Rel

ativ

e m

RN

A ex

pres

sion

MDA-MB-231 Cells

D

IRE1

ACTIN

GAPDH

shScr

shMYC-1

shMYC-2

MYC

A

shScr

shMYC-1

shMYC-2

0.000

0.002

0.004

0.006

0.008

0.010**

***

Rel

ativ

e m

RN

A ex

pres

sion

MDA-MB-231 Cells

IRE1

shScr

shMYC-1

shMYC-2

0.0

0.4

0.8

1.2

Rel

ativ

e XB

P1 s

/t ra

tio

******

XBP1 s/t

Rel

ativ

e m

RN

A ex

pres

sion

SUM159 Cells

B

shScr

shMYC-1

shMYC-2

NCL

0.00

0.05

0.10

0.15

0.20 *****

CDK4

0.00

0.01

0.02

0.03 ******

shScr

shMYC-1

shMYC-2

ODC1

0.000

0.002

0.004

0.006

0.008 ******

shScr

shMYC-1

shMYC-2

0.000

0.005

0.010

0.015

0.020

0.025***

***

DDX18

shScr

shMYC-1

shMYC-2

HSPD1

0.000.010.020.030.040.050.060.07 ns

**

shScr

shMYC-1

shMYC-2

Rel

ativ

e m

RN

A ex

pres

sion

BT549 Cells

C

NCL

0.00

0.05

0.10

0.15

0.20

0.25 ******

shScr

shMYC-1

shMYC-2

CDK4

0.000

0.004

0.008

0.012

0.016 *ns

shScr

shMYC-1

shMYC-2

ODC1

0.0000

0.0005

0.0010

0.0015

0.0020 nsns

shScr

shMYC-1

shMYC-2

HSPD1

0.00

0.02

0.04

0.06

0.08***

***

shScr

shMYC-1

shMYC-2

DDX18

0.000

0.005

0.010

0.015

0.020 *****

shScr

shMYC-1

shMYC-2

NCL

0.0

0.1

0.2

0.3***

***

shScr

shMYC-1

shMYC-2

CDK4

0.000

0.005

0.010

0.015

0.020ns

ns

shScr

shMYC-1

shMYC-2

ODC1

0.000

0.001

0.002

0.003

0.004****

shScr

shMYC-1

shMYC-2

HSPD1

0.00

0.02

0.04

0.06***

***

shScr

shMYC-1

shMYC-2

DDX18

0.000

0.004

0.008

0.012

0.016

0.020******

shScr

shMYC-1

shMYC-2

Supplemental Figure 1. MYC is necessary for activation of the IRE1/XBP1 pathway.(A) MDA-MB-231 cells were infected with lentiviruses encoding control shRNA (shScr) or two distinct MYC shRNAs (shMYC-1 and shMYC-2). Left, Immunoblot of MYC and IRE1 in whole cell lysates. ACTIN and GAPDH were used as loading control. Middle, qRT-PCR analysis of IRE1 expression. Data are presented relative to ACTIN and shown as mean ± s.d. of technical triplicates. Right, qRT-PCR analysis of XBP1 splicing. XBP1 s/t: the ratio of XBP1s to total XBP1 (XBP1t). The XBP1 s/t ratio was normalized to that of the scramble (shScr) control. Data are presented as mean ± s.d. of technical triplicates. (B-D) qRT-PCR analysis of MYC and MYC target genes in SUM159 (B), BT549 (C) and MDA-MB-231 (D) cells infected with lenti-viruses encoding control shRNA (shScr) or two distinct MYC shRNAs (shMYC-1 and shMYC-2). Data are presented relative to ACTIN and shown as mean ± s.d. of technical triplicates. All results shown are representative of three independent experiments. p value was calculated using one-way ANOVA with Turkey’s multiple comparison test. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.

MYC

shScr

shMYC-1

shMYC-2

0.00

0.05

0.10

0.15

0.20**

***

MYC

shScr

shMYC-1

shMYC-2

0.00

0.05

0.10

0.15

0.20***

***

MYC

shScr

shMYC-1

shMYC-2

0.00

0.05

0.10

0.15

0.20

0.25 ******

0 1 10 1000.00

0.05

0.10

0.15 ns

*****

DNAJC10

0 1 10 1000.0

0.2

0.4

0.6 ns

*****

PDIA3

0 1 10 1000.0000

0.0002

0.0004

0.0006 ns

nsns

ERN2

0 1 10 1000.0

0.2

0.4

0.6

0.8 ns

*****

NCL

0 1 10 1000.0

0.5

1.0

1.5

2.0 *

***ns

HSPD1

0 1 10 1000.00

0.05

0.10

0.15 ns

****

DDX18

0 1 10 1000.00

0.01

0.02

0.03

0.04

0.05 ns

***ns

SEC61A1

0 1 10 1000.000

0.002

0.004

0.006

0.008 ns

*ns

ODC1A

C

4-OHT (nM)

4-OHT (nM) 4-OHT (nM) 4-OHT (nM)

4-OHT (nM) 4-OHT (nM) 4-OHT (nM)

Rel

ativ

e m

RN

A ex

pres

sion

Rel

ativ

e m

RN

A ex

pres

sion

Supplemental Figure 2. MYC induces the expression of XBP1 target genes.

4-OHT (nM)

0 1 10 1000.0

0.5

1.0

1.5

2.0

2.5

4-OHT (nM)

Rela

tive

IRE1

ban

d in

tens

ity

B

D DGAT2

0 1 100.000

0.001

0.002

0.003

0.004ns

ns***

4-OHT (nM)

CD59

0 1 100.0

0.5

1.0

1.5

2.0 nsns

ns

4-OHT (nM)

Rela

tive

mRN

A ex

pres

sion

PMP22

0 1 100.00

0.01

0.02

0.03 nsns

ns

4-OHT (nM)

100 100 100

(A) qRT-PCR analysis of MYC target genes expression in MCF10AMYC-ER cells treated with different doses of 4-OHT for 24 h. (B) Intensity analysis of IRE1 immunoblot band in Figure 2B using ImageJ software. The IRE1 band intensity was divided by the sum of ACTIN and GAPDH band intensities and normalized to that of the vehicle-treated group. (C, D) qRT-PCR analysis of UPR genes (C) and RIDD targets (D) expression in MCF10AMYC-ER cells that were treated with different doses of 4-OHT for 24 h. In (A, C, D), data are presented relative to ACTIN and shown as mean ± s.d. of technical triplicates. All results shown are representative of three independent experiments. p value was calculated using one-way ANOVA with Turkey’s multiple comparison test. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.

