Nature - USA (2020-09-24)

sgNC

AGPS-sg1

FAR1-sg2

PEX3-sg1

AGPAT3-sg1

786-O xenograft tumors

0714 21 28 35 42

0

200

400

600

800

786-O xenograft

Dayspost injection

Tumor volume (mm

3 ) sgNCAGPS-sg 1

FAR1-sg2

PEX3-sg1

AGPAT3-sg

d

0123

0

2

4

6

8

Days

Relative

viabilit

y

OVCAR-8

sgNC

AGPS-sg2 bulk

AGPS-sg2 SCC9

FAR1-sg1 bulk

FAR1-sg1 SCC9

sgNC

OVCAR-8 Ovary fat pad xenograft

FAR1-sg1 bulk

FAR1-sg1 SCC9

AGPS-sg2 bulk

AGPS-sg2 SCC9

sgNC

AGPS-sg2 (bulk

)

AGPS-sg2-SCC9FA

R1-sg1 (bulk

)

FAR1-sg1-SCC9

0.0

0.5

1.0

1.5

Tumor

We

ight (g)

nsns ns

p=0.0083

Ovary fat pad xenograft

a

e

b c

ACSL4

FAR1

AGPS

GPX4

β-tubulin

AGPS-sg2:

FAR1-sg2:

PEX3-sg1:

GPX4-/- SCC2:

sgNC:++---

-+ + ++

OVCAR- 8

--+ --

--- +-

----+

-1.5 -1.0 -0.5 0.00.5 1.01.5 2.0

0

2

4

6

-Log

adj. p 10

Ether-phospholipids

PUFA-TAGs

Lipidomics

C38:7 ePE

C38:7 ePCC40:7 ePC

C34:2 ePE

C38:6 ePEC38:6 ePC

C34:2 ePC

C34:1 ePC

C48:5TAG

C50:6TAG

C52:6TAG

C54:8TAG

C54:7TAG

C56:9TAG

C56:8TAG

C58:11TAG

C58:10TAG

C60:12TAG

4 -202 02468 Lo2 FCLFreS1CCCCCC-202 402468 Lo2 FCLFreS1CCCCCC

-2 024

0

2

4

6

8

Log 2 (FR#2a / WT)

Free fatty acids

S1P

C20:2n 6

C20:4n 6

C20:3n 6

C22:5n 6

C22:6n 6

-Log C20:5n^3

10

adj. p

f

i

g

jk

-1.5

-1.0

-0.5

0

0.5

1.0

TMEM189 1.5

AGPS

ACSL4

AGPAT 3

FAR1

LPCAT 3

AIFM2(FSP1)

PEX3

PEX10

GPX4+/+

WT

GPX4-/-

FR2#a

GPX4-/-

FR2#d

Log

fold change 2

50

37

β-ACTIN

37

AIFM2/

FSP1

MW(KDa) LR

Tumor isolated cells (786-O)

GPX4 +/+ (WT)FR2#d

LRLR

FR2#a

h

Log 2 (FR#2a / WT)

-5 -4 -3 -2 -1 0123

Log 2 (FR2 #d / WT)

acetylcholine

C3-DC-CH3 carnitine

N-acetylputrescine

C3 carnitineaspartate

alpha-glycerophosphocholine

C14 carnitinC18:1 eLPCC16 carnitinee

C18:2 LPC

C22:4 LPC

C18:1 LPC

C16:0 LPC

C16:1 eLPCC20:1 LPC

C18:0 LPE_BC20:0 LPE

C20:1 LPE

-5 -4-3 -2-1 01234

0

1

2

3

4

5

Log 2 (FR2 #a / WT)

-Log

10

adj. p

HILIC-Positivemetabolomics

acetylcholine

C3-DC-CH3 carnitine

N-acetylputrescine

aspartate

C20:4 carnitine

C18:1 eLPC

anthranilic acidC16:1 eLPC

C20:1 LPC

guanidinoacetic acid

C20:0 LPC18:0 LPE_BE

C5 carnitine

C20:1 LPE

C18:2 carnitine

C20 carnitine

N1-acetylspermidine

0

1

2

3

4

50 μm

LLL

RRR

GPX4+/+ (WT) GPX4-/- FR2#a GPX4-/- FR2#d

PTS1-GFP (Peroxisomes)Nucleus

LR

WT FR2#d

LRLR

FR2#a

0

5

10

15

20

20

110

200

#P

uncta

in each

cell

Peroxisomes

(786-O xenograft derived cells)

-2 -1 0123

-25

-20

-15

-10

-5

0

Log 2 (FR2 #a / WT)

TMEM189

AGPS

ABCD1

l

Extended Data Fig. 10 | See next page for caption.