Repression of the transcriptional activity of ERRα with sequence-specific DNA-binding polyamides

Repression of the transcriptional activity of ERR

with

sequence-specific DNA-binding polyamides

Chien-yu Chen

1,3

Yang Li

Tiezheng Jia

2,4

Lina He

Alissa A. Hare

2,5

Amanda

Silberstein

2,6

John Gallagher

Thomas F. Martinez

2,7

Joseph W. Stiles

1,8

Bogdan

Olenyuk

1,9

Peter B. Dervan

Bangyan L. Stiles

Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern

California, Los Angeles, CA 90033, USA

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,

CA 91125, USA

Present address: Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA

Present address: SUSTECH, 1088 Xueyuan Avenue, Shenzhen, Guangdong Province 518055,

P. R. China

Present address: Vanderbilt, Nashville, TN 37240, USA

Present address: Westmont College, 955 La Paz Rd, Santa Barbara, CA 93108, USA

Present address: Salk Institute, 10010 N Torrey Pines Rd, La Jolla, CA 92037, USA

Present address: Princeton University, Princeton, NJ 08544, USA

Present address: PRISM, 505 Coast Blvd. S., Suite 206, La Jolla, CA 92037, USA

Abstract

The orphan nuclear receptors estrogen-related receptors (ERRs) bind to the estrogen-related

receptor response element (ERRE) to regulate transcriptional programs in cellular metabolism

and cancer cell growth. In this study, we evaluated the potential for a pyrrole-imidazole polyamide

to block ERR

binding to ERREs to inhibit gene expression. We demonstrated that the ERRE

targeted polyamide 1 blocked the binding of ERR

to the consensus ERRE and reduced

the transcriptional activity of ERR

in cell culture. We further showed that inhibiting ERR

transcriptional activity with polyamide 1 led to reduced mitochondrial oxygen consumption, a

primary biological effect regulated by ERR

. Finally, our data demonstrated that polyamide 1 is

an inhibitor for cancer cell growth.

Keywords

ERR

; Pyrrole-imidazole polyamide; Mitochondria

Bangyan L. Stiles, bstiles@usc.edu.

Conflict of interest The authors declare that they have no conflict of interest.

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Med Chem Res

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Published in final edited form as:

Med Chem Res

. 2020 April ; 29(4): 607–616. doi:10.1007/s00044-019-02493-4.

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Introduction

The estrogen-related receptors (ERR), also known as nuclear receptor 3B (NR3B), are

orphan nuclear receptors that play crucial roles in metabolic homeostasis. The orphan

nuclear receptor subfamily comprises three members referred to as NR3B1 (ERR

NR3B2 (ERR

), and NR3B3 (ERR

). The ERRs display constitutive transcriptional activity

independent of natural estrogen ligands and do not directly take part in classic estrogen

signaling pathways (

Zhang et al. 2015

). ERR

is abundantly expressed in organs with

high oxidative activity and is recognized as a key regulator of adaptive energy metabolism

in response to environmental stimuli and energy demands (

Villena and Kralli 2008

ERR

has a vital role in cell fate determination and pluripotency by interacting with

OCT4-SOX2 complex and NANOG (

van den Berg et al. 2008

). ERR

works to switch

bioenergetic responses to hypertrophic stress primarily in the heart (

Kwon et al. 2013

Both ERR

and ERR

orchestrate mitochondrial functions with the coactivator peroxisome

proliferator-activated receptor gamma coactivator 1-alpha (PGC1-

), either by directly

activating genes encoding proteins needed to maintain the mitochondrial components, or

by indirectly activating major transcription factors governing mitochondrial biogenesis

(

Kamei et al. 2003

;

Gleyzer et al. 2005

;

Gaillard et al. 2007

;

Takacs et al. 2013

). In

addition, ERR

directly regulates the transcription of genes encoding proteins involved in

the citric acid cycle, mitochondrial oxidative phosphorylation (OXPHOS) and respiratory

chain, such as the citrate synthase, succinate dehydrogenase, cytochrome

(CytC), and

NADH dehydrogenases (

Giguere 2008

ERR contain two highly conserved zinc finger motifs that preferentially bind to the

consensus estrogen-related response element (ERRE) sequence 5

′

-AAGGTCA-3

′

(Fig.