B CA

D

IB: XBP1s

IB: MYC

IgG IP

XBP1s IP

Input

BT549 Cells

IB: MAX

MAX

GAPDH

BT549

SUM159

293T

E

J

XBP1u

ACTIN

SUM159 Cells

75 KD50 KD

34 KD25 KD

15 KD

100 KD150 KD200 KD

TAD central region HLH/LZ

Transactivation domain central region

TAD 1aa 150aa

143aa 360aacentral region

HLH/LZ337aa 439aa

1

2

3

4 1 2 3 4 1 1 2 3 4 1

75 KD50 KD

34 KD

25 KD

15 KD

IB: Flag

F

G H I

75 KD50 KD

34 KD

25 KD

15 KD

100 KD150 KD200 KD Basic ZIP C-terminal (XBP1s)

Basic ZIP

Basic ZIP C-terminal (XBP1u)

1aa 75aa 133aa 164aa 376aa

1aa 75aa 133aa 164aa 261aa

1

2

3

4

1aa 75aa 133aa 164aa

1aa 75aa

DNA binding and dimerization

1 2 3 4 1 1 2 3 4 1

75 KD50 KD

34 KD25 KD

15 KD

IB: Flag

s

s

SERP1

XBP1s C

hIP

IgG re

ChIP

MYC reChIP

0

50

100

150

N.D.

**Fo

ld e

nric

hmen

t

HSPA5

XBP1s C

hIP

IgG re

ChIP

MYC reChIP

0

200

400

600

N.D.

**

PDIA3

XBP1s C

hIP

IgG re

ChIP

MYC reChIP

0

10

20

30

40

50

N.D.

***

Supplemental Figure 3. MYC interacts with XBP1.(A) ChIP-re-ChIP assay of tunicamycin-treated SUM159 cells was performed using the anti-XBP1s antibody first (XBP1s ChIP), the eluents were subjected to a second ChIP assay using IgG (IgG reChIP) or anti-MYC antibody (MYC reChIP) to detect enriched gene promoter fragments. N.D., not detected by qPCR assay. Data are shown as mean ± s.d. of technical triplicates. p value was calculated using two-tailed unpaired Student’s t-test. ** p<0.01; *** p<0.001. (B) Immunoblot of MAX in 293T, BT549 and SUM159 cell lines. GAPDH was used as loading control. (C) Nuclear extracts from tunicamycin-treated BT549 cells were subjected to co-IP with anti-XBP1s antibody. The immunoblot was probed with anti-XBP1s, anti-MYC, and anti-MAX antibodies. Normal IgG was used as negative control. (D) Coomassie blue staining of GST protein and GST-tagged XBP1s protein. (E) Schematic diagram of full-length (1) and truncated forms (2, 3, 4) of Flag-tagged MYC proteins. (F) GST pull-down assay was performed using GST-tagged XBP1s or GST protein and 293T cell lysates overexpressing Flag-tagged full-length (1) and truncated forms (2, 3, 4) of MYC proteins. Immunoblot-ting was performed with anti-Flag antibody. (G) Coomassie blue staining of GST protein and GST-tagged MYC protein. (H) Schematic diagram of full-length and truncated forms of Flag-tagged XBP1s and XBP1u proteins. (I) GST pull-down assay was performed using GST-tagged MYC protein or GST protein and 293T cell lysates overexpressing Flag-tagged full-length XBP1s (1), full-length XBP1u (4) and truncated forms XBP1 (2, 3) proteins. Immunoblotting was performed with anti-Flag antibody. (J) Immunoblot of XBP1u in SUM159 cells treated with DMSO or 10 μM MG132 for 6 h. ACTIN was used as loading control.

B

A

SUM159 Cells

DNAJC3 DNAJB9 HSPA50.00

0.05

0.10

0.15

SUM159 Cells

shLacZshMYCshLacZshMYC

IgG ChIP

XBP1s ChIP

% o

f Inp

ut

0 0 30 60 120 180

shLacZshMYC

CHX (min)

XBP1s

ACTINGAPDH

0 0 30 60 120 180TM - + + + + + - + + + + +

n.s.

MYC

shLa

cZ

shMYC

0.0

0.2

0.4

0.6

0.8

1.0 ***

Rela

tive

RNA

expr

essi

on

XBP1

shLa

cZ

shXBP1

0.0

0.2

0.4

0.6

0.8 **

Rela

tive

RNA

expr

essi

on

D

C

SUM159 Cells (whole cell lysates)

0 15 30 60 90 120

shLacZ

0 15 30 60 90 120

shXBP1CHX (min)

MYC

ACTINGAPDH

SUM159 Cells (whole cell lysates)

**

*

*

DANJC3

DANJB9

HSPA5

DANJC3

DANJB9

HSPA5

DANJC3

DANJB9

HSPA5

DANJC3

DANJB9

HSPA50.00

0.05

0.10

0.15

0.20

0.25

shLacZshMYC

IgG ChIP P300 ChIP SRC3 ChIP CBP ChIP

% o

f Inp

ut

Supplemental Figure 4. MYC promotes XBP1s binding to its target genes. (A) SUM159 cells were infected with lentiviruses encoding control shRNA (shLacZ) or MYC shRNA (shMYC). ChIP assays were performed using anti-XBP1s antibody to detect enriched gene promoter fragments. (B) SUM159 cells were infected with lentiviruses encoding control shRNA (shLacZ) or MYC shRNA (shMYC). ChIP assays were performed using anti-P300, anti-SRC3 or anti-CBP antibodies to detect enriched gene promoter fragments. In (A, B), IgG was used as mock control. Data are presented relative to input and shown as mean ± s.d. of technical triplicates. (C) SUM159 cells were infected with lentiviruses encoding control shRNA (shLacZ) or MYC shRNA (shMYC) and treated with 100 μg/ml Cycloheximide (CHX) for various periods in the presence of DMSO or 5 µg/ml tunicamycin (TM). Left, qRT-PCR analysis of MYC expression. Right, immunoblot of XBP1s. n.s. indicates non-specific band. (D) SUM159 cells were infected with lentiviruses encoding control shRNA (shLacZ) or XBP1 shRNA (shXBP1) and treated with 100 μg/ml CHX for various periods. Left, qRT-PCR analysis of XBP1 expression. Right, immunoblot of MYC. In (C, D), ACTIN and GAPDH were used as loading control in immunoblot assays. qPCR Data are presented relative to ACTIN and shown as mean ± s.d. of technical triplicates. In (A, C, D), p value was calculated using two-tailed unpaired Student’s t-test. * p<0.05; ** p<0.01; *** p<0.001.

