Echinomycin inhibits adipogenesis in 3t3-l1 cells in a hif-independent manner

ABSTRACT Obesity is a risk factor for many diseases including diabetes, cancer, cardiovascular disease, and chronic kidney disease. Obesity is characterized by the expansion of white adipose

tissue (WAT). Hypertrophy and hyperplasia of adipocytes cause tissue hypoxia followed by inflammation and fibrosis. Its trigger, preadipocyte differentiation into mature adipocytes, is

finely regulated by transcription factors, signal molecules, and cofactors. We found that echinomycin, a potent HIF-1 inhibitor, completely inhibited adipogenesis in 3T3-L1 WAT preadipocytes

by affecting the early phase of mitotic clonal expansion. The dose required to exert the effect was surprisingly low and the time was short. Interestingly, its inhibitory effect was

independent of HIF-1 pathways. Time-course DNA microarray analysis of drug-treated and untreated preadipocytes extracted a major transcription factor, CCAAT/enhancer-protein β, as a key

target of echinomycin. Echinomycin also inhibited adipogenesis and body weight gain in high fat diet mice. These findings highlight a novel role of echinomycin in suppressing adipocyte

differentiation and offer a new therapeutic strategy against obesity and diabetes. SIMILAR CONTENT BEING VIEWED BY OTHERS GRK5 IS REQUIRED FOR ADIPOCYTE DIFFERENTIATION THROUGH ERK

ACTIVATION Article Open access 21 January 2025 IL-27 ALLEVIATES HIGH-FAT DIET-INDUCED OBESITY AND METABOLIC DISORDERS BY INHIBITING ADIPOGENESIS VIA ACTIVATING HDAC6 Article Open access 19

March 2025 HMGB2 ORCHESTRATES MITOTIC CLONAL EXPANSION BY BINDING TO THE PROMOTER OF C/EBPΒ TO FACILITATE ADIPOGENESIS Article Open access 02 July 2021 INTRODUCTION Obesity is a global

health burden and serves as a significant risk factor for many diseases such as diabetes, hypertension, cancer, cardiovascular disease, and chronic kidney disease1, 2. Expansion of white

adipose tissue (WAT), which is caused by the proliferation and differentiation of white adipocytes, is responsible for obesity development. Elucidating the underlying mechanism and

abrogating it have been of great interest in this field. Obesity exacerbates tissue hypoxia due to increased adipocyte oxygen consumption and insufficient angiogenesis3, 4. Hypoxia inducible

factor (HIF)-1, the master regulator of cellular adaptation to hypoxia consisting of α and β subunits, is induced early in the course of diet-induced obese WAT4 and contributes to glucose

intolerance and insulin resistance4, 5. Previous reports on genetic deletion of HIF-1 have both positive and negative effects on adipogenesis of WAT4, 5. Echinomycin is a cyclic peptide

belonging to a family of quinoxaline antibiotics isolated from _Streptomyces echinatus_ 6. It reversibly intercalates into double-stranded DNA sequences such as 5′-CGTACG, 5′-[d(ACGTACGT)2]

or 5′-[d(TCGATCGA)2]. The target sequence includes a hypoxia responsive element (HRE) sequence, making echinomycin a potent HIF-1 inhibitor7, 8. Antitumour activity was confirmed in several

preclinical studies, which led to a phase II clinical trial of echinomycin in metastatic soft tissue sarcoma patients9. We decided to investigate the effect and mechanism of echinomycin as

an HIF-1 inhibitor of WAT adipogenesis in 3T3-L1 cells, a preadipocyte cell line that is widely used as a model of adipocyte differentiation with a well characterized adipogenic cascade1, 2,

as well as in high fat diet (HFD) mice that is commonly used as an obesity model. RESULTS ECHINOMYCIN INHIBITS ADIPOGENESIS IN 3T3-L1 CELLS The conventional hormone cocktails containing MDI

lead 3T3-L1 preadipocytes to robustly differentiate into mature adipocytes as shown by a substantial amount of lipid accumulation in mature adipocytes in oil red O staining (Fig. 1A and B).

