Ve h 0 100 300 500 C16:0 (µM) BSA (µM)

Transcription

Ve h 0 100 300 500 C16:0 (µM) BSA (µM)
0
100
300
500
Veh
A
C16:0 (µM)
50
10
30
50
BSA (µM)
BSA
Veh
B
0
1
2.5
5
7.5
C16:0 (500 µM)
10 DHA (µM)
A
0
3
6
9
12
C16:0 (150 µM)
16
24
Hours
C
3
10 Ve
a- 0 n h
C g/
N 16 ml
a- 25
C
µ
1
N
a- 6 5 M
C
0
1
µ
N
a- 6 1 M
C 0
B 16 0 µ
SA 1 M
B -C 50
SA 1
6 µM
B -C1 50
SA 6
µ
- 1 M
B C1 00
SA 6
µM
-C 300
16
µ
50 M
0
µM
N
Pa
m
A
B
D
A
0
100
LPS
0
50 100
C16:0
0 100
MDP
0 100
C12-DAP
0 100
Pam3
Tak-242 (nM)
Supplemental Legends
Supplemental Figure 1: BSA-solubilized palmitic acid induces but Docosahexaenoic
acid (DHA) inhibits IL-1ȕ secretion in THP-1 monocytes. (A) THP-1 cells were serum
starved in 0.25% FBS-RPMI-1640 for 12 hours then treated with BSA-solubilized C16:0
for 24 hours. Cell culture supernatants were analyzed for IL-1ȕ by ELISA. (B) Serum
starved THP-1 cells were incubated with DHA for 1 hour then treated with BSAsolubilized C16:0 for 24 hours. Cell culture supernatants were analyzed for IL-1ȕ by
ELISA. Data are expressed as mean {plus minus} s.d. and are representative of two
independent experiments with similar results.
Supplemental Figure 2: Palmitic acid induces expression of NALP3 in THP-1
monocytes. (A) THP-1 cells were serum starved in 0.25% FBS-RPMI-1640 for 12 hours
then treated with C16:0 (150 ȝM) for the indicated times. Cell lysates were
immunoblotted for NALP3 and analyzed by densitometry. Data are presented as mean
{plus minus} SEM and were calculated from three independent experiments.
Significance was determined by ANOVA (*P < 0.05, **P < 0.01 significantly different
from time 0).
Supplemental Figure 3: Palmitate and Pam3CSK4 (TLR2 ligand) produce similar levels
of cell death. THP-1 cells were serum starved in 0.25% FBS/RPMI-1640 for 12 hours
then treated with vehicle, 100 ng/ml Pam3CSK4, 25-150 μM sodium palmitate (Na-C16),
or 50-500 μM BSA-complexed palmitic acid (BSA-C16) for 24 hours (A). THP-1 cells
were serum starved as in A then treated with vehicle, 100 ng/ml Pam3CSK4, 50-150 μM
sodium palmate, or 100-500 μM BSA-complexed palmitic acid for 1-12 hours (B-D).
Equivalent concentrations of BSA were used as controls. Adenylate kinase (AK) activity
was measured in culture supernatants using the Toxilight bioassay kit (Lonza, Rockland,
ME) following the supplier's procedures. IL-1ȕ levels were measured in culture
supernatants by ELISA.
Supplemental Figure 4: Palmitic acid-induced IL-1ȕ secretion is inhibited by a specific
inhibitor of TLR4. (A) THP-1 cells were serum starved in 0.25% FBS-RPMI-1640 for
12 hours then incubated with TLR4-specific small molecule inhibitor TAK-242 for 1
hour. Cells were treated with TLR4 ligand LPS (100 ng/ml), C16:0 (100 ȝM), NOD2
ligand MDP (1 ȝg/ml), NOD1 ligand C12-iE-DAP (1 ȝg/ml), or TLR2/1 ligand
Pam3CSK4 (10 ng/ml) for 24 hours. Cell culture supernatants were analyzed for IL-1ȕ by
ELISA. Data are presented as mean percentages of control {plus minus} SEM and were
calculated from three independent experiments. Significance was determined by ANOVA
(**P < 0.01, ***P < 0.001 significantly different from control).

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