Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images -Figure- 1A

HT29 HT29
HT29
kDa kDa
VC RSL3
VC RSL3 VC RSL3
kDa
150 IRE1a
150 PERK
100
100

50
b-actin
37

HT29
HT29
kDa
kDa
VC RSL3

100 ATF6-FL VC RSL3

75 ATF6-P 50
50 ATF6-N b-actin
37
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.
Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images -Figure- 1B

kDa SW620 SW620

VC RSL3 kDa VC RSL3

150 PERK
100

50 b-actin
37

SW620 SW620
kDa kDa SW620
kDa
VC RSL3 VC RSL3
VC RSL3
150 IRE1a
100 100
75 ATF6-P
ATF6-N 50
50 b-actin
37
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.
Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images -Figure- 1C

DLD1 DLD1
kDa
VC RSL3
kDa VC RSL3

150 PERK
100

50
b-actin
37

DLD1 DLD1
kDa DLD1
kDa
VC RSL3 kDa
VC RSL3 VC RSL3

150 IRE1a
100 100 ATF6-FL
75 ATF6-P
ATF6-N 50
50 b-actin
37
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.
Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images -Figure- 1D

HCT116 HCT116
kDa
VC RSL3
kDa

150 PERK VC RSL3

100

50
b-actin
37

HCT116 HCT116 HCT116

kDa kDa
VC RSL3 kDa VC RSL3
VC RSL3
150 IRE1a
100 100 ATF6-FL
75 ATF6-P
ATF6-N 50
50 b-actin
37
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images -Figure- 1E

HT29
VC RSL3
kDa HT29
15 kDa GPx4 VC RSL3
50
37 SLC7A11
25

HT29
kDa
kDa HT29 VC RSL3
50
VC RSL3 37 b-actin

75 ACSL4
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images -Figure- 1F

HT29 HT29
kDa
kDa 50
EV PERK KD EV PERK KD
37
150 PERK
SLC7A11
100

HT29 HT29 HT29

kDa kDa
EV PERK KD
kDa
EV PERK KD
20 50 EV PERK KD
GPx4
b-actin
37
100
75 ACSL4
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images -Figure- 1G

HT29
HT29 kDa
HT29 kDa EV ATF6 KD
EV ATF6 KD
kDa
50
EV ATF6 KD 37 SLC7A11
20
25 GPx4
150
100 ATF6

HT29
kDa
EV ATF6 KD
HT29
50 b-actin
37
kDa EV ATF6 KD

150
100 ACSL4
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images -Figure- 1H

HT29 HT29
kDa
kDa

EV IRE1a KD
IRE1a 150 EV IRE1a KD
100 100
75
ACSL4

HT29 kDa
HT29 HT29
kDa kDa
EV IRE1a KD
EV IRE1a KD
50 50
b-actin
SLC7A11 37
37
EV IRE1a KD
25
GPx4 20
15
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images -Figure- 1I

HT29 RSL3
PERK PERK
EV EV
kDa KD KD
HT29
kDa
RSL3 50
SLC7A11
EV
PERK
EV
PERK 37
KD KD
150 PERK 25
100

HT29
RSL3
kDa EV
PERK
EV
PERK
KD KD
50 b-actin
37
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images -Figure- 1J

HT29 HT29

kDa PERK
VC inhibitor
kDa PERK b-actin
VC inhibitor HT29 50
50 SLC7A11 kDa
PERK 37
37
VC inhibitor
150 PERK
100
25
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images -Figure- 1K

HT29
HT29
kDa
kDa

VC RSL3 RSL3+ 50
VC RSL3 RSL3+ inhibitor
inhibitor 37
SLC7A11
PERK 150

100

HT29
kDa
VC RSL3 RSL3+
inhibitor
50
b-actin
37
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images -Figure- 1L

