Biochemical and Biophysical Research Communications 511 (2019) 855e861

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

A time-resolved proteomic analysis of transcription factors regulating

adipogenesis of human adipose derived stem cells
Sen Li a, Ting Xue c, Feng He b, Zhuo Liu b, Shengrong Ouyang b, Dingding Cao b,
Jianxin Wu a, b, *
a
Department of Biochemistry & Immunology, Capital Institute of Pediatrics-Peking University Teaching Hospital, NO. 2, Yabao Road, Chaoyang District,
Beijing, 100020, China
b
Beijing Municipal Key Laboratory of Child Development and Nutriomics, Capital Institute of Pediatrics, NO. 2, Yabao Road, Chaoyang District, Beijing,
100020, China
c
Omicsolution Co, Ltd, Shanghai, 201101, China

a r t i c l e i n f o a b s t r a c t

Article history: Adipogenesis is one of the key processes during obesity development. Better understanding of this
Received 15 February 2019 process could advance our knowledge on obesity and its treatment. Transcription factors (TFs) are master
Accepted 25 February 2019 regulators during adipogenesis, however, a system-wide analysis of TFs dynamic proteome during adi-
Available online 6 March 2019
pogenesis is lacking. Here, we profiled 472 TFs and systematically elucidated their roles during the first 7
days of adipogenesis of human adipose-derived stem cells (hADSCs) on proteome scale. We identified
Keywords:
two main and four sub-phases during adipogenesis. The commitment phase (0 he8 h) mainly mediated
Obesity
stem cell proliferation, differentiation and chromatin remodeling, in which proteins of SWI/SNF family
Adipogenesis
Transcription factors
are the key centroid nodes. The determination phase (1D-7D) predominately regulated fat cell differ-
Proteomics entiation and response to lipid and oxygen, which could be associated with terminal differentiation of
adipocyte and responsible for maturation. PPARg, CREB1 and MYC are the centroid nodes of this phase.
Remarkably, we identified and verified three TFs (BATF3, MAFF and MXD4) as novel regulators of adi-
pogenesis, whose over-expression could inhibit adipogenesis of hADSCs in vitro. Overall, our study
provided a valuable TFs resource to understand the complex process of adipogenesis.
© 2019 Elsevier Inc. All rights reserved.

1. Introduction expression. Since transcription factors (TFs) are master regulators

Obesity has become one of the worlds’ most important public
health problems, however, there is no effective way to reverse this
issue to date [1]. Generation of new adipocytes through differen-
tiation (adipogenesis) is one of the most striking features during
obesity development [2]. Hence, better understanding the process
of adipogenesis could advance our knowledge on obesity devel-
opment and benefit in developing promising strategies for obesity
prevention and treatment [3].
Adipogenesis is a tightly regulated cellular differentiation pro-
cess, which is accompanied by extensive reprogramming of gene
* Corresponding author.Capital Institute of Pediatrics-Peking University Teaching
Hospital, NO. 2, Yabao Road, Chaoyang District, Beijing, 100020, China.
E-mail addresses: albertleesen@bjmu.edu.cn (S. Li), xueting221314@126.com
(T. Xue), hefengfeng2001@163.com (F. He), liuzhuozhuo2005@163.com (Z. Liu),
cipbiochem@163.com (S. Ouyang), cddzlm@163.com (D. Cao), cipbiolab@163.com
(J. Wu).
to precisely control-turn on and off-genes and drive various bio-
logical processes, it is of interest to investigate their roles in adi-
pogenesis. Indeed, dozens of TFs that play important roles during
adipogenesis have been figured out through in vitro knockdown or
ectopic over-expression [4]. However, numerous studies have
shown that TFs would modulate each other and constitute complex
network to precisely guide adipogenesis [5]. Hence, systematically
studying the remodeled TFs profiles will help to better investigate
the process of adipogenesis and find out potential targets for
obesity intervention.
Researches on transcriptome have identified transcripts of TFs at
a large-scale. Wang has utilized gene expression data to analysis
TFs’ network of adipogenesis in hADSCs and 3T3-L1 cells [6].
However, the correlation of mRNA and protein expression is low,
suggesting the post-transcriptional regulation that accounts for the
proteome [7]. In addition, limited by the very low concentration of
TFs, it is a big challenge to quantitatively identify TFs at proteome
scale through general approaches [8]. Hence, there is no systematic