1) (

Sladek et al. 1997

). This signature motif is also found within the regulatory regions

of genes encoding for carnitine/acylcarnitine translocase (SLC25A29), medium chain acyl

CoA dehydrogenase and fatty acid binding protein 3 (

Dufour et al. 2007

). All three genes

are involved in fatty acid

-oxidation (

Shekhawat et al. 2005

;

Gutierrez-Aguilar and Baines

2013

) and their expression is reduced when ERR

is absent in adipocytes, intestinal

epithelia and hepatocytes (

Sladek et al. 1997

;

Carrier et al. 2004

;

Alaynick et al. 2007

Genetic studies in mice also suggest a role for ERRs in lipid metabolism. Mice lacking

ERR

were reported to be resistant to high fat diet-induced obesity (

Luo et al. 2003

Published ChIP-on-chip data (

Charest-Marcotte et al. 2010

) show that ERR

bound to the

regulatory domain of lipogenic genes and positively regulated their expression. In agreement

with its putative function in lipogenesis, ERR

along with its coactivator PGC1-

were

found to be essential for adipogenic differentiation induced by glucocorticoid, cAMP and

insulin (

Ijichi et al. 2007

). Furthermore, ERR

was positively regulated by insulin-mediated

PI3K/AKT signaling in hepatocytes (

Li et al. 2013

). Taken together, these results support a

role of ERR

in promoting lipogenesis.

Given the diverse role of ERRs in the regulation of mitochondria and lipid metabolism, it is

proposed that ERRs may play a role in tumor metabolism (

Conzen 2008

;

Bernatchez et al.

2013

;

Fradet et al. 2016

;

Ye et al. 2019

). Transformed cells are known to reprogram their

energy homeostasis and metabolism with various strategies to meet energetic and anabolic

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demands (

Zheng 2012

;

Cheng et al. 2018

). Recent studies have highlighted the role of

dysfunctional lipid metabolism in promoting tumor cell transformation and growth (

Liu et

al. 2018

;

Long et al. 2018

;

Nakagawa et al. 2018

). In order to understand the function

of ERR

in metabolic regulation and explore the potential therapeutic benefit of targeting

ERR

, we designed a pyrrole–imidazole (Py-Im) polyamide inhibitor to disrupt ERR

DNA binding. Py-Im polyamides are a class of synthetic DNA minor groove-binding ligands

with programmable sequence selectivity, and have been shown to modulate gene expression

by inhibiting transcription factor-DNA binding (

Dervan and Edelson 2003

;

Olenyuk et al.

2004

;

Nickols and Dervan 2007

;

Nickols et al. 2013

;

Taniguchi et al. 2017

;

Wei et al.

2018

). Here, we report the design and validation of an ERRE-targeting Py-Im polyamide

that inhibits ERR

-mediated cellular functions.

Materials and methods

Cell culture

Mouse hepatocytes were isolated from mouse livers as previously described (

Zeng et al.

2011

). Mouse and human hepatocytes (SNU398 and Huh7 cells purchased from ATCC)

were cultured in Dulbecco’s Modified Eagle’s Medium (Mediatech) supplemented with 10%

FBS (Atlas Biologicals), 5 μg/ml insulin (Sigma-Aldrich), and 10 ng/ml epidermal growth

factor (Invitrogen). The C4–2b human prostate cancer cell line was cultured in RPMI-1640

medium (Mediatech) supplemented with 10% FBS. All cell culture was supplemented with

1% of penicillin–streptomycin and incubated at 37 °C with 85% relative humidity/5% CO

Py-Im polyamide synthesis

Py-Im polyamides 1 and 2 were provided by the Dervan Group (California Institute of

Technology) and were synthesized on Oxime resin as described (

Puckett et al. 2012

Electromobility shift assay (EMSA)

Oligonucleotide containing the 5

′

-NNNNAAGGTCAN NNN-3

′

consensus sequence

corresponding to the ERRE binding site was synthesized by and purchased from Integrated