B

D

A

IRE1

GAPDH

ACTIN

XBP1s

TM

Scr LacZ

IRE1-K

O-1

IRE1-K

O-2

XBP1-KO-1

XBP1-KO-2

SUM159 Cells

NCL

ScrLa

cZ

IRE1-K

O1

IRE1-K

O2

XBP1-KO1

XBP1-KO2

0.0

0.2

0.4

0.6

ns ns nsns

CDK4

ScrLa

cZ

IRE1-K

O1

IRE1-K

O2

XBP1-KO1

XBP1-KO2

0.00

0.02

0.04

0.06

ns ns ns ns

ODC1

ScrLa

cZ

IRE1-K

O1

IRE1-K

O2

XBP1-KO1

XBP1-KO2

0.000

0.002

0.004

0.006

0.008

nsns ns

ns

HSPD1

ScrLa

cZ

IRE1-K

O1

IRE1-K

O2

XBP1-KO1

XBP1-KO2

0.0

0.5

1.0

1.5

ns nsns

**

Rel

ativ

e m

RN

A e

xpre

ssio

n

DDX18

ScrLa

cZ

IRE1-K

O1

IRE1-K

O2

XBP1-KO1

XBP1-KO2

0.00

0.05

0.10

0.15

nsns ns

ns

SUM159 Cells

Rel

ativ

e m

RN

A e

xpre

ssio

n

CDKN1A

ScrLa

cZ

IRE1-K

O1

IRE1-K

O2

XBP1-KO1

XBP1-KO2

0.0000

0.0005

0.0010

0.0015

ns ns ns *

CDKN2B

ScrLa

cZ

IRE1-K

O1

IRE1-K

O2

XBP1-KO1

XBP1-KO2

0.0000

0.0001

0.0002

0.0003

0.0004

ns ns nsns

CSUM159 Cells

empty

3×E-bo

x0

2

4

6

8 shLacZshXBP1

ns

ns

Rela

tive

luci

fera

se a

ctivi

ty

IRE1pG

L3-ba

sic

prom

oter

0

50

100

150

200 ns**

Rela

tive

luci

fera

se a

ctivi

ty

pGL3

-prom

oter

enha

ncer

IRE1

0

1

2

3shLacZshXBP1shMYC

ns***

Rela

tive

luci

fera

se a

ctivi

ty

SUM159 Cells

Supplemental Figure 5. The IRE1/XBP1 pathway does not affect MYC and MYC-MIZ1 transcriptional program in SUM159 cells. (A) 3×E-Box luciferase reporter was transfected into SUM159 cells infected with lentiviruses encoding control shRNA (shLacZ) or XBP1 shRNA (shXBP1), and luciferase activity was measured 48 h post transfection. (B) IRE1 promoter or enhancer luciferase reporter was transfected into SUM159 cells infected with lentiviruses encoding control shRNA (shLacZ), XBP1 shRNA (shXBP1), or MYC shRNA (shMYC), and luciferase activity was measured 48 h post transfection. pGL3-basic or pGL3-promoter is empty vector control for IRE1 promoter or enhancer reporter, respectively. In (A, B), data are presented relative to Renilla readings and shown as mean ± s.d. of biological triplicates. (C) SUM159-iCas9 cells were infected with lentiviruses encoding control gRNA (Scr or LacZ), two distinct IRE1 double gRNA constructs (IRE1-KO-1, IRE1-KO-2), or two distinct XBP1 double gRNA constructs (XBP1-KO-1, XBP1-KO-2). Immunoblot of IRE1 and XBP1s were performed in multiclonal cells treated with 5 µg/ml tunicamycin (TM) for 4 h. ACTIN and GAPDH were used as loading controls. (D) qRT-PCR analysis of MYC and MYC-MIZ1 target genes in SUM159-iCas9-KO multiclonal cells. Data are presented relative to ACTIN and shown as mean ± s.d. of technical triplicates. All results shown are representative of two independent experi-ments. p value was calculated using two-tailed unpaired Student’s t-test (A) or one-way ANOVA with Turkey’s multiple comparison test (B, D). ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.

B

D

A

C

IRE1

ACTIN

GAPDH

XBP1s

Scr IRE1-K

O

Scr XBP1-KO

MDA-MB-231 Cells

TM

Rela

tive

mRN

A ex

pres

sion

NCL

Scr

IRE1-K

O

XBP1-KO

0.0

0.1

0.2

0.3

0.4

ns**

ODC1

Scr

IRE1-K

O

XBP1-KO

0.0000

0.0005

0.0010

0.0015

nsns

CDK4

Scr

IRE1-K

O

XBP1-KO

0.000

0.005

0.010

0.015

0.020 ns**

HSPD1

Scr

IRE1-K

O

XBP1-KO

0.00

0.05

0.10

0.15

0.20

0.25

nsns

Rela

tive

mRN

A ex

pres

sion

DDX18

Scr

IRE1-K

O

XBP1-KO

0.00

0.02

0.04

0.06

0.08

ns*

CDKN1A

Scr

IRE1-K

O

XBP1-KO

0.0000

0.0005

0.0010

0.0015

nsns

CDKN2B

Scr

IRE1-K

O

XBP1-KO

0.0000

0.0000

0.0001

0.0001

0.0002

0.0002

***

Rel

ativ

e m

RN

A e

xpre

ssio

n

CDK4

GFPXBP1

0.00

0.02

0.04

0.06

0.08ns

NCL

GFPXBP1

0.0

0.1

0.2

0.3

0.4

0.5 ns

ODC1

GFPXBP1

0.000

0.005

0.010

0.015 *

HSPD1

GFPXBP1

0.0

0.5

1.0

1.5

2.0

2.5 ns

Rela

tive

mRN

A ex

pres

sion

DDX18

GFPXBP1

0.00

0.02

0.04

0.06

0.08 **

CDKN1A

GFPXBP1

0.000

0.001

0.002

0.003

0.004 ns

CDKN2B

GFPXBP1

0.0000

0.0005

0.0010

0.0015 **

GAPDH

XBP1s

GFPXBP1s

MDA-MB-231 Cells

MCF10A Cells

MCF10A Cells

Supplemental Figure 6. The IRE1/XBP1 pathway does not affect MYC and MYC-MIZ1 transcriptional program in MDA-MB-231 and MCF10A cells. (A) Immunoblot of IRE1 and XBP1s in whole cell lysates of pooled MDA-MB-231 KO cells. To detect XBP1s, cells were treated with 5 µg/ml tunicamycin (TM) for 4 h. ACTIN were used as loading controls. (B) qRT-PCR analysis of MYC and MYC-MIZ1 target genes in pooled MDA-MB-231 KO cells. (C) Immunoblot of XBP1s in MCF10AMYC-ER cells infected with lentiviruses encoding GFP or XBP1s. GAPDH were used as loading controls. (D) qRT-PCR analysis of MYC and MYC-MIZ1 target genes in MCF10AMYC-ER cells infected with lentiviruses encoding GFP or XBP1s. In (B, D), data are presented relative to ACTIN and shown as mean ± s.d. of technical triplicates. p value was calculated using one-way ANOVA with Turkey’s multiple comparison test (B) or two-tailed unpaired Student’s t-test (D). ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.