HT29
kDa Cystine-
Cystine+
HT29
kDa 50 EV PERK KD EV PERK KD

Cystine+ Cystine- 37 SLC7A11

EV PERK KD EV PERK KD

150 PERK
100

HT29
kDa
50
b-actin
37
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images -Figure- 1M

SW620
kDa Cystine+ Cystine- SW620
EV PERK KD EV PERK KD kDa
Cystine+ Cystine-
PERK
150 50 EV PERK KD EV PERK KD

100 SLC7A11
37

SW620
Cystine+ Cystine-
kDa EV PERK KD EV PERK KD
50
b-actin
37
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images -Figure- 1N

HT29
kDa Cystine-
Cystine+
HT29 PERK PERK
kDa 50 VC Inhibitor EV Inhibitor

Cystine+ Cystine- 37 SLC7A11

PERK PERK
VC Inhibitor EV Inhibitor

150 PERK
100

HT29
Cystine+ Cystine-
PERK PERK
kDa VC Inhibitor EV Inhibitor

50 b-actin

37
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images -Figure- 1O

SW620
kDa Cystine+ Cystine- SW620
PERK PERK
VC Inhibitor EV Inhibitor kDa Cystine+ Cystine-
PERK PERK
150 PERK 50 VC Inhibitor EV Inhibitor
100
37 SLC7A11

SW620
Cystine+ Cystine-
kDa PERK PERK
VC Inhibitor EV Inhibitor
50
b-actin
37
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images -Figure- 2J

HT29 HT29 HT29
kDa kDa HT29
kDa
EV PERKKD
EV PERK KD 250-
150
PERK EV PERK KD 150- EV PERK KD
100
50 100-
b-actin 75- 4-HNE
37 50
50-
b-actin
37
37-

25-
20-

HT29 HT29
EV PERK KD

250-
150-
100-
75- EV PERK KD
50- 50 b-actin
37- MDA 37
25-
20-
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images -Figure- 2K

HT29
HT29

VC PERK
Inhibitor
250- VC PERK
150- Inhibitor

100- 50 b-actin
75-
37
50-

37- 4-HNE

25-
20-

HT29

HT29
-250
VC PERK -150
Inhibitor -100 VC PERK
Inhibitor
-75
-50 b-actin 50

-37 37

MDA -25
-20
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India
Un-cropped Immuno-blot Images -Figure- 3C

HT29
HT29 kDa RSL3
kDa VC FER-1 VC FER-1
RSL3 HT29
VC FER-1 VC FER-1 RSL3
VC FER-1 VC FER-1
150 kDa
PERK
100 75 50
GRP78 b-actin
50
37

HT29
HT29 RSL3
kDa
RSL3 VC FER-1 VC FER-1
VC FER-1 VC FER-1

kDa
50
ATF4
50 b-actin
37
37
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images -Figure- 3D

kDa kDa
HT29 HT29
Thapsigargin - + + -
kDa HT29 PERK inhibitor - - + + Thapsigargin - + + -
50 ATF4 PERK inhibitor - - + +
Thapsigargin - + + - 50
PERK inhibitor - - + + 37 SLC7A11
37
150 PERK
25
100

kDa HT29
Thapsigargin - + + -
PERK inhibitor - - + +
50
b-actin
37
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images -Figure- 3E

HT29
kDa
EV PERK
KD kDa HT29
PERK EV PERK
150
100 KD
50 ATF4

37

kDa HT29
HT29
kDa
EV PERK
KD
EV PERK 50
KD b-actin
50 37
SLC7A11
37
25
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images -Figure- 3F

HT29
kDa kDa
HT29
EV ATF4
KD EV ATF4
50 50 KD
ATF4
37 SLC7A11
37

25

kDa
HT29
EV ATF4
HT29 50 KD
kDa
EV ATF4 37 b-actin
KD
150 PERK
100
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images -Figure- 3O