To investigate the effect of echinomycin on adipocyte differentiation, 3T3-L1 preadipocytes were treated with various concentrations and/or durations of echinomycin simultaneously with MDI

(Fig. 1B). Echinomycin inhibited adipogenesis in a time- and dose-dependent manner. Even under re-stimulation with MDI 6 hr after MDI plus echinomycin treatment, cells failed to

differentiate into mature adipocytes (Fig. 1B _left upper panel_). Treatment with 6 nM for 6 hr was sufficient to fully inhibit adipogenesis, and we used this condition in the following

studies unless otherwise indicated. This treatment concentration and time is within the range of previously reported usage of echinomycin in cancer cells (e.g., U-251 and HeLa cells)7, 8,

10. Accordingly, the altered expression levels of mature adipogenic marker genes such as fatty acid binding protein 4 (_Fabp4_), peroxisome proliferator-activated receptor γ (_Pparg_), and

CCAAT/enhancer-binding protein α (_Cebpa_) were blunted by echinomycin treatment (Fig. 1C). On the other hand, the addition of echinomycin from 24 hr after induction was insufficient to

abolish adipocyte differentiation (data not shown), which suggested that echinomycin exerts its effect on the early stage of adipogenesis. ECHINOMYCIN INHIBITS ADIPOGENESIS INDEPENDENTLY OF

HIF ACTIVITY Echinomycin intercalates DNA and binds to sequences including HRE sequences, that is, GCGTG or ACGTG, inhibiting HIF transcriptional activity. In 3T3-L1 cells, 6 nM echinomycin

treatment significantly reduced the increased HREluc reporter activity from MDI treatment (Fig. 2A), as well as the expression levels of representative HIF-1 target genes such as hexokinase

2 (_Hk2_), vascular endothelial growth factor A (_Vegfa_) (Fig. 2B), and glucose transporter 1 (GLUT1) (Fig. 2C). Although HIF-1α was induced at 2 hr after MDI treatment, which was probably

a PI3K/AKT-mediated response to insulin, this induction was very modest compared with that by hypoxic stimuli (Fig. 2D). Although echinomycin is a DNA intercalater, it also affects the

expression level of HIF-1α protein depending on the cell line11; in 3T3-L1 cells, echinomycin reduced HIF-1α protein expression (Fig. 2D _left panel_). HIF-2α was not induced at this early

time point (Fig. 2D _right panel_), which is in accordance with previous reports that HIF-2α is induced in much later phase of adipogenesis12, 13. These results demonstrate that the

conventional effect of echinomycin as a HIF-1 inhibitor both in function and quantity also applies to 3T3-L1 cells. On the other hand, hypoxia (1% O2) inhibited adipocyte differentiation,

which was demonstrated by Oil red O staining and its quantification (Fig. 2E). qRT-PCR for the mature adipocyte marker genes (e.g., _Pparg_, _Fabp4_, and _Cebpa_) at day 8 after MDI exposure

also remained low under hypoxic condition (Fig. 2F). Previous studies have also reported that 3T3-L1 cells with knockdown of HIF-1α undergo adipogenic differentiation even under hypoxia,

which implies that HIF-1 functions as an inhibitory signal in the cascade12, 14. Thus, if an effect of echinomycin on adipogenesis is mediated by the HIF pathway, echinomycin should have

stimulated adipogenesis. Our results suggest that echinomycin inhibits adipogenesis independently of HIF pathways. TRANSCRIPTOME ANALYSIS OF EARLY ADIPOGENESIS WITH OR WITHOUT ECHINOMYCIN To

investigate the molecular mechanism that underlies the effect of echinomycin on adipogenesis, gene expression profiling of 3T3-L1 cells during adipogenesis was performed with DNA

microarrays at times 0, 2, 6, and 24 hr following treatment either with MDI plus DMSO or MDI plus echinomycin (6 nM for 6 hr). First, the 2,204 gene set probes that increased more than