DNA technologies. NE-PER Nuclear and Cytoplasmic Extraction Reagents, Biotin 3

′

End

DNA Labeling and LightShift Chemiluminescent EMSA kits were purchased from Thermo

Fisher Scientific. In brief, two complementary oligonucleotides were labeled separately

with biotin according to manufacturer’s instruction and allowed to reanneal. For binding

and competition reactions, 20 fmole biotin-labeled ERRE dsDNA was incubated with 10

μg nuclear protein extract from ERR

overexpressing mouse hepatocytes with or without

unlabeled ERRE dsDNA, ERR

antibody (ab76228, Abcam), and indicated concentrations

of two different polyamides (final concentration: 1 nM of biotin-labeled ERRE dsDNA, 0.5

μg/μl of protein extract and 10 pM to 1 μM polyamides). The mixture was then separated

on polyacrylamide gel following incubation and DNA transferred into membrane. Biotin

labeled DNA-protein complexes were detected using X-ray film following the instructions of

the EMSA kit.

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Luciferase reporter assay

For the luciferase reporter plasmid, the −686 to +55 human genomic sequences relative

to the transcription initiation site of CytC, containing ERRE (

Schreiber et al. 2004

), was

amplified and cloned upstream of the luciferase coding sequences of pGL4 (Promega).

Subsequently, the pGL4-CytC (−686/+55) reporter plasmid was introduced to mouse

hepatocytes using Lipofectamine 2000 (Invitrogen) and selected with puromycin. Prior to

polyamide treatment, the mouse hepatocytes stably expressing pGL4-CytC luciferase were

seeded in six-well plates (1 × 10

cells per well) and allowed to attach for 24 h. These

cells were then treated with the indicated concentration of polyamides for another 24 h. All

experiments were repeated at least three times. Cell lysate preparation and luminescence

detection was performed according to manufacturer’s instructions (Dual-Luciferase Reporter

Assay System, Promega).

RNA interference, RNA isolation and quantitative PCR (qPCR)

siRNAs for ERR

were purchased from Santa Cruz Bio-technology (sc-44707, siERR

−1)

and OriGene (SR414015, siERR

−2). Control siRNA was from Santa Cruz Bio-technology

(sc-37007). Plasmids and siRNA were delivered using Lipofectamine 2000 according

to the manufacturer’s instruction. Overall, 4 μg of DNA or 100 pmol siRNA and

10 μl of Lipofectamine 2000 were added to cells growing at 70–90% confluence

in six-well plates in triplicate and incubated for 24 h. Total RNA was isolated

using TRIzol reagent (Invitrogen) following manufacturer’s instruction. cDNA was

produced by reverse transcription from RNA samples using the Reverse Transcription

System (Promega). Quantitative PCR was performed using SYBR green qPCR mix

(Thermo) and 7900 HT fast real-time PCR system (Applied Biosystems). Gene-specific

primers used for qPCR: mouse CytC forward 5

′

-CCAGTGCCACACCGTTGAA-3

′

and reverse 5

′

-TCCCCAGATGATGCCTTTGTT-3

′

. Mouse GAPDH: forward 5

′

GCACAGTCAAGGCCGAGAAT-3

′

and reverse 5

′

-GCCTTCTCCATGGTGGTGAA-3

′

Human CytC forward 5

′

-TCAGGCCCCTGGATACTCTT-3

′

and

reverse 5

′

-GCTATTAAGTCTGCCCTTTCTTCC-3

′

. Human GAPDH 5

′

GAAGGTGAAGGTCGGAGTC-3

′

and reverse 5

′

-GAAGATGGTGATGGGA TTTC-3

′

Western blot

Cell lysates were prepared in lysis buffer (50 mM Tris-HCl pH 7.4, 1 mM EDTA, 150 mM

NaCl, 1% NP40, 5 mM NaF, 0.25% sodium deoxycholate and 2 mM NaVO3) supplemented

with phosphatase inhibitors and protease inhibitors (Roche). Protein electrophoresis and

Western blotting were performed using Mini Trans-Bolt Cell system (Bio-Rad). Blots

were probed with anti-ERR

(ab76228, Abcam) and anti-

-actin (a2228, Sigma-Aldrich)

antibodies. The protein blot membranes were visualized with ECL Western Blotting

Substrate (Thermo Fisher Scientific) and images were taken with X-ray film or ChemiDoc

Imaging System (Bio-Rad).