C

MCF10AMYC-ER Cells

DNAJB9

EtOH 4-OHT0.00

0.02

0.04

0.06*** ***

***

DNAJC10

EtOH 4-OHT0.00

0.05

0.10

0.15

0.20*** ***

***

Rela

tive

mRN

A ex

pres

sion

HSPA5

EtOH 4-OHT0.0

0.1

0.2

0.3 ****

**

SEL1L

EtOH 4-OHT0.00

0.02

0.04

0.06

0.08*** ***

***

Rela

tive

mRN

A ex

pres

sion

SYVN1

EtOH 4-OHT0.00

0.05

0.10

0.15shLacZshXBP1shIRE1

*** ******

DNAJC3

EtOH 4-OHT0.00

0.05

0.10

0.15

0.20*** ***

***

EDEM1

EtOH 4-OHT0.00

0.02

0.04

0.06

0.08

0.10***

*****

Rela

tive

mRN

A ex

pres

sion

PDIA3

EtOH 4-OHT0.0

0.2

0.4

0.6

0.8***

*****

SEC61A1

EtOH 4-OHT0.00

0.05

0.10

0.15*** ***

***SEC63

EtOH 4-OHT0.00

0.05

0.10

0.15

0.20

0.25***

*****

IRE1

shLa

cZ

shIR

E10.000

0.002

0.004

0.006

0.008

0.010 ***

Rela

tive

mRN

A ex

pres

sion

A XBP1

shLa

cZ

shXBP1

0.00

0.01

0.02

0.03

0.04 **

Rel

ativ

e m

RN

A e

xpre

ssio

n

MCF10AMYC-ER Cells

PDIA2

EtOH 4-OHT0.0000

0.0002

0.0004

0.0006 *******

MCF10AMYC-ER Cells

B

Supplemental Figure 7. MYC promotes IRE1/XBP1 dependent UPR gene expression. (A, B) qRT-PCR analysis of XBP1 (A) or IRE1 (B) expression in MCF10AMYC-ER cells infected with lentiviruses encoding control shRNA (shLacZ), XBP1 shRNA (shXBP1) or IRE1 shRNA (shIRE1). (C) qRT-PCR analysis of UPR downstream gene expression in MCF10AMYC-ER cells infected with lentiviruses encoding control shRNA (shLacZ), XBP1 shRNA (shXBP1) or IRE1 shRNA (shIRE1) and treated with EtOH or 100 nM 4-OHT for 24 h. Data are presented relative to ACTIN and shown as mean ± s.d. of technical triplicates. p value was calculated using two-tailed unpaired Student’s t-test (A, B) or one-way ANOVA with Turkey’s multiple comparison test (C). ** p<0.01; *** p<0.001.

A

CPERK

shLa

cZ

shPERK

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007 ***

Rel

ativ

e R

NA

exp

ress

ion

0 5 10 20 50 100-60

-40

-20

0

20

40

60

80

4-OHT (nM)

** *** ***ns**shLacZshPERK

Cha

nge

in c

olon

y nu

mbe

r (%

)

ATF6

shScr

shATF6-1

shATF6-2

0.00

0.01

0.02

0.03

0.04 ******

Rela

tive

mRN

A ex

pres

sion

0 5 10 20 50 100-50

0

50

100

4-OHT (nM)

****

**

shLacZshATF6-1

**

***** shATF6-2****

***

** *

Cha

nge

in c

olon

y nu

mbe

r (%

)

F

shLacZ

shATF6-1

shATF6-2

4-OHT (nM) 0 50105 10020

4-OHT (nM) 0 50105 10020

shLacZ

shPERK

0 5 10 20 50 100 4-OHT (nM)

XBP1s (NE)

ACTIN

eIF2α

p-eIF2α

ATF6

PERK

IRE1

p-IRE1IRE1

GAPDH

ATF6 (p)

MCF10AMYC-ER Cells

B

MCF10AMYC-ER Cells

Rela

tive

mRN

A ex

pres

sion

CHOP

0 1 100.00

0.01

0.02

0.03

0.04

0.05 ns

ns

4-OHT (nM)

100

***

GADD34

0.0000

0.0005

0.0010

0.0015

0.0020 ns****

0 1 10

4-OHT (nM)

100

D E

G H

Supplemental Figure 8. Impact of PERK and ATF6 on the clonogenic growth of MYC-hyperactivated cells.(A) Immunoblot of IRE1 phosphorylation (phos-tag SDS-PAGE), XBP1s, ATF6 cleavage (ATF6p), PERK phosphorylation, and eIF2α phosphorylation in MCF10AMYC-ER cells treated with different doses of 4-OHT for 24 h. XBP1s was detected from nuclear extracts (NE). ACTIN and GAPDH were used as loading control for whole cell lysates. (B) qRT-PCR analysis of CHOP and GADD34 expression in MCF10AMYC-ER cells treated with different doses of 4-OHT for 24 h. (C-H) Clonogenic survival and growth of MCF10AMYC-ER cells infected with lentiviruses encoding shRNAs against LacZ control, PERK (C-E), or ATF6 (F-H). The cells were treated with different doses of 4-OHT (to induce MYC-ER translocation). Ethanol was used as vehicle control for 4-OHT. (C, F) qRT-PCR analysis of knockdown efficiency in MCF10AMYC-ER cells. (D, G) Representative images of cell colonies. (E, H) Quantification of changes in colony number. Changes in colony number were compared with vehicle-treated MCF10AMYC-ER cells expressing shLacZ. Data are presented as mean ± s.d. of biological triplicates. In (B, C, F), qRT-PCR data are presented relative to ACTIN and shown as mean ± s.d. of technical triplicates. All data shown are representative of two indepen-dent experiments. p value was calculated using one-way ANOVA with Turkey’s multiple comparison test (B, F, H) or two-tailed unpaired Student’s t-test (C, E). ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.

A

B

DNAJB9

DMSO 0 1 2 5 100.00

0.05

0.10

0.15

Tunicamycin

*** ***

******

Rela

tive

mRN

A ex

pres

sion

SEC61A1

DMSO 0 1 2 5 100.00

0.05

0.10

0.15

0.20

0.25

*** ns*****

***

Rela

tive

mRN

A ex

pres

sion

µM 8866µM 8866Tunicamycin

293T Cells

D

-0.5 0.0 0.5 1.0 1.5 2.00

50

100

150 WTIRE1-KO

EC50 = 6.16 µMEC50 = 18.99 µM**

*

Log [8866] (µΜ)

Res

pons

e (%

max

)

SUM159 Cells

F

SUM159 Cells

CD59

0 1 2 5 100.00

0.02

0.04

0.06

0.08

0.10

Rela

tive

mRN

A ex

pres

sion

8866 (µM)

DGAT2

0 1 2 5 100.000

0.002

0.004

0.006

0.008

0.010

8866 (µM)

PMP22

0 1 2 5 100.000

0.005

0.010

0.015

8866 (µM)