2-fold compared with 0 hr among the time-course DMSO-treated control groups were selected and clustered into three groups (Fig. 3A and Table S1). Different subsets of genes responded at

different time points to MDI stimulation, consistent with previous reports15; cluster 1 with genes responded as early as 2 hr after MDI treatment [e.g., CCAAT/enhancer-binding protein

(_Cebp_) _b_, _Cebpd_, Kruppel like factor (_Klf_) _4_, _Klf5_, and _Klf9_]; cluster 2 with genes such as fos-like antigen 1 (_Fosl1_) and integrin subunit alpha 5 (_Itga5_); and cluster 3

with genes such as a disintegrin, metallopeptidase domain 8 (_Adam8_) and hepatocyte growth factor (_Hgf_). To focus on the gene expression profiles and their changes at the very early phase

of adipogenesis, we compared gene expression profiles of cells treated with MDI plus DMSO and MDI plus echinomycin at 2 hr and 6 hr after induction. The 14,005 probes were distributed in

all of the four quadrants at either 2 hr or 6 hr, which signified that some of the genes are even upregulated under echinomycin treatment, ruling out the possibility that echinomycin

globally suppresses gene transcription (Fig. 3B). The microarray analysis was validated by qRT-PCR for representative genes in adipogenesis (e.g., _Cebpb_, _Cebpd_, _Klf4_, and _Klf5_) (Fig.

 3C). The probes induced in both the 2 hr- and 6 hr- MDI plus DMSO treated groups more than 2-fold compared with 0 hr were extracted (Fig. 3B-c). The resulting 564 probes with annotation

(out of 592 probes) were further clustered by centred average linkage (Fig. 3D and Table S2). Because a 2 hr treatment with echinomycin was insufficient to inhibit adipogenesis (Fig. 1B),

expression of the target genes of echinomycin was expected to be suppressed at both 2 hr and 6 hr. The cluster that met this criterion included 111 probes (Fig. 3D and Table S2). C/EBPΒ AS A

KEY TARGET OF ECHINOMYCIN During early differentiation (24–48 hr following MDI treatment), 3T3-L1 cells are known to re-enter the cell cycle and undergo two to three cycles of clonal

expansion before terminal differentiation1, 16, 17. The fact that re-stimulation with MDI 6 hr after echinomycin treatment failed to induce adipocyte differentiation supports the hypothesis

that echinomycin inhibited this critical phase of clonal expansion. We observed that cell proliferation was inhibited in an echinomycin-dose-dependent manner in this period, supporting our

hypothesis (Fig. 4A and B). Gene ontology analysis of the above 111 probes by DAVID also showed significant enrichment (enrichment score 4.65, _P_ < 4.5 × 10−7) in transcriptional

regulation (Fig. 4D). Representative genes included _Cebpb_, _Klf5_, _Klf9_, B-cell lymphoma 3-encoded protein (_Bcl3_), Glis Family Zinc Finger 1 (_Glis1_), _Smad6_, and _Etv6_. These

results suggest that the key transcription factors critical in the adipogenic cascade are targets of echinomycin. Three members of the C/EBP family, C/EBPβ, C/EBPδ and C/EBPα, play crucial

roles in adipocyte differentiation18, 19. In the very early phase of adipogenesis, C/EBPβ and C/EBPδ are induced, and these in turn activate PPARγ and C/EBPα, which play central roles in the

rest of the regulatory cascade in adipocyte differentiation20,21,22. Mice lacking C/EBPβ display reduced depots of WAT, a phenotype exacerbated in C/EBPβ−/−/C/EBPδ−/− mice23. Whereas

_Cebpb_ was extracted as one of the top candidate target genes of echinomycin, the expression level of _Cebpd_ did not change following echinomycin treatment (Fig. 3C and Table S2). Other

adipogenic cascade regulators such as KLF5 and KLF9, which are listed as candidate genes in Fig. 4D, are known to be downstream of C/EBPβ and thus are not expected to be a direct target of

echinomycin. In addition, genes upstream of _Cebpb_ in the cascade, such as _Creb_, _Klf4_, and _Krox20_, were not affected by echinomycin treatment either (Figs 3C and 4C, and Table S2)24.