Seahorse oxygen consumption rate (OCR) assay

Cells treated with polyamides or transfected with siERR

were harvested and replated (2

× 10

and 4 × 10

cells per well for mouse hepatocytes and human hepatocytes Huh7,

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respectively) on XF24 cell assay plates (Agilent technologies). After being allowed to

attach for 16 h, cells were washed twice with Mito Assay Medium and preincubated in a

non-CO

incubator for 1 h before the assay. During the Seahorse OCR assay, four baseline

respiration rates were recorded for each condition followed by sequential injections of the

four mitochondrial inhibitors, oligomycin (1 μM), FCCP (1 μM), and rotenone/antimycin A

(1 μM) to measure OCR.

Cell proliferation assay

Cells were seeded (1 × 10

cells per dish) onto 60 mm dishes with complete medium

supplemented with polyamides. Medium and polyamides were replaced daily. To measure

cell proliferation rate, the cells were completely trypsinized and counted using a

hemocytometer for five consecutive days. Each experiment was repeated at least three times.

Statistical analysis

Data in this study are presented as mean ± standard error of the mean. For multiple

group comparisons, multivariance ANOVA was used to determine if there were intergroup

differences. This was followed by the post hoc Tukey’s test. For two-group comparison,

data were analyzed by the two-tailed Student’s

test.

values < 0.05 and

p <

0.01 were

considered statistically significant and denoted by * and **, respectively.

Results and discussion

Inhibition of ERR

DNA-binding activity by a Py-Im polyamide

To inhibit transcription activation by ERR

, we sought to inhibit the binding of ERR

ERREs. We used a Py-Im polyamide that was previously shown to inhibit the binding of the

estrogen receptor to the estrogen-response element, which comprises two half-sites that bear

resemblance to the ERRE sequence of 5

′

-AAGGTCA-3

′

(Fig. 1) (

Nickols et al. 2013

). In

addition to Py-Im polyamide 1, which targets the ERRE, we also included Py-Im polyamide

2, which targets the sequence 5

′

-WGWWCW-3

′

, as a mismatch control (Fig. 2).

To evaluate the ability of the two Py-Im polyamides to inhibit ERR

binding to DNA

oligos containing the consensus ERRE, we employed the EMSA assay (Fig. 3). In this

assay, the biotin-labeled DNA probe containing ERRE (ERRE dsDNA) was incubated alone

(lane 1) or with nuclear extract from cultured hepatocytes that over-expressed ERR

protein

(lane 2). As expected, a band migrating at higher molecular weight than the probe alone

(lane 1) was observed when nuclear extract was incubated with the free probe (lane 2),

indicating the complex of ERRE dsDNA with ERR

(ERR

+ ERRE). When unlabeled

competitor (cold probe) was added in lane 3, the cold probe displaced the biotin-labeled

probe and diminished the observed ERR

+ ERRE complex band, suggesting that the upper

band observed in lane 2 was indeed complexed with the ERRE dsDNA probe. When the

monoclonal antibody for ERR

was added (lane 4), the antibody bound to the complex of

ERR

+ ERRE and caused further upwards shift of the complex due to increased overall

molecular weight, suggesting that the complex indeed contained the ERR

protein. For

lanes 5 to 10, increasing dose of polyamide 1 (10 pM to 1 μM in log scale) was added to

the lysate and probe mixture. The data showed that incubation with polyamide 1 at 0.1 μM

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concentration and beyond completely blocked the binding of ERR

to ERRE dsDNA as

demonstrated by the diminishing band of ERR

+ ERRE in lanes 9 and 10. In contrast, a

tenfold higher dose of polyamide 2 (1 μM) was needed before any loss of complex formation

was observed (lane 16). This result suggested that polyamide 1 was able to disrupt the

ERR

–ERRE complex and establishes a dose range for subsequent evaluations.

