Science - USA (2020-09-25)

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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