Enhanced C/EBPβ protein expression was suppressed by echinomycin continuously from 6 hr after adipogenic induction (Fig. 5A). The result that 2 hr treatment with echinomycin was insufficient

to inhibit adipogenesis (Fig. 1B), and the fact that the expression of C/EBPβ in this period is a prerequisite for mitotic clonal expansion in adipogenic differentiation, both support the

hypothesis that C/EBPβ is the target. MDI treatment increased the activity of the mouse _Cebpb_ promoter, which was significantly decreased by echinomycin treatment, which indicated that

echinomycin binds to _Cebpb_ promoter and suppresses its transcription (Fig. 5B). To rule out the possibility that C/EBPβ induction by MDI stimuli is dependent on HIF-1, loss-of-function

mutations of HIF-1 transcriptional activity was introduced to 3T3-L1 cells through CRISPR/Cas9 system25. CRISPR/Cas9-mediated stable knockout 3T3-L1 cells of the _Hif1a_ gene was generated

by puromycin screening and selecting positive clones using the HREluc assay (Fig. 5C _left panel_). Knockout of _Hif1a_ did not suppress the induction of C/EBPβ under MDI stimuli, which

indicates that C/EBPβ induction is independent of HIF-1, and that the effect of echinomycin on C/EBPβ is not mediated by HIF-1. In addition, 3T3-L1 cells with retrovirus transduction of

C/EBPβ (Fig. 5D-a) escaped from the suppression of adipogenesis by echinomycin (Fig. 5D-b). These results strongly point to C/EBPβ as the main target of echinomycin. ECHINOMYCIN INHIBITS

ADIPOGENESIS _IN VIVO_ To further investigate the effect of echinomycin on adipogenesis _in vivo_, male C57BL/6Jcl mice were fed with high-fat diet (HFD) for 2.5 weeks. Echinomycin was

intraperitoneally injected at two doses and frequency for the entire period based on the previous reports in mice and humans26, 27: 10 μg/kg for 5-days on/2-days off (EC-Low), or 50 μg/kg

every alternate day (EC-High). These regimens are much milder than those used in human clinical trials for anti-cancer treatment. Each regimen effectively inhibited the HIF-1 activity (shown

by the qRT-PCR for _Vegfa_) in the epididymal white adipose tissue (eWAT) of both normal fat diet (NFD) and HFD mice, which demonstrated that echinomycin was effectively delivered to eWAT

(Fig. 6A). Each echinomycin treatment inhibited the body weight gain (Fig. 6B), without effects on the food intake (data not shown). eWAT/gBW and qRT-PCR of the established HFD-induced

gene28, _leptin_, were analysed among each group, which confirmed that echinomycin inhibited adipogenesis _in vivo_ (Fig. 6C and D). H&E staining of eWAT accordingly demonstrated that

echinomycin efficiently suppressed the adipocyte size in HFD mice (Fig. 6E). These results are in accordance with the _in vitro_ results. DISCUSSION Our current study demonstrates that

echinomycin, a DNA intercalater, completely inhibits adipogenesis in the 3T3-L1 adipocyte cell line of WAT, as well as in eWAT of HFD mice. A DNA transcriptome array extracted one of the

essential adipogenic transcription regulators, C/EBPβ, as its major target (Fig. 7). An advantage of this small molecule is that a relatively small dose and short-term treatment are

sufficient to exhibit its effect. Echinomycin is traditionally known as a HIF-1 inhibitor7, 8. It binds to HRE sequences and decreases HIF-1 transcriptional activity. Whether echinomycin

affects the expression of HIF-1α protein depends on the cell line; in U251, HepG2, and HeLa cells, HIF1-α protein level is not affected7, 8, 10; in MCF-7 cells, it is decreased11. In 3T3-L1

cells, echinomycin inhibited HIF-1α protein expression and HIF-1 activity. The _in vivo_ functional role of HIF-1 in adipogenesis of WAT is controversial. Some reported that

adipocyte-specific genetic deletion of HIF-1α protects obese mice from insulin resistance and inflammation, whereas constitutively active expression of HIF-1α results in increased insulin

resistance and tissue fibrosis3. In these knockout mice, white adipose tissue mass and mean adipocyte size are increased4, 12, which implies that HIF-1 in the end inhibits adipogenesis in