Uncleaved 5’FAM RNA

Uncleaved 5’FAM RNA

Uncleaved 5’FAM RNA

Cleaved 5’FAM RNA

Cleaved 5’FAM RNA

Cleaved 5’FAM RNA

RNA alon

e

no 2-

5ADMSO

0.5 nM5 n

M50

nM

500 n

M

5000

nM

50,00

0 nM

[8866]

5 min

10 min

20 min

-4 -2 0 20

10000

20000

30000

40000

50000

0

3000

6000

9000

12000

15000

IC50 = 86.2 nM R2 = 0.93mIRE1IC50 = NDscIRE1

Vo (F

I uni

ts /

min

) (m

IRE1

)

Vo (FI units / min) (scIR

E1)

C

Log [8866] (µΜ)

E CD59

DMSO TM0.00

0.02

0.04

0.06

******

*********

Rela

tive

mRN

A ex

pres

sion

DGAT2

DMSO TM0.0000

0.0005

0.0010

0.0015

****

*********

Rela

tive

mRN

A ex

pres

sion

293T Cells

PMP22

DMSO TM0.000

0.005

0.010

0.015

0.020

0.025012510

******

********* 8866 (µM)

Rela

tive

mRN

A ex

pres

sion

Supplemental Figure 9. Characterization of IRE1 RNase inhibitor 8866.(A) qRT-PCR analysis of XBP1 target genes in 293T cells that were treated with different doses of 8866 in the presence of DMSO or 5 µg/ml tunicamycin for 6 h. (B) Concentration-response curves of 8866 in WT or multiclonal IRE1-KO SUM159 cells. The EC50 values were estimated using nonlinear regression analysis. (C) Effect of 8866 on the RNase activity of RNase L using an RNA substrate labeled with 6-FAM and BHQ1 on its 5’ and 3’ termini, respectively. Cleavage reactions were monitored at 5, 10 and 20 min and the samples were run on a UREA-TBE RNA gel. No RNase L activator (no 2-5A) or no enzyme (RNA alone) groups served as negative controls. (D) Inhibition profiles of murine IRE1 (mIRE1) and yeast IRE1 (scIRE1) RNase activity by 8866 using FRET assay. IC50 values were estimated using nonlinear regression analysis. (E) qRT-PCR analysis of RIDD targets expression in 293T cells treated with different doses of 8866 in the presence of DMSO or 5 µg/ml tunicamycin (TM) for 6 h. (F) qRT-PCR analysis of RIDD targets expression in SUM159 cells treated with different doses of 8866 for 24 h. In (A, E, F), qRT-PCR data are presented relative to ACTIN and shown as mean ± s.d. of technical triplicates. p value was calculated using one-way ANOVA with Turkey’s multiple comparison test (A, E), or extra sum-of-squares F test (B). ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.

G

8866

Vehicle

Liver Heart Kidney LungMC1 PDX

2147 PDX

8866

Vehicle

Liver Heart Kidney Lung

C

E 4913 PDX

8866

Vehicle

Liver Heart Kidney Lung

I

Days of Treatment

Days of Treatment

Days of Treatment

MC1 PDX

0 5 10 15 200

4

8

12

16

20

Vehicle (n=6)8866 (n=7)ns ns ns ns ns

Body

wei

ght (

g)

4913 PDX

0 5 10 15 20 25 30 35 40 450

4

8

12

16

20

24Vehicle (n=5)8866 (n=5)

ns ns ns ns nsns ns ns ns ns

Body

wei

ght (

g)

2147 PDX

0 5 10 15 20 25 300

4

8

12

16

20

Vehicle (n=8)8866 (n=7)

ns ns ns nsns

* *

Body

wei

ght (

g)

Days of Treatment

4195 PDX

0 5 10 15 20 250

4

8

12

16

20

Vehicle (n=7)8866 (n=7)ns ns ns ns

nsns

Body

wei

ght (

g)

A B

Actin

Xbp1sXbp1u

0 100 200 300 8866 (mg/kg)

Dnajb9

0 100 200 3000.00

0.01

0.02

0.03

0.04p=0.07

Rela

tive

mRN

A ex

pres

sion

Sec61a1

0 100 200 3000.00

0.05

0.10

0.15

0.20

0.25

*

8866 (mg/kg) 8866 (mg/kg)

n=3 n=5 n=5

n=5

n=3 n=5 n=5

n=5Liver from TM-injected mouse

Liver from TM-injected mouse

D

F

H

Supplemental Figure 10. The 8866 treatments do not cause obvious toxicity in PDX tumor-bearing mice. (A, B) 8866 inhibits Xbp1 splicing in vivo. RT-PCR analysis of Xbp1 splicing (A) and Xbp1 targets expression (B) in livers tissue of C57BL/6 mice that were treated with either vehicle or 8866 through oral gavage for 24 h. All mice received tunicamycin (TM) through i.p. injection 6 h prior to sacrifice. qPCR data are presented relative to Actin and shown as mean ± s.d. of biological replicates. p value was calculated using two-tailed unpaired Student’s t-test. * p<0.05. (C-I) Toxicity analysis of MC1 (C, D), 4913 (E, F), 2147 (G, H) and 4195 (I) PDX-bearing SCID/Beige mice treated with vehicle or 8866 via daily oral gavage at 300 mg/kg dose. (C, E, G) H&E staining of liver, heart, kidney and lung of treated mice harvested at the end of the experiments. Scale bar, 50 µm. (D, F, H, I) Body weight of mice treated with 8866. In D, F, H and I, data are presented as mean ± s.e.m of biological replicates. p value was calculated using two-way ANOVA with Bonferroni posttest. ns, not significant; * p<0.05.

A B4195 PDX

0 2 5 7 9 110

200

400

600 Vehicle (n = 5)8866 (n = 5)

Days of Treatment

CHIR99021 (n=5)8866+CHIR99021 (n=5)

**

% c

hang

e in

tum

or v

olum

eVehicle CHIR99021

4195 PDX

MYC

ACTIN

GAPDH

Supplemental Figure 11. Increasing MYC level in 4195 PDX model sensitizes the tumors to 8866 treatment. (A) Immunoblot of MYC in tissue lysates of 4195 PDX tumors treated with vehicle or CHIR99021 (30 mg/kg, i.p. injection every two days). ACTIN and GAPDH were used as loading control. n=3 in each group. (B) Quantifica-tion of tumor volume change of established 4195 PDX tumors in SCID/Beige mice treated with vehicle, 8866, CHIR99021 or 8866 + CHIR99021 (n=5). Data are presented as mean ± s.e.m of biological replicates. p value was calculated using two-way ANOVA with Bonferroni posttest. ** p<0.01.

