
independent experiments.
Supplementary Figure 4: HaCaT cells were pre-exposed to 10 µM -napthoflavone (-NF)
for 6 h and subsequently treated with 3.5 µM BP (white bars) or 0.1% DMSO (black bars) for
48 h. This was followed by a 24 h treatment with CD95L or TRAIL as indicated. Cell viability
was then determined by crystal violet staining. Values represent means ± S.D. of three
independent experiments.
Supplementary Figure 5: Expression of NHEK differentiation markers in cells exposed
to medium lacking growth supplements
NHEK were initially cultured in keratinocyte growth medium (KBM). After reaching confluence,
media were changed, using again KBM (KBM) or KBM medium lacking all supplements
except Bovine Pituitary Extract and CaCl2 (KBM―) to trigger differentiation. Cultures were
maintained for at least 24 h. Expression of keratinocyte cell markers was analysed by realtime PCR (see Supplemental Table 1 for primer sequences)
Supplementary Figure 6: Effects of AhR agonists on cytokine expression in HaCaT
cells
(A) HaCaT AhR parental (empty vector) control cells and AhR shRNA knock-down cells were
treated with 3.5 µM BP, 10 µM β-NF or 0.1 % DMSO for 48 h and expression of cytokoines
was carried out using real-time PCR (see Supplemental Table 1 for primer sequences).
Supplementary Figure 7: AhR-dependent ubiquitination does not affect the level of
apoptosis in BP-treated HaCaT cells
(A) HaCaT AhR empty vector (e.v.) control cells were pretreated with 3.5 µM BP (white bars)
or 0.1% DMSO (black bars) for 48 h and at t=45 h 1 µM MG-132 was added (right). Cells
7
were subsequently exposed to different concentrations of CD95L or TRAIL for further 24 h
without (left) or with (right) 1 µM MG-132. Cell viability was then determined by crystal violet
staining as described in the section Material and Methods.
(B) HaCaT shRNA AhR knock-down (k.d.) cells were pretreated with 3.5 µM BP (white bars)
or 0.1% DMSO (black bars) for 48 h and at t=45h 1 µM MG-132 was added (right). Cells
were subsequently exposed to different concentrations of CD95L or TRAIL for further 24 h
without (left) or with (right) 1 µM MG-132. Cell viability was then determined by crystal violet
staining as described in the section Material and Methods.
Supplemental Table 1: Primers used for real-time PCR analysis
Gene
Accession
Fw primer
Rev Primer
Involucrin
NM_005547.2
ttgcttcctgtagagcacca
tagcggacccgaaataagtg
Keratin 1
NM_006121.3
atttctgagctgaatcgtgtgatc
ctgatggactgctgcaagtt
Keratin 5
NM_000424.3
caacccactagtgcctggtt
gacacacttgactggcgaga
Keratin 10
NM_000421.3
atgagctgaccctgaccaag
tcacatcaccagtggacaca
CD95L
NM_000639.1
ggcctgtgtctccttgtgat
tgccagctccttctgtaggt
TRAIL
NM_001190942.1
aaggaagggcttcagtgacc
tgcaggagcactgtgaagat
Interferon-
NM_000619.2
gtccaacgcaaagcaataca
atattgcaggcaggacaacc
Tumor necrosis
NM_000594.2
cctgtgaggaggacgaacat
ggttgagggtgtctgaagga
Interleukin 1-β
NM_000576.2
ggagaatgacctgagcacct
cgtgcacataagcctcgtta
Interleukin 6
NM_000600.3
attctgcgcagctttaagga
atctgaggtgcccatgctac
Interleukin 10
NM_000572.2
gagaacagctgcacccactt
actctgctgaaggcatctcg
Interleukin 17A
NM_002190.2
gcccctcagagatcaacaga
gccctaggagtgttgcttga
factor-α
8