1634 25 SEPTEMBER 2020•VOL 369 ISSUE 6511 sciencemag.org SCIENCE
Fig. 1. DMF inhibits py-
roptosis.(AtoF) BMDM
treated as indicated and
LDH, IL-1b, or TNF-a
measured with [(A) to
(C)] enzyme-linked
immunosorbent assay
(ELISA), (D) GSDMD/
b-actin by immunoblotting,
or [(E) and (F)] perme-
ability of cells to SYTOX
Orange. (G) GSDMD
oligomerization in native
and reduced cell lysates
treated as above. (Hand
I) Time course of perme-
abilization to SYTOX
Orange or GSDMD-N for-
mation in cells treated as
indicated. (J) Survival
rates of WT mice after
50 mg/kg of LPS (n=
10 mice) or LPS+DMF
(n=9 mice). (KandL)
Serum IL-1bor TNF-a
levels of WT mice 5 hours
after intraperitoneal
injection with 5 mg/kg
LPS (n=6 mice) or
phosphate-buffered saline
(PBS) (n=3 mice).
(A) to (C) are pooled from
three independent
experiments. (D) to (I) are representative from three independent experiments. In (J) to (L), data points indicate individual mice. P< 0.05; P< 0.01; P< 0.001; ****P<
0.0001) [(J), Mantel–Cox survival analysis; (A) to (C) and (K), two-way analysis of variance (ANOVA)]. Error bars indicatemeans ± SEM.
100
75
50
25
0
% Cell Death (LDH)
Vehicle Vehicle
_- Ketoglutarate
Succinate
Methyl PyruvateEthyl Pyruvate
Malonate
DMFMMFCitrate
5000
4000
3000
1000
IL-1
` (pg/ml) 2000
TNF-
_ (pg/ml)
12000
9000
6000
3000
0
****
Vehicle
DMF
0 20 40 60
Survival %
0
100
75
50
25
IL-1
` (pg/ml)
TNF-
_ (pg/ml)
800
600
400
200
0
800
600
400
200
0
1000
Vehicle LPS Vehicle LPS
Vehicle Vehicle _- Ketoglutarate SuccinateMethyl PyruvateEthyl PyruvateMalonateDMFMMFItaconateCitrate
LPS+Nig
50- GSDMD
35-
GSDMD
GSDMD-N
fumarase
`-actin
Vehicle LPS+Nig
FHIN1 - + - +
GSDMD
`-actin
GSDMD
Oligomer
Native
Reduced
50-
35-
35-
35-
70-
LPS+Nig
DMF - + - +
100-
130-
50-
35-
35-
35-
****
A
`-actin
Vehicle
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Cell Death (SYTOXOge+/Hoechst)^05 Reads^101520
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Cell Death (SYTOXOge+/Hoechst)
LPS/Nig Pam3/T.f LPS
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 5 10 15 20
Reads
Vehicle
DMF
FHIN1 *
LPS/Nig
Hours
0 ****
Itaconate
Vehicle
DMF
0 5 10 15 20
Reads
Vehicle
Cell Death (SYTOXOge+/Hoechst)
BC
DEF G
HIJ K L
Vehicle Vehicle
_- Ketoglutarate
Succinate
Methyl PyruvateEthyl Pyruvate
Malonate
MMFCitrate
Itaconate Vehicle Vehicle
_- Ketoglutarate
Succinate
Methyl PyruvateEthyl Pyruvate
Malonate
MMFCitrate
Itaconate
LPS+Nig LPS+Nig LPS+Nig
DMF DMF
DMF
GSDMD-N
GSDMD-N
100
50
0
Relative Intensity %
1000 m/z 2000
2-Monomethyl Succinyl-Cysteine at Cys192
Relative Intensity %
100
0
2-Dimethyl Succinyl-Cysteine at Cys192
1000 m/z^2000
EV
GSDMD-N
DMF+GSDMD-N
50
40
30
20
0
% Cell Death (LDH)
10
80
60
40
20
0
% Cell Death (LDH)
EV
GSDMD-N
GSDMD-N-C192A
EV
GSDMD-N
GSDMD-N-C192A
0 2 4 6810
0
0.1
0.2
0.3
0.4
0.5
Cell Death (SYTOXOge+/Hoechst)Reads
*** ***
Cu2+ Biotin-Azide
GSDMD
`-actin
GSDMD
`-actin
GSDMD
Streptavidin
IR-Dye
Streptavidin
IR-Dye
Streptavidin
IR-Dye
GSDMD
LPS+Nig - + + +
MMF-Yne - 5 10 20 μM
Strept. Beads
Post-click Input
50-
35-
100-
50-
35-
100-
50-
35-
50-
35-
35-
35-
35-
50-
50-
35-
100-
50-
Strept. Beads35-
Post-click Input
Cu2+ Biotin Azide
LPS+Nig + + +
MMF-Yne + + + DMF - - +
GSDMD-N - - + -
GSDMD - + - -
GSDMD-N-C192A - - - +
MMF-Yne + + + +
Cu2+ Biotin-Azide
Strept.
Input
50-
35-
50-
35-
35-
Vehicle DMF
Input
IP GSDMD IP
Input
GSDMD
CASP1
GSDMD
LPS/Nig - 15 30 60 - 15 30 60 mins CASP1 Beads
50-
35-
50-
Rec.GSDMD - + +
GSDMD
GSDMD
`-actin
25- DMF - - +
50-
50-
35-
35-
35-
15-
GSDMD
GSDMD
CASP1 P20
CASP1 P20
`-actin
ABC
D
E
FGH
IJ
GSDMD-N
GSDMD-N
GSDMD-N
GSDMD-N
GSDMD-N
GSDMD-N
Fig. 2. Succination of GSDMD inhibits pyroptosis.(AandB) Immunoblot
of GSDMD in streptavidin pulldown from clicked lysates treated as indicated.
(CandD) Representative mass spectrometry spectra of 2-monomethyl and
2-dimethylsuccination of GSDMD immunoprecipitated from DMF-treated BMDMs.
(E) Immunoblot of GSDMD in clicked lysates from transfected HEK293T cells.
(FtoH) Cell death of HEK293T transfected as indicated. (I) Immunoblots of
CASP1p20 and GSDMD in BMDMs. (J) Immunoblots of an in vitro binding
assay of succinated and nonsuccinated GSDMD incubated with caspase 1 beads.
[(A), (B), (F), (J), and (K)] Representative images from three independent
experiments. [(C) to (E)] Representative mass spectrometry spectra from two
independent experiments. [(H) and (I)] Pooled data from three independent
experiments. ***P< 0.001 (one-way ANOVA). Error bars indicate means ± SEM.
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