Polyamide 1 inhibits ERR

transcriptional activity

To evaluate the ability of polyamide 1 to inhibit ERR

-mediated transcriptional activity,

we utilized a dual-luciferase reporter assay where the transcription of firefly luciferase

was controlled by ERR

. CytC is a component in mitochondrial OXPHOS, and the CytC

promoter contains an ERRE under the transcriptional regulation of ERR

(

Schreiber et

al. 2004

). A pGL4-CytC construct containing a promoter of CytC from codon −686 to

+55 was introduced to and stably expressed in mouse hepatocytes (

Li et al. 2013

). In the

CytC-driven luciferase expressing hepatocytes, treatment with 1 μM polyamide 1 reduced

the ERR

-dependent luciferase expression by 55%. Comparatively, cells treated with 0.1

and 1 μM polyamide 2 showed no significant reductions (Fig. 4).

Next, we tested the endogenous expression of CytC in response to polyamide 1 treatment

in human and mouse hepatocytes. In human hepatocellular carcinoma SNU398 cells and

mouse hepatocytes (

Li et al. 2013

) where high ERR

expression was found, we showed that

polyamide 1 significantly reduced the expression of CytC (Fig. 5a). This observation was

consistent with the effects of ERR

knockdown using the siRNA approach (Fig. 5b). Taken

together, these results demonstrate the ability of polyamide 1 to modulate ERR

-mediated

expression of CytC.

ERR

-targeting reduces OCR

In mammalian cells, ATP is generated through mitochondrial OXPHOS. ERR

were

initially characterized as a transcriptional activator that promoted mitochondria biogenesis

and respiration in collaboration with PGC1-

(

Luo et al. 2017

). Thus, inhibiting

ERR

function in cells is expected to decrease mitochondrial respiration, hence oxygen

consumption of the cells. We used the Seahorse XF24 analyzer to measure OCR in cultured

cells in order to assess polyamide 1 as an ERR

inhibitor in real time. Mitochondrial

respiration was monitored as basal, ATP production-linked, maximal, and proton leak-linked

OCR with the use of four mitochondrial inhibitors. Oligomycin blocked ATP synthase,

thus allowing measurement of ATP production-linked OCR (

Plitzko et al. 2017

). The

mitochondrial OXPHOS uncoupler, FCCP, permeabilized the inner mitochondrial membrane

for protons and allowed maximum electron flux through the electron transport chain (

Dranka

et al. 2011

). Thus, the OCR measured after FCCP treatment represented the maximal

mitochondrial respiratory capacity of the cells. Antimycin A and rotenone together inhibited

complexes I and III, respectively (

Chen et al. 2003

). Any OCR measured after treatment

with rotenone and antimycin A are nonmitochondrial based oxygen consumption.

To assess the effect of polyamide 1 on ERR

-regulated mitochondrial respiration, cells were

pretreated with polyamide 1 or siRNA against ERR

for 24 h followed by 16 h incubation

before the OCR assay run (Fig. 6a). In mouse hepatocytes, the introduction of siERR

1 and

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2 that target different sequences of ERR

gene led to 70 and 60% reduction of basal and

maximal respiration, respectively (Fig. 6b). In the dose-dependency experiment, polyamide

1 treatment at 0.2 μM led to 52 and 54% reduction in basal and maximal respirations,

respectively; treatment of poly-amide 1 at 1 μM led to 70 and 72% reduction in basal and

maximal respiration, respectively (Fig. 6c). This effect of polyamide 1 on the mitochondrial

respiration was comparable with that induced by siRNA treatment targeting ERR

. On the

other hand, the same concentration of the mismatched polyamide control (polyamide 2, 1

μM) only caused 9 and 10% reduction on the basal and maximal respiration, respectively

(Fig. 7a). Polyamide 2 at 1 μM also has little impact on the protein levels of ERR

(Fig.

7b) or endogenous expression of CytC expression (Fig. 7c). Furthermore, the effect of

polyamide 1 is more potent than that of XCT-790, an inverse agonist for ERR

(

Willy et al.

2004

;

Eskiocak et al. 2014

), on reducing the mitochondrial OCR.