Vehicl

e88

66 Doc

8866

+Doc

0

5

10

15

20

25

Cl.

casp

ase3

+ cel

lspe

r tum

or a

rea

Vehicl

e88

66 Doc

8866

+Doc

0

5

10

15

CD

31+ m

icro

vess

els

per t

umor

are

a

Vehicle 88

66 Doc

8866

+Doc

0

20

40

60

80

100

******

***

BrdU

+ cel

lspe

r tum

or a

rea

D

Cleaved Caspase-3CD31BrdUB

MC1 PDX

8866

Vehicle

Liver Heart Kidney Lung

8866+Docetaxel

Docetaxel

Pancreas Small intestine

A

BrdU

CD31

CleavedCasp-3

Vehicle 8866 Docetaxel 8866+DocetaxelMC1 PDX - Day 4 Tumor

Total

Cleaved

Caspase-3

Caspase-3

Veh 8866 Doc 8866+Doc

Total

Cleaved

Caspase-3

Caspase-3

ACTINGAPDH

ShortExposure

LongExposure

MC1 PDX - Day 20 Tumor

C

Supplemental Figure 12. The effect of 8866 +/- docetaxel treatments on MC1 PDX tumor. (A) BrdU, cleaved Caspase-3 and CD31 immunostaining images of MC1 PDX tumors treated with vehicle, 300 mg/kg 8866 (daily oral gavage), docetaxel (10 mg/kg, weekly i.p. injection), or the combination of 8866 and docetaxel for 4 days. Scale bar, 50 µm. (B) Quantification of BrdU positive cells, CD31 positive microvessels or cleaved Caspase-3 positive cells on tumor sections stained in (A). More than 10 tumor areas from each group were counted. Doc: docetaxel. p value was calculated using one-way ANOVA with Turkey’s multiple comparison test. *** p<0.001. (C) Immunoblot of Caspase-3 in tissue lysates of MC1 PDX tumors treated with vehicle, 300 mg/kg 8866 (daily oral gavage), docetaxel (10 mg/kg, weekly i.p. injection), or the combination of 8866 and docetaxel for 20 days. Mice undergoing monotherapy also received vehicle. n=3 in each group. (D) H&E staining of liver, heart, kidney, lung, pancreas and small intestine of MC1 PDX-bearing SCID/Beige mice treated as in (C). Scale bar, 50 µm.

A B

Cleaved

Total

Cleaved

p-

Total Caspase-3

Caspase-3

P-eIF2α

eIF2α

Gapdh

Veh 8866 Doc 8866+Doc TM

Perk

Liver

Xbp1sXbp1u

ActinRT-PCR

Atf4

Ire1

Atf6

Atf6 (p)

Ire1p-Ire1

Caspase-3

Caspase-3

eIF2α

eIF2α

Veh 8866 Doc 8866+Doc TM

Perk

Pancreas

ActinRT-PCRXbp1s

Xbp1u

Gapdh

Atf4

Ire1

Atf6

Atf6 (p)

Ire1p-Ire1

C

Liver TUNEL+DAPI

Vehicle 8866 Docetaxel 8866+Docetaxel

Pos CtrlNeg Ctrl

Vehicle 8866 Docetaxel 8866+Docetaxel

Pos CtrlNeg Ctrl

Pancreas TUNEL+DAPI

D

Vehicl

e88

66 Doc

8866

+Doc

0

2

4

6

8

10

TUN

EL+

cel

l cou

nt

Vehicl

e88

66 Doc

8866

+Doc

0

2

4

6

8

TUNE

L+ c

ell c

ount

Supplemental Figure 13. The effect of 8866 +/- docetaxel treatments on the liver and pancreas of MC1 PDX tumor-bearing mice.(A, B) Immunoblot and Xbp1 splicing assays of liver (A) and pancreas (B) tissues from MC1 PDX tumor-bearing SCID/Beige mice treated with vehicle, 300 mg/kg 8866 (daily oral gavage), docetaxel (10 mg/kg, weekly i.p. injection), or the combination of 8866 and docetaxel. Mice undergoing monotherapy also received vehicle. Phos-tag SDS-PAGE gels was used to detect Ire1 phosphorylation. Gapdh was used as loading control for immunoblot and Actin was used as loading control for RT-PCR. n=3 in each group. NIH3T3 cells treated with 5 µg/ml tunicamycin (TM) for 6 h were used as control. (C, D) TUNEL staining of liver (C) and pancreas (D) tissues from MC1 PDX tumor-bearing SCID/Beige mice treated as in (A, B). Tissue sections incubated with TUNEL reaction buffer without dTd enzyme served as negative control. Tissue sections treated with DNase I served as positive control. Scale bar, 100 µm. TUNEL positive cells were quantitated from more than 15 random views. No statistically significant differences were observed among the groups using one-way ANOVA with Turkey’s multiple comparison test.

MC1 PDX

0 5 10 15 20 25 30 35 40 45 50 550

4

8

12

16

20

24Vehicle (n=4)8866 (n=4)Docetaxel (n=4)8866 + Docetaxel (n=4)

Body

wei

ght (

g)

Days of Treatment

Glucose

Vehicl

e88

66

Doceta

xel

8866

+Doc

etaxe

l0

40

80

120

160ns *

ns

Glu

cose

(m

g/dL

)

A BFood intake

Vehicl

e88

66

Doceta

xel

8866

+Doc

etaxe

l0.00

0.05

0.10

0.15

0.20ns

nsns

Food

inta

ke(g

/day

/mou

se/g

BW

)

C

Supplemental Figure 14. The effects of 8866 +/- docetaxel treatments on MC1 PDX tumor-bearing mice. (A, B) Food intake (A) and blood glucose levels (after overnight fasting) (B) of MC1 PDX tumor-bearing SCID/Beige mice treated with vehicle, 300 mg/kg 8866 (daily oral gavage), docetaxel (10 mg/kg, weekly i.p. injection), or the combination of 8866 and docetaxel for 20 days. Mice undergoing monotherapy also received vehicle. (C) Body weight of mice treated as in Figure 7B. All data are presented as mean ± s.e.m of biological replicates. p value was calculated using one-way ANOVA with Turkey’s multiple comparison test (A, B). ns, not significant; * p<0.05.