WAT. Others, on the other hand, reported the opposite results using the same _aP2_ promoter-driven adipocyte-specific _Hif1a_ deleted mice29, 30. HIF-1 deficiency improved HFD-induced

insulin resistance and reduced WAT adipocity. _In vitro_ studies using 3T3-L1 cells show that hypoxia inhibits adipogenesis through HIF-112, 14. Our results also demonstrate that while

HIF-1α is induced as early as 2 hr and again from 12 hr after adipogenic induction (data not shown), adipogenesis is suppressed under hypoxia. These findings suggest that although the

temporal induction of HIF-1 in the early course of adipogenesis may have some functional roles in regulating the growth arrest and differentiation of preadipocytes, it acts as an inhibitory

signal for adipogenesis. Of note, while HIF-2α is known to promote adipogenesis in 3T3-L1 cells, previous reports suggest that HIF-2α is induced at later phase of adipogenesis, from 4 day

after MDI induction12, 13. We confirmed that HIF-2α was not induced at least until 24 hr after MDI induction (data not shown), and that the inhibitory effect of echinomycin on adipogenesis

was unlikely to be mediated by HIF-2. In contrast to our original expectation, our results demonstrated that echinomycin exerted its inhibitory effect on adipogenesis independently of the

HIF pathway. Adipocyte differentiation is precisely controlled by a complex network of transcription factors, cofactors and signalling molecules. A comprehensive DNA microarray analysis was

performed, and C/EBPβ was identified as a key target of echinomycin in the adipogenic cascade. C/EBPβ is known as an indispensable adipogenic transcription factor, and ectopic overexpression

of C/EBPβ is sufficient to induce PPARγ and subsequent adipocyte differentiation. Continuous suppression of C/EBPβ was observed both at mRNA and the protein level, while C/EBPδ remained

unaffected. An _in silico_ search predicted several HRE sequences in the promoter region of _Cebpb_, which are potential binding sites for echinomycin, and the loss of this echinomycin

effect in _Cebpb_ promoters smaller than 1 kbp suggests that echinomycin inhibits the _Cebpb_ transcription and its binding site exists between 2 k and 1 k in the promoter region. None of

their mutations, however, cancelled the effect of echinomycin on the promoter activity (data not shown), which prevented us from drawing an unequivocal conclusion on its binding site. These

observations have two implications for the mechanism of action of echinomycin on C/EBPβ; Echinomycin binds to a _Cebpb_ promoter region which is yet to be identified, or, echinomycin binds

to an upstream gene of _Cebpb_, which results in the transcriptional inhibition of C/EBPβ. Although our microarray results and _Cebpb_ promoter assay favour the former story, the exact

mechanism of action of echinomycin on C/EBPβ remains to be a subject of future research. There are some other reports on inhibitory molecules against adipogenesis. Rapamycin, an inhibitor of

mammalian target of rapamycin, suppresses adipogenic differentiation through its anti-proliferative activity31. Pyridinyl imidazoles also block 3T3-L1 adipogenesis by targeting p38

mitogen-activated protein kinase32. Our study gives an additional insight into how adipogenesis could be blocked by a temporal intervention. It is also unique that it affects the very early

phase of transcriptional cascade. Taken together, our results demonstrate a novel effect of echinomycin beyond its anti-tumour activity, by acting on adipocyte differentiation. Echinomycin

inhibits adipogenesis of 3T3-L1 cells in a HIF-independent manner. Time-course DNA microarray analysis of drug-treated and untreated preadipocytes extracted a major transcription factor,