EV PERK KD
SLC7A11 SLC7A11
kDa Control OE Control OE

50
37 SLC7A11

150 PERK
100

50
b-actin
37
HT29
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India
Un-cropped Immuno-blot Images -Figure- 4D

HT29 tumor tissues

kDa EV KD EV KD EV KD EV KD HT29 tumor tissues
kDa
150 PERK
100

EV KD EV KD EV KD EV KD
50 EV KD EV KD EV KD EV KD 50
SLC7A11 37
37

25

HT29 tumor tissues

EV KD EV KD EV KD EV KD
50
EV KD EV KD EV KD EV KD
37 b-actin
50
37

Continue…
 kDa kDa

HT29 Mice tumors HT29 Mice tumors

EV KD EV KD EV KD EV KD EV KD EV KD EV KD EV KD
37 37 GAPDH
25 4-HNE 25
20
15

HT29 Mice tumors

EV KD EV KD EV KD EV KD
150
100
75
HT29 Mice tumors
50
MDA
37 EV KD EV KD EV KD EV KD
25 37 GAPDH
20
15 25
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.
Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images –Supplementary Figure- 1

SW620
HT29 HT29 HT29 SW620
VC RSL3
VC RSL3 VC RSL3 VC RSL3 VC RSL3 kDa
kDa kDa
kDa

100 GRP78 100 GRP78

75 75
50 ATF4 b-actin 50 ATF4
37 37

SW620 DLD1 DLD1 DLD1 DLD1

VC RSL3 kDa kDa kDa
VC RSL3 VC RSL3 VC RSL3 VC RSL3
kDa

100
75 GRP78
b-actin
50 ATF4 50
50
b-actin b-actin
37 37 37

Continue…
 HCT116 HCT116 HCT116
HCT116
kDa kDa
VC RSL3 kDa VC RSL3 VC RSL3
kDa VC RSL3

100
75 GRP78
50 ATF4 50 b-actin
37 50 b-actin 37
37
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images – Supplementary Figure- 2

SW620 DLD1
kDa VC RSL3 HCT116
kDa VC RSL3 VC RSL3
GPx4 kDa
15
15 GPx4
15 GPx4

50 50
37 SLC7A11 37
50 SLC7A11
37 SLC7A11
25

75 ACSL4 75
ACSL4

75 ACSL4
50 50
b-actin
50 37 37 b-actin
37 b-actin
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India
Un-cropped Immuno-blot Images - Supplementary Figure- 3

SW620
RSL3
PERK PERK
EV EV
KD KD
kDa

PERK
150
100

kDa

50
37 SLC7A11

kDa

50
b-actin
37
Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images - Supplementary Figure- 4

SW620 DLD1 HCT116

PERK
PERK PERK
VC inhibitor
VC inhibitor kDa VC inhibitor
kDa kDa
50
37 SLC7A11
50 37 SLC7A11
SLC7A11
37

PERK PERK 150 PERK

150 150
100
100 100

kDa
50 b-actin
50 b-actin 37
37 37 GAPDH
 Loss of PERK function promotes ferroptosis by downregulating System Xc⁻(SLC7A11) in colorectal cancer Saini et al.

Division of Cancer Biology Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, 226031, India

Un-cropped Immuno-blot Images - Supplementary Figure- 11

SW620 DLD1 HCT116 HCT116

SW620 DLD1
VC PERK PERK VC PERK
PERK PERK VC PERK
Inhibitor VC Inhibitor Inhibitor
VC Inhibitor VC Inhibitor Inhibitor

75-
100- 100- 100- 100-
75- 75- 75- 75-
75-
50-
50- 4-HNE 50- 50- 50-
4-HNE 50- MDA
MDA 37- 37- 37- 4-HNE 37-
MDA
37- 37-
25- 25- 25- 25-
25- 20- 20- 20- 20-
25-
20-
20-

50
b-actin
50 37 50 b-actin
50 b-actin
b-actin 37 50 50
37
37 b-actin b-actin
37 37