Polyamide 1 inhibits cell proliferation

Recent clinical oncology studies show that elevated ERR

expression is significantly

associated with unfavorable clinical outcome and increases the risk of recurrence in human

cancers including prostate and liver cancer, and ERR

may serve as a potent predictive

biomarker of cancer therapy (

Fujimura et al. 2007

;

Xia et al. 2019

). We determined cell

growth rate in mouse and two cancer cell lines by counting cell numbers for five consecutive

days with sustained polyamide treatment (Fig. 8). In both mouse and human hepatocytes,

polyamide 1 treatment led to a dose dependent reduction in cell number as compared to

vehicle and polyamide 2 control (Fig. 8 top and middle). The observed inhibition was

sustained throughout the entire duration of the experiment. In addition to hepatocytes,

incubation with polyamide 1 in the high ERR

expression human prostate cancer lines

C4–2b (

Cheung et al. 2005

) resulted in similar growth repression after 5 days of treatment

(Fig. 8 bottom). These findings were consistent with the observation that XCT-790 treatment

in HepG2 hepato-carcinoma cells and its multidrug resistant subline led to apoptosis (

Wu et

al. 2009

Conclusions

ERR

is a pivotal transcriptional factor that coordinates mitochondrial and lipid homeostasis

by mediating the expression of genes vital for maintaining mitochondrial function and lipid

metabolism (

Vega and Kelly 1997

;

Eichner and Giguere 2011

). Here, we evaluated a Py-Im

polyamide as a potential inhibitor of ERR

. Our data indicated that polyamide 1 blocked

the ERR

binding to the consensus ERRE sequence that is necessary for transcriptional

activation (

Sladek et al. 1997

). We demonstrated that polyamide 1 treatment is comparable

with siRNA knockdown at downregulating ERR

-mediated transcription, and at inhibiting

the primary biological effects governed by ERR

, i.e., mitochondrial function. Finally, we

showed that polyamide 1 inhibits cell growth/survival. Whether this effect was dependent on

its function on mitochondria and lipid metabolism remains to be further explored.

Acknowledgements

BLS was supported by R01CA154986. This work is partially supported by a technology development grant from

the University of Southern California.

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Fig. 1.

DNA-binding motif sequence for nuclear receptor ERR

. Sequence logos of consensus

DNA-binding sites, designated as estrogen-related receptor response element (ERRE). The

consensus sequence of ERR

is variable with a number of possible bases at certain

positions in the motif, whereas other positions have a fixed base. The height of the letter

represents how frequently that nucleotide is observed in that position. AAGGTCA is the

most commonly occurring ERRE, for ERR

. The logos were generated from information

obtained from the MEME database (

meme-suite.org

)

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Fig. 2.

Structure of the two polyamides that target the ERRE site. Polyamide 1 and 2 are consisted

of two polyamide strands covalently linked by a

-aminobutyric acid (

-turn) linker. Within

this structure design, the Py/Im pair target C/G; Im/Py pair target G/C; Py/Py target A/T

or T/A. The

-turn linker has demonstrated selectivity for A, T over G, C. For each panel:

top, chemical structure of the polyamides; bottom, the ball-and-stick models with the target

sequence of polyamides shown together. Solid circle, imidazole; Open circle, pyrrole. IPA

isophthalic acid

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Fig. 3.

Polyamide 1 reduces ERR

binding to the ERRE motif. Electromobility shift assay

performed with the polyamide 1 and polyamide2. From left, lane 1, biotin-labeled

ERRE dsDNA only; lane 2, labeled ERRE dsDNA (10 nM) + nuclear extract containing

overexpressed ERR

; lane 3, unlabeled competitor added; lane 4, antibody for ERR

(ERR

-Ab) added; lanes 5–10, increasing dose (10 pM to 1 μM in log scale) of polyamide

1; lanes 11–15, increasing dose (10 pM to 1 μM in log scale) of polyamide 2. Arrows point

to the complex of ERR

+ ERRE or ERR

+ ERRE + antibody used for ERR

detection

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Fig. 4.