CCAAT/enhancer-protein β, as a key target of echinomycin. The effect of echinomycin also applied to eWAT of experimental obesity model, HFD mice. This study provides further molecular

insight into the spatially and temporally regulated cascade of WAT adipocytes. MATERIALS AND METHODS REAGENTS Insulin, dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX), and oil red O

were purchased from Sigma (Sigma-Aldrich, St Louis, MO). Echinomycin (Calbiochem, Gibbstown, NJ) was dissolved in DMSO (Wako, Tokyo, Japan) according to the manufacturer’s instructions. CELL

CULTURE, DIFFERENTIATION, AND DRUG TREATMENT 3T3-L1 cells (American Type Culture Collection, Rockville, MD) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nissui, Tokyo, Japan)

supplemented with 10% calf serum (CS). The differentiation protocol is as described previously33. Briefly, two days after cells reached confluence (Day 0), the medium was replaced with a

mixture consisting of 10% FCS and MDI (0. 5 mM IBMX, 1 μM DEX, and 10 μg/ml insulin). After 48 h, the medium was changed to DMEM containing 10% FCS and 10 μg/ml insulin. The medium was

replenished at 2-day intervals, and the appearance of cytoplasmic triglycerides was monitored by microscopy and confirmed by staining with oil red O. For hypoxic stimulation, cells were

exposed to 1% O2/5% CO2, with nitrogen as the balance, in a multigas incubator, APM-30D (ASTEC, Fukuoka, Japan). OIL RED O STAINING At day 8, 3T3-L1 cells were rinsed with PBS twice and

fixed with 10% formaldehyde in H2O for 10 min. The cells were treated with 60% isopropanol in H2O for 1 min and then stained in freshly diluted oil red O solution (0.18% (w/v) oil red O in

60% isopropanol) for 20–40 mins until the cells were stained. The cells were then washed with 60% isopropanol in H2O and twice with PBS. The cells were observed in PBS under a microscope and

photographed. Oil red O quantification was performed by extracting the dye by 100% isopropanol and OD 490 nm was subsequently measured in 96-well dishes. LUCIFERASE REPORTER ASSAY HIF-1

transcriptional activity (HREluc activity) was measured by dual-luciferase reporter assays (Promega, Madison, WI) in 24-well culture dishes. Five-hundred nanograms of pGL3 (Promega) vector

driven by 7× hypoxia-responsive elements (pHREluc)34 and 25 ng of _Renilla_ luciferase vector (pRL-TK or pRL-CMV) were co-transfected using Lipofectamine LTX (Invitrogen, Carlsbad, CA); 2

days after full confluence, the cells were differentiated as above and exposed to hypoxia (1% O2) for 8 hr. The cells were processed in fixed protein aliquots, and HREluc activity was

measured using a Lumat 9507 luminometer (EG and Berthold, Bad Wildbad, Germany). The relative light unit (RLU) value of firefly luciferase was divided by that of _Renilla_ luciferase to

correct for the transfection efficiency. To measure _Cebpb_ promoter activity, a similar method to the above was used. _Cebpb_ promoter fragments of 3 kb, 2 kb, 1 kb, or 0.5 kb (mCEBPB3K, 2 

K, 1 K, or 0.5 K promoter) were PCR amplified from mouse genomic DNA using PrimeStar HS DNA polymerase (Takara Bio, Shiga, Japan) and primers with _KpnI_ and _XhoI_ overhangs (see Table 1

for primers). Digested and purified fragments were inserted into the pGL3basic reporter vector (Promega). PLASMIDS AND STABLE OVEREXPRESSION 3T3-L1 clones, which stably overexpress C/EBPβ,

were generated with retrovirus transduction (Platinum Retrovirus Expression System, Pantropic, Cell Biolabs, San Diego, CA). pcDNA-mC/EBPb was a gift from Jed Friedman (Addgene plasmid

#49198). The plasmid was digested with _XhoI_ and _PmeI_, and subcloned into the retroviral vector, pMXs-IRES-Puro using the _XhoI_-_SnaBI_ site. The plasmids were transfected to the

packaging cell line, and the culture supernatants were collected 24 and 48 hr later, passed through 0.22-μm filters, and added to the cells. Drug selection by 2 μg/ml puromycin was initiated