A

B

Vehicl

e88

66 Doc

8866

+Doc

0

10

20

30

TIL

nsns

ns

% o

f CD

45+

cells

Vehicl

e88

66 Doc

8866

+Doc

0

10

20

30

40

50

MDSC

nsns

**

MD

SC

% o

f TIL

Vehicl

e88

66 Doc

8866

+Doc

0

20

40

60

80

Macrophage

nsns

ns

Mac

roph

age

% o

f TIL

Vehicl

e88

66 Doc

8866

+Doc

0

5

10

15

20

25

Dendritic cell

nsns

ns

CD

11c+ %

of T

IL

Vehicl

e88

66 Doc

8866

+Doc

0

10

20

30

40

CD3+ T cells

nsns

***

CD

3+

% o

f TIL

Vehicl

e88

66 Doc

8866

+Doc

0

5

10

15

20

CD8+ T cells

nsns

***

CD

8+ % o

f TIL

Vehicl

e88

66 Doc

8866

+Doc

0

2

4

6

8

CD4+ T cells

nsns

**

CD

4+ % o

f TIL

Vehicl

e88

66 Doc

8866

+Doc

0

5

10

15

20

B220+ B cells

nsns

ns

B22

0+ % o

f TIL

Vehicl

e88

66 Doc

8866

+Doc

0

5

10

15

TIL

nsns

ns

% o

f CD

45+

cells

Vehicl

e88

66 Doc

8866

+Doc

0

20

40

60

80

MDSC

nsns

ns

MD

SC

% o

f TIL

Vehicl

e88

66 Doc

8866

+Doc

0

20

40

60

80

100

Macrophage

nsns

nsM

acro

phag

e %

of T

IL

Vehicl

e88

66 Doc

8866

+Doc

0

20

40

60

Dendritic cell

*****

**

CD

11c+ %

of T

IL

Vehicl

e88

66 Doc

8866

+Doc

0

10

20

30

40

CD3+ T cells

nsns

ns

CD

3+ % o

f TIL

Vehicl

e88

66 Doc

8866

+Doc

0.0

0.5

1.0

1.5

2.0

CD8+ T cells

nsns

ns

CD

8+ % o

f TIL

Vehicl

e88

66 Doc

8866

+Doc

0.0

0.5

1.0

1.5

2.0

2.5

CD4+ T cells

nsns

ns

CD

4+ % o

f TIL

Vehicl

e88

66 Doc

8866

+Doc

0

1

2

3

4

B220+ B cells

nsns

ns

B22

0+ % o

f TIL

2153L GEM Tumors

T11 GEM Tumors

2153L

0 3 6 90

4

8

12

16

20 Vehicle (n = 6)8866 (n = 6)

Days of treatment

Docetaxel (n=6)8866+Docetaxel (n=6)

Bogy

wei

ght (

g)

T11

0 3 6 9 12 160

4

8

12

16

20 Vehicle (n = 5)8866 (n = 5)

Days of Treatment

Docetaxel (n=5)8866+Docetaxel (n=5)

Bogy

wei

ght (

g)

C D

Supplemental Figure 15. The effects of 8866 +/- docetaxel treatments on the immune surveillance in the syngeneic GEM tumors.(A, B) Immunophenotyping of p53-null GEM tumors 2153L (A) and T11 (B) that were transplanted into BALB/c mice and treated with vehicle, 300 mg/kg 8866 (daily oral gavage), docetaxel (10 mg/kg, weekly i.p. injection), or the combination of 8866 and docetaxel. Mice undergoing monotherapy also received vehicle. Tumor-infiltrating leukocytes (TIL) were defined as CD45+ cells. Percentages of MDSCs (myeloid-derived suppressor cells, CD11b+ Gr-1+), macrophage (CD11b+ Gr-1- F4/80+), CD11c+ dendritic cells, CD3+ T cells, CD8+ T cells, CD4+ T cells, or B220+ B cells in CD45+ TILs are shown. Data are presented as mean ± s.e.m. of biological replicates. p value was calculated using one-way ANOVA with Turkey’s multiple comparison test. ns, not significant; ** p<0.01; *** p<0.001. (C, D) Body weight of BALB/c mice bearing 2153L (C) or T11 (D) GEM tumors treated as in (A, B). All data are presented as mean ± s.e.m of biological replicates.

MDA-MB-231-LM2-Luc

Vehicl

e (n=

5)

8866

(n=5

)

Doceta

xel (n

=5)

8866

+ Doc

etaxe

l (n=5

)5

6

7

8

9

10 ns ****

*

Met

asta

si le

sion

s(P

hoto

n flu

x lo

g 10)

Vehicle

8866

Docetaxel

8866+Docetaxel

A B

Supplemental Figure 16. The effects of 8866 treatments on lung metastasis.MDA-MB-231-LM2-Luc cells were injected into the athymic nude mice through tail vein. Mice were then treated with vehicle, 300 mg/kg 8866 (daily oral gavage), docetaxel (10 mg/kg, weekly i.p. injection), or the combination of 8866 and docetaxel for 5 weeks. Mice undergoing monotherapy also received vehicle. n=5 in each group. (A) Representative bioluminescent images of lung metastasis formed by MDA-MB-231-LM2-Luc cells. Bioluminescent images were obtained 5 weeks after treatment. (B) Quantifi-cation of imaging studies as in A. Data are presented as mean ± s.e.m. p value was calculated using one-way ANOVA with Turkey’s multiple comparison test. ns, not significant; * p<0.05; *** p<0.001.

Name Sense Strand / Sense Primer (5'-3') Antisense Strand / Antisense Primer (5'-3')

shLacZ CCGGCGCTAAATACTGGCAGGCGTTCTGCAGAACGCCTGCCAGTATTTAGCGTTTTT

AATTAAAAACGCTAAATACTGGCAGGCGTTCTGCAGAACGCCTGCCAGTATTTAGCG

shXBP1 CCGGGACCCAGTCATGTTCTTCAAACTCGAGTTTGAAGAACATGACTGGGTCTTTTTG

AATTCAAAAAGACCCAGTCATGTTCTTCAAACTCGAGTTTGAAGAACATGACTGGGTC

shIRE1 CCGGGCAGGACATCTGGTATGTTATCTCGAGATAACATACCAGATGTCCTGCTTTTTG

AATTCAAAAAGCAGGACATCTGGTATGTTATCTCGAGATAACATACCAGATGTCCTGC

shPERK CCGGTAGCAGCAATCCCTAATATATCTCGAGATATATTAGGGATTGCTGCTATTTTTG

AATTCAAAAATAGCAGCAATCCCTAATATATCTCGAGATATATTAGGGATTGCTGCTA

shATF6 -1 CCGGGCAGCAACCAATTATCAGTTTCTCGAGAAACTGATAATTGGTTGCTGCTTTTTG

AATTCAAAAAGCAGCAACCAATTATCAGTTTCTCGAGAAACTGATAATTGGTTGCTGC

shATF6 -2 CCGGCAGACAGTACCAACGCTTATGCTCGAGCATAAGCGTTGGTACTGTCTGTTTTTG

AATTCAAAAACAGACAGTACCAACGCTTATGCTCGAGCATAAGCGTTGGTACTGTCTG

Primers for luciferase reporter generationIRE1- promoter ATATAACGCGTCCTAGGATAAGTTAGATAC TATATCCATGGCGAGGACTCGGCCCTGGIRE1- enhancer ATATAACGCGTGGTCTGCATGTCTTGTTTCT TATATAGATCTTTCTGCAAGAGCAGGCATTA