Polyamide 1 suppresses the ERR

-dependent luciferase expression. Mouse hepatocytes

stably expression cytochrome

-driven luciferase were treated with polyamide 1 or 2 for

24 h at indicated dose. The cells were then lysed and used to determine the luciferase

activity using a luminometer. Polyamide 1 treatment at 1 μM reduced the luciferase activity

whereas polyamide 2 did not. Neg con, the mouse hepatocyte without luciferase expression

vector; Naive, untreated cells expressing the luciferase reporter; Veh con, cells expressing

the luciferase reporter treated with vehicle.

= 3. ** statistically significant at

< 0.01 when

compared to vehicle control group

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Fig. 5.

Polyamide 1 suppresses the ERR

downstream gene expression.

Polyamide 1 treatment

reduced the ERR

downstream target cytochrome

(CytC) gene expression. Mouse and

human (SNU398) hepatocytes were treated with polyamide 1 for 24 h at 0.2 and 1 μM.

Cells were then lysed and mRNA prepared for qPCR analysis of CytC expression.

siERR

introduction reduced expression of CytC. siR NAs designed for ERR

was introduced to

mouse and human hepatocytes. After 24 h, cells were lysed, mRNA was prepared and used

for qPCR analysis to measure relative CytC expression. Top, mRNA expression of CytC;

bottom, immunoblotting analysis shows protein expression of ERR

was inhibited with the

introduction of siERR

= 3; *

< 0.05; **

< 0.01 difference between experimental

and control groups. SNU398, a human hepatocyte cells line. Actin was detected as loading

control for immunoblotting analysis

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Fig. 6.

Polyamide 1 dose-dependently suppresses the ERR

-regulated mitochondrial respiration

similar to siERR

Treatment protocol. Polyamide or siRNA was introduced to mouse

hepatocytes and human hepatocytes Huh7 to inhibit ERR

activity. After 24 h of the

treatment, cells were harvested and plated into Seahorse oxygen consumption rate (OCR)

assay plates and allowed for growth for 16 h. The cellular OCR date was then recorded in

real-time for a duration of ~2 h.

siERR

robustly inhibited OCR in mouse hepatocytes.

Two independent siRNAs for ERR

were used to inhibit the expression of ERR

. Both

siERR

−1 and siERR

−2 led to decreased OCR in mouse hepatocytes. siScr, scrambled

siRNA as control.

Mouse hepatocytes were treated with indicated dose of polyamide 1

for 24 h before assaying for OCR. Polyamide 1 treatment dose-dependently inhibited OCR

in mouse hepatocytes. Con, vehicle-treated cells.

Top, real-time plots for OCR levels,

each data point was replication of four readings for each sample with three samples each

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group for each experiment; bottom left, the basal respiration was calculated by averaging

the baseline OCR minus the nonmitochondrial OCR; bottom right, the maximal respiratory

capacity was calculated by averaging the maximal OCR minus the nonmitochondrial OCR.

Oligomycin, an ATPase inhibitor; FCCP, a mitochondrial uncoupler; antimycin A and

rotenone, inhibitors of complexes I and III respectively of the electron transport chain.

Arrows indicate time points when these chemicals were added.

= 3. **

< 0.01 difference

between experimental and control groups

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Fig. 7.

Specificity for Polyamide 1 at reducing mitochondrial respiration.

The effect of polyamide

1 (1 μM) was compared with that of the mismatch polyamide 2 (1 μM) and an ERR

inverse agonist, XCT-790 (2 μM). Con, vehicle-treated cells. Arrows indicated time points

when these chemicals were added.

= 3. Top, real-time plots for OCR levels, each data

point was replication of four readings for each sample with three samples each group for

each experiment. Bottom left, the basal respiration was calculated by averaging the baseline

OCR minus the nonmitochondrial OCR; bottom right, the maximal respiratory capacity was

calculated by averaging the maximal OCR minus the nonmitochondrial OCR.

Comparison

of ERR

levels in Huh7 cells treated with polyamide 1 vs. polyamide 2 and controls. **

< 0.01 difference between experimental and control groups.

Comparison of cytochrome

(CytC) gene expression in Huh7 cells treated with polyamide 1 vs. polyamide 2 and controls

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