72 hr after infection. For CRISPR-Cas9 system, pX330-U6-Chimeric_BB-CBh-hSpCas9 was a gift from Feng Zhang (Addgene plasmid #42230)24. Hif1a target sgRNAs were designed by using

CRISPRdirect35, and ligated into pX330 plasmid by following the procedures from Zhang lab (http://www.genome-engineering.org/crispr). Sequences are listed in Table 1. 3T3-L1 cells were

co-transfected with the two pX330-Hif1a and a puromycin resistant plasmid which facilitates selection with puromycin (2 μg/ml). Selected colonies went further selection by HREluc activity

under hypoxia. MICROARRAY ANALYSIS A transcriptome microarray analysis of 3T3-L1 cells was conducted at 0 hr, 2 hr, 6 hr, and 24 hr of treatment with either MDI plus DMSO or MDI plus

echinomycin (6 nM). For the 24-hr samples, cells were washed and re-incubated with MDI 6 hr after induction. Total cellular RNA was isolated using RNAiso plus (Takara Bio). cRNA was

synthesized from cellular RNA and hybridized to high density Affymetrix microarray gene chips containing 45,102 probe sets. The expression value for each mRNA was obtained using the Robust

Multi-array Analysis (RMA) method. Probes with normalized intensity lower than 100 in all seven arrays (31,097 probes) were excluded from the analyses, which left 14,005 probes for further

analysis. To identify the transcripts that were increased by MDI treatment, all probes with a fold change >= 2.0 in expression (either 2, 6, or 24 hr after MDI + DMSO treatment) compared

with the control (time 0 hr) were selected (2,204 probes), and hierarchical clustering analysis was performed using the average linkage and relative correlation as a measure of similarity

for the selected genes. To identify transcripts that are significantly induced by MDI and suppressed both at 2 hr and 6 hr after echinomycin treatment, the following selection procedures

were performed. All probes that had a fold change >= 2.0 in expression compared with the control (time 0 hr) at both two time points, 2 hr and 6 hr, were first selected (592 probes).

After excluding probe sets that did not have gene annotation, the remaining 564 probes were used for further analyses. Hierarchical clustering analysis of a log 2-fold change from the

control (time 0 hr) was performed using the average linkage and relative correlation as a measure of similarity for the selected genes. The Database for Annotation, Visualization and

Integrated Discovery (DAVID)36, 37, an online gene database provided by the NIH (version 6.8; http://david.abcc.ncifcrf.gov/), was used to investigate biological functions associated with

the gene lists. RNA ISOLATION AND REAL-TIME QUANTITATIVE RT-PCR (QRT-PCR) RNA isolation and real-time quantitative (q) RT-PCR assays were performed as previously described38. The data were

calibrated to the β-actin value. The primers are described in Table 1. IMMUNOBLOTTING ANALYSIS The cells were collected for immunoblotting analysis as previously described38. The primary

antibodies were as follows: anti-HIF1α (Novus Biologicals, Littleton, CO); anti-HIF2α (Novus Biologicals, Littleton, CO); anti-GLUT1 (Abcam plc, Cambridge, UK); anti-C/EBPβ (Santa Cruz

Biotechnology, Santa Cruz, CA); and anti-α-tubulin (Cell Signaling Technology, Danvers, MA). HRP-conjugated anti-rabbit IgG (Bio-Rad Laboratories, Hercules CA) or anti-mouse IgG (Bio-Rad

Laboratories) antibodies were used as secondary antibodies. The signals were detected with the ECL Plus reagent (Thermo Fischer Scientific, Waltham, MA) using the chemiluminescence protocol.

