Download Supplemental fig. 1. TNFα increases HSD11B1 mRNA levels and

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Supplemental fig. 1. TNFα increases
HSD11B1 mRNA levels and activity in HMC.
HepG2 cells (A) HMC were treated with 5
nM of TNFα for 0-30 h. The amount of
HSD11B1 mRNA was quantified by real-time
PCR. The HSD11B1 mRNA values in treated
cells were calculated relative to the amount
found in untreated cells, which was defined as
1. Results are the mean of relative amount of
HSD11B1 mRNA values ± S.E. of triplicate
measurements from at least three independent
experiments. (B). Specific activity was
expressed as percent of converted substrate.
The activity assay in HMC was performed as
described in Fig. 2B. Results are means ± S.E.
of triplicates measured in three independent
experiments. *, p<0.05.
Supplemental fig. 4. Kinetics of activation of
p38 MAPK, CREB and ATF-2 in HepG2 cells
treated with TNFα. After the indicated times,
cells were washed with 1xPBS and lysed in
RIPA buffer. Fifteen μg of each sample were
loaded on a10% gel and separated by SDSPAGE. Phosphorylation levels of the proteins
were analyzed by Western blotting using
phospho-specific antibodies (Cell Signaling),
following transfer of total proteins onto
nitrocellulose membranes. Antibodies against
p38, CREB (Cell Signaling) and ATF-2 were
used to normalize the amount of protein
loaded onto each lane.
Supplemental fig. 5. Partial nucleotide
sequences of human HSD11B1 promoter and
first exon. Consensus sites for C/EBP and
NFκB transcriptional factors are presented as
boxes, for AP-1-with ellipses. Numbers refer
to the position relative to the transcription
start in the liver transcript (+1).
Supplemental fig. 2. HepG2 cells were cotransfected with NFκB-luciferase plasmid
together with different amounts of IκBα
plasmid for 24 h. After treatment of cells
without or with TNFα for another 24h
HSD11B1 mRNA levels were measured. Data
are means ± S.D. from one experiment
performed in triplicate. *, p<0.05.
Supplemental fig. 6. Copmosition of C/EBPα,
β and δ proteins bound to wtC/EBP site.
EMSA analysis was performed with [γ-32P]
wtC/EBP probe in the presence of nuclear
extracts from HepG2 cells over-expressing
C/EBPs transcription factors. Complex
compositions were analyzed using antibodies
against C/EBPα, β and δ. C/EBPα/β/δ
indicates specific DNA binding complexes
and
ns
non-specific
bands.
Supplemental fig. 3. HMC were incubated
without or with TNFα in the presence or
absence of 2 μM SB 202190 for 24 h. The
HSD11B1 mRNA values in treated cells were
calculated relative to the amount found in
untreated cells, which was defined as 1.
Results are means ± S.E. of triplicates
measured in three independent experiments.*,
p<0.05.
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