Nature - USA (2020-09-24)

-2 -1 01

0.00

0.25

0.50

0.75

1.00

1.25

Log 10 (M ML210)

-3 -2 -1 0

Log 10 (M RSL3)

sgNC

TMEM189-sg 1

TMEM189-sg 2

TMEM189-sg 3

786-O-Cas9

Relative viability

Dox –++ –+–

50

TMEM189 (2)

MW(KDa)

TMEM189-TetOnshRNAsh1 sh2 sh3

OE

37

20

TMEM189 (1)

50

37

β-ACTIN

786-O

50

37

β-ACTIN

50

37

TMEM189 (1)

sg1sg2sg3

sg-TMEM189

MW(KDa) sgNC

-2 -1 012

0.0

0.4

0.8

1.2

-3 -2 -1 01 -3 -2 -1 01 -3 -2 -1 01

sh1 -Dox

sh1 +Dox

sh2 -Dox

sh2 +Dox

sh3 -Dox

sh3 +Dox

Log (M ML210) Log (M RSL3) Log 10 (M RSL3) Log 10 (M RSL3)

TMEM189-TetOn-shRNA

786-O786-O OVCAR- 8 HuH-7

Relative viability

cd

50

TMEM189 (2)

MW(KDa)

37

TMEM189 (1)

50

37

β-ACTIN

EV TM

786-O-WT

e

fg

b

-2 -1 01

0.0

0.4

0.8

1.2

-3 -2 -1 0

Vector

TMEM189

Vector+Fer-1

TMEM189+Fer-1

Vector+Lip-1

TMEM189+Lip-1

Relative viability

Log 10 (M ML210) Log 10 (M RSL3)

786-O

Gene PCE

FAR1 0.42

PEX7 0.40

MYB 0.38

ACSL4 0.37

SEC14L1 0.37

TMEM189

Gene PCE

PEX7 0.41

FAR1 0.36

TMEM189 0.35

GCLC 0.29

SEC14L1 0.29

AGPS

a

25

Extended Data Fig. 11 | ER-resident enzyme plasmanylethanolamine
desaturase/TMEM189 is dispensible for ferroptosis sensitivity in selected
cancer cells. a, Top genes that show co-dependency with TMEM189 (left) or
AGPS (right) using the cancer dependency map (DepMap) database. PCE,
Pearson correlation coefficient. b, Immunoblotting analysis of TMEM189
levels in 786-O cells expressing sgNC or TMEM189-targeting sgRNAs.
Representative results of experiment performed twice. c, Viability curves for
786-O cells expressing sgNC or TMEM189-targeting sgRNAs and treated with
indicated concentrations of ML210 or RSL3 for 48 h. n = 4 biologically
independent samples. Representative results of experiment performed twice.
d, Immunoblotting analysis of TMEM189 levels in 786-O cells expressing
doxycycline (dox)-inducible TMEM189-shRNAs. β-Actin was used as a loading
control. Results from experiment performed once. e, Viability curves for

786-O, OVCAR-8 and HuH-7 cells expressing dox-inducible TMEM189-targeting

shRNAs, pretreated with vehicle (DMSO) or dox, and then treated with

indicated concentrations of ML210 or RSL3. n = 4 biologically independent

samples. Representative results of experiment performed twice.

f, Immunoblotting analysis of TMEM189 protein levels in 786-O cells expressing

empty vector (EV) or human TMEM189 cDNA construct. β-Actin was used as a

loading control. Results from experiment performed once. g, Viability curves

for 786-O cells expressing empty vector or TMEM189 cDNA and treated with

indicated concentrations of ML210 or RSL3 for 48 h. n = 4 biologically

independent samples. Representative results of experiment performed twice.

β-Actin was used as a loading control in immunoblots. See Supplementary

Information for uncropped immunoblot images. For viability curves, data are

mean ± s.d.