For detecting the signals of GLUT1 and HIF-2α, SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fischer Scientific) was used. ANIMAL EXPERIMENTS Male C57BL/6NJcl mice aged 7

weeks (Nippon Seibutsu Zairyo Center, Tokyo, Japan) were fed with either normal fat diet or high fat diet (HFD 60) containing 60% of fat (Oriental Yeas Co. Ltd., Tokyo, Japan). Echinomycin

was dissolved in DMSO at 5 mg/ml and subsequently diluted with 10 mg/ml BSA/saline to a concentration of 0.0293 mg/ml. Either 1)10 μg/kg for 5-days on/2-days off, or, 2) 50 ug/kg alternate

day, was intraperitoneally injected until the day of sacrifice. At sacrifice, the mice were euthanized and the epididymal white adipose tissue (eWAT) were harvested, measured, fixed in

formalin for immunohistochemical analysis, snap-frozen in OCT, or stored at −80 °C for qRT-PCR analysis. All mice were housed in the animal care facility of the University of Tokyo under

standardized conditions (25 °C, 50% humidity, 12 hr light/dark cycle) with food and water available at libitum. All the experiments were carried out in accordance with the guidelines and

regulations of the Committee on Ethical Animal Care and Use at the University of Tokyo Graduate School of Medicine, as well as with the guidelines of the National Institutes of Health for

the use of animals in research. All experimental protocols were approved by the animal care and use committee at the University of Tokyo Graduate School of Medicine (Approval Number:

M-P16-104). For adipocyte size measurement, eWAT was processed for paraffin embedding, and 3-μm tissue sections were stained with hematoxylin and eosin. The adipocyte areas were manually

traced and analyzed using the Image J software. STATISTICAL ANALYSIS All data are reported as the mean ± SEM or the mean ± SD. The data for two groups were analysed using Student’s unpaired

_t_ test. The differences among more than two groups were analysed using one-way ANOVA with Tukey’s post-test or two-way ANOVA with Bonferroni’s post-test. Differences with a _P_ value < 

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Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by a Grant-in-Aid for Scientific Research (C) 26461215 (T.T.), Grant-in-Aid

for Scientific Research on Innovative Areas 26111003 (M.N.), and Grants-in-Aid for Scientific Research (B) 15H04835 (M.N.) by Japan Society for the Promotion of Science (JSPS). The authors

thank Kahoru Amitani (The University of Tokyo) for technical support. The authors appreciate Hideko Nagasawa (Gifu Pharmaceutical University) and Kent Fukui (Japan Tobacco Inc.) for helpful

discussions. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Division of Nephrology and Endocrinology, The University of Tokyo Graduate School of Medicine, Tokyo, Japan Junna Yamaguchi, 

Tetsuhiro Tanaka, Hisako Saito & Masaomi Nangaku * Genome Science laboratory, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan Seitaro Nomura 

& Hiroyuki Aburatani * Department of Diabetes and Metabolic Diseases, Department of Molecular Science on Diabetes, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

Hironori Waki * Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Takashi Kadowaki Authors * Junna Yamaguchi View author

publications You can also search for this author inPubMed Google Scholar * Tetsuhiro Tanaka View author publications You can also search for this author inPubMed Google Scholar * Hisako

Saito View author publications You can also search for this author inPubMed Google Scholar * Seitaro Nomura View author publications You can also search for this author inPubMed Google

Scholar * Hiroyuki Aburatani View author publications You can also search for this author inPubMed Google Scholar * Hironori Waki View author publications You can also search for this author

inPubMed Google Scholar * Takashi Kadowaki View author publications You can also search for this author inPubMed Google Scholar * Masaomi Nangaku View author publications You can also

search for this author inPubMed Google Scholar CONTRIBUTIONS J.Y., and M.N. designed research; J.Y., and S.N. conducted the _in vitro_ experiments; J.Y., and H.S. conducted the animal

experiments; J.Y., T.T., S.N., H.A., H.W., T.K., and M.N. analysed the results; J.Y., T.T., and M.N. wrote the manuscript. All authors reviewed the manuscript. CORRESPONDING AUTHOR

Correspondence to Masaomi Nangaku. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare that they have no competing interests. ADDITIONAL INFORMATION PUBLISHER'S NOTE: Springer

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Tanaka, T., Saito, H. _et al._ Echinomycin inhibits adipogenesis in 3T3-L1 cells in a HIF-independent manner. _Sci Rep_ 7, 6516 (2017). https://doi.org/10.1038/s41598-017-06761-4 Download

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