Primers for RT-qPCRIRE1 AGTATGTGGAGCAGAAGGAC GTTGTGTGGCTTTAGGTCTCXBP1s CTGAGTCCGCAGCAGGTG TCCAAGTTGTCCAGAATGCCXBP1 total GGCATCCTGGCTTGCCTCCA GCCCCCTCAGCAGGTGTTCCACTIN GCGAGAAGATGACCCAGATC CCAGTGGTACGGCCAGAGGDNAJB9 TCGGCATCAGAGCGCCAAATCA ACCACTAGTAAAAGCACTGTGTCCAAGSEC63 CCTCCACTTACCTGCCCATA GGTTCCGGGCCATTACTATTHSPA5 GACGGGCAAAGATGTCAGGA GCCCGTTTGGCCTTTTCTACDNAJC3 CTCAGTTTCATGCTGCCGTA TTGCTGCAGTGAAGTCCATCMYC GCCACGTCTCCACACATCAG TCTTGGCAGCAGGATAGTCCTTSEC61A1 TGGAAGTCATCAAGCCCTTC AGAGGGTGATAGCGGTCCACNCL GCGAAGGCAGGTAAAAATCA GACGACCTCTTCTCCACTGCCDK4 TACTGAGGCGACTGGAGGCT AGTTCGGGATGTGGCACAGODC1 CAAAGGCAACTCTCCAGGAA GGCTGCGACTCAGGCTC HSPD1 GTTGAAGGATCTTTGATAGTTG TTCTCTTCTTTAGGAATTTCTGDDX18 TTCATGGGCTTTTGTTTTGA GATCGAGAAGCGGAACCTCDNAJC10 GGCCAGTATGAAAGCTGGAA GTGAACAGCCTGGGGAGTAAPDIA3 AAGCTCAGCAAAGACCCAAA CACTTAATTCACGGCCACCTERN2 CGTGCTGTGAGGAACAAGAA GGGAAGCGGTTTGTGAAGTACHOP CTGCTTCTCTGGCTTGGCTG GCTCTGGGAGGTGCTTGTGAGADD34 CCCAGAAACCCCTACTCATGATC GCCCAGACAGCCAGGAAATEDEM1 TTGACAAAGATTCCACCGTCC TGTGAGCAGAAAGGAGGCTTCPDIA2 GATCAGCGGCCAGTTAAGAC GATGTCCTCGTGGTCTTGGTSEL1L AGTTGGACAGAGTGGGCTTG ATCCACCCAGCCTTGTTCAGSYVN1 TACGCCGTCACAGAGACTTG TGCGTTCCATAAAGTCCACACD59 GGAATCCAAGGAGGGTCTGT TGCAGTCAGCAGTTGGGTTADGAT2 CATACGGCCTTACCTGGCTA ATTGCCACTCCCATTCTTTGPMP22 CCTGTCGATCATCTTCAGCA AGCACTCATCACGCACAGACCDKN1A GCCATTAGCGCATCACAGT TGCGTTCACAGGTGTTTCTGCDKN2B ATGCGCGAGGAGAACAAG CTCCCGAAACGGTTGACTCSec61a1 (mouse) CTGGCGGTAGAATGCCTCT TGAGACCATTGTGTGGAAGGDdit3 (mouse) GTCCCTAGCTTGGCTGACAGA TGGAGAGCGAGGGCTTTGDnajb9 (mouse) CCCCAGTGTCAAACTGTACCAG AGCGTTTCCAATTTTCCATAAATTHspa5 (mouse) TCATCGGACGCACTTGGA CAACCACCTTGAATGGCAAGAGadd34 (mouse) AGGACCCCGAGATTCCTCTA CTTCGATCTCGTGCAAACTGActin (mouse) TACCACCATGTACCCAGGCA CTCAGGAGGAGCAATGATCTTGAT

Primers for shRNA construct generation

Supplemental Table 1. Sequences of DNA Oligonucleotides

Primers for XBP1 splicing assayXBP1 splicing CCTGGTTGCTGAAGAGGAGG CCATGGGGAGATGTTCTGGAGACTIN GCGAGAAGATGACCCAGATC CCAGTGGTACGGCCAGAGGXbp1 splicing (mouse) ACACGCTTGGGAATGGACAC CCATGGGAAGATGTTCTGGGActin (mouse) TACCACCATGTACCCAGGCA CTCAGGAGGAGCAATGATCTTGAT

Primers for ChIP-qPCRDNAJB9 CTCGTCTGTCGACTCACTTC TGGAAAACTGTTGTTGCTGCP4HB TTGGGGTGACGCAATTTCC CATGCCTCGGTGAGTGTCTDNAJC10 TCAACCTAAACCTACACCGC GGTACCGGTAGGTTCCTCADNAJC3 ATCAAATAGTCCCACTCGCC GATAACGTGGAAGCACGAGAEDEM1 TTCCTTAAAGGGGAAGCGAG AAGACGAGCCACAATACTCCSERP1 TTGCCTTTGAGAACTACCCG CCGATTGCCACGCTATATCAHERUPD1 CCAAACATGGCCACCGAA CAGAACGCTCTGTGGCTGHSPA5 CGAAACACCCCAATAGGTCA ATATAAGCCGAGTAGGCGACPDIA3 TAGAACTCACGGACGACAAC AAGAACTCGACGAGCATGAGSEC61A1 CTGTTACGTGTATGGGAGCC CAGACTTGGTAACGCGAGAGERN2 CTTATGGAGAACTTGCCACG CTGGTGCAATGATAAAGAGCGMBTPS1 GGGGCTGTTTACTCCCAAC AAATGTCGCGAGACCTTCGTVEGFA (Ctrl) TGAGGGTTCATCAAGCTGGTGTCT TTGGAGAGGGCAGTGCTTAACTCASEL1L ATGTCCGTGAGTCGTGGTC GCTTCTGGTCCAATCACCATSYVN1 CCGTAGACATCACCCCAGAT GGGTCAGACACCTCACTTCCTSEN34 CCAAAGAGGATGAGACCAGTG TGGTGGGGAGTACAGAGAAGAATP50 TACTGCCGCAGAGTTTGATCT CCCTTCCTGGCATCTTAGGTANDUFA3 TCAAGAATGCCTGGGACAAG ATGGGGATAGATAAGGGGATGIRE1 -P1 TAACTGTCCAGGCTTCTTGG GTGACCCTGTAACATCAGCAIRE1 -P2 TTTCCCAGCCACAGACAAG GGCTGTAAAGGACCAGGCIRE1 -P3 CCTCATGAGCGAAGCCAG CTATAAGCGCTGGGTGCCIRE1 -P4 GAGACGAATGATAGGGCCTG ACGCTCACATCCTGAACAATIRE1 -E1 GAGGCTTATTCCAGGTGCTT GATTCAGGACATTGGCAGGAIRE1 -E2 TGAGTTGTTTGTTTGCTGCC CCCAGCTCCTATACAAAGGCIRE1 -E3 TCTGTCACCATAAGCAGAGC AGCGATGAGGTATTTCCAGCIRE1 -E4 ATGTAGAGCTACTCCAGGCA GGATGGATGCAGAACGAAGA