https://doi.org/10.1016/j.bbrc.2019.02.134
0006-291X/© 2019 Elsevier Inc. All rights reserved.
856 S. Li et al. / Biochemical and Biophysical Research Communications 511 (2019) 855e861
research of TFs on adipogenesis at proteomics level to date.
Here, based on a recently developed approach to enrich TFs by
Qin Lab [9], we first systematically quantified the dynamic prote-
ome of endogenous TFs during adipogenesis of hADSCs. Enriching
TFs by a synthetic DNA made of a concatenated tandem-array of the
consensus response elements (catTFRE) and analyzing by label-free
mass spectrometry, we finally quantitatively identified 472 TFs
during adipogenesis of hADSCs. We found that this process could be
divided into two main and four sub-stages based on the dynamic
TFs proteome. We also found and validated three TFs as novel tar-
gets to regulate adipogenesis, whose over-expression could effi-
ciently inhibit adipogenesis of hADSCs in vitro.
2. Materials and methods
2.1. Cell culture and differentiation
hADSCs purchased from ScienCell Research Laboratories were
seeded at 2
104/cm2 density and proliferated in Mesenchymal
Stem Cell Growth Media (Cyagen). Two days after confluence, cells
were induced to differentiate with hADSCs Adipogenesis Basal
Medium (Cyagen), and collected at 0 h, 0.5 h, 2 h, 8 h, 1 day, 3 day
and 7 day, respectively. Three biological replicates were conducted
for each time point. All cells were cultured at 37 Cand 5% CO2 in a
humidified atmosphere.
2.2. catTFRE pull-down and trypsin digestion
catTFRE DNA was obtained from Beijing Proteome Research
Center (Beijing, China). The details of catTFRE pull-down and
trypsin digestion could refer to their previous work [10]. Briefly,
nuclear proteins were extracted using NE-PER kit (Thermo Fisher
Scientific) according to the manufacturer's instruction. Afterwards,
2 mg of biotinylated DNA was immobilized with 40 mL of Dynabeads
(Thermo Fisher scientific), and incubated with 100 mg nuclear ex-
tracts in 4  C for 1.5 h. After incubation, the supernatant was dis-
carded, and the NETN buffer (100 mM NaCl, 20 mM Tris-HCl,
0.5 mM EDTA, and 0.5% [vol/vol] Nonidet P-40) and PBS were used
to wash the Dynabeads twice respectively. Beads were digested
overnight with 1.5 mg trypsin at 37  C in 50 mmol NH4HCO3.
2.3. LC-MS/MS using Q Exactive HF
Peptides were analyzed on a Q Exactive HF mass spectrometer
(Thermo Fisher Scientific) connected to an Easy-nLC 1000 liquid
chromatography system (Thermo Fisher Scientific). Peptides were
loaded to a trap column (100 mm
2 cm, homemade; particle size,
3 mm; pore size, 120 Å; SunChrom, USA) in Solvent A (0.1% formic
acid), and separated on a home-made 150 mm
30 cm silica
microcolumn (particle size, 1.9 mm; pore size, 120 Å; Dr. Maisch
GmbH) with a gradient of 5%e35% Solvent B (100% acetonitrile and
0.1% formic acid) at a flow rate of 600 nL/min for 150 min. MS
analysis was performed with one full scan (300e1400 m/z,
R ¼ 120,000 at 200 m/z) with an AGC of 3e6, followed by up to 30
data-dependent MS/MS scans (R ¼ 15,000 at 200 m/z) with an AGC
of 2e4, maximum injection time of 40 ms, isolation window of
1.6 m/z, and normalized collision energy of 27%. Dynamic exclusion
time was enabled at 18 s.
2.4. MS data processing
Raw MS files were managed by MaxQuant software (version
1.6.0.16). MS/MS-based peptide identification was carried out with
the Andromeda search engine in MaxQuant. Andromeda uses a
target-decoy approach to identify peptides and proteins at an FDR
<1%. As a forward database, Uniprot Human Protein Database was
used. A reverse database for the decoy search was generated
automatically in MaxQuant. Enzyme specificity was set to Trypsin,
and a minimum number of seven amino acids was required for
peptide identification. Variable modification: acetylation (Protein-
N terminus) and oxidation (methionine); fixed modification: car-
bamidomethylation (cysteine).
2.5. Overexpression of target genes and determination of
adipogenesis
Lentiviral vectors, which are over-expressing EGFP only (pLV
[Exp]-EGFP:T2A:Puro-Null), over-expressing MAFF and EGFP
(pLV[Exp]-EGFP:T2A:Puro-EF1A > hMAFF[NM_152878.1]), over-
expressing BATF3 and EGFP (pLV[Exp]-EGFP:T2A:Puro-
EF1A > hBATF3[NM_018664.2]), over-expressing MXD4 and EGFP
(pLV[Exp]-EGFP:T2A:Puro-EF1A > hMXD4[NM_006454.2] were
purchased from Cyagen Biosciences Inc (Guangzhou, China) and
were used to infect hADSCs according to manufacturer's in-
structions, respectively. EGFP expression was observed by fluores-
cence microscopy (Olympus) after infected for 48 h to evaluate the
transfection efficiency. If the efficiency of transfection was more
than 80%, the cells were split equally for subsequent determination
experiment of adipogenesis. Cells were stained by Oil red O (Sigma)
at 7 day after exposed to differentiation stimuli. Cells were visual-
ized and photographed by digital microscope (Olympus). The area
dyed by Oil red O is calculated by ImageJ (National Institutes of
Health, USA).
2.6. RT-PCT analysis for target gene expression
After infected for 48 h, cells were harvested for quantitative
real-time PCR analysis to determine gene expression. Total RNA was
extracted using TRIzol reagent (Ambion). Reverse transcription was
completed using Reverse Transcription System (promega). TOYOBO
SYBR Green Realtime PCR master mix (Toyobo) was used for
quantitative real-time polymerase chain reaction. b-actin gene was
used as the internal control.
hMAFF: F:50 -AGGAGGTGACACGGCTCAAG-3’; R:50 -CCAGCTCC-
GACTTCTGCTTC-3’.
hBATF3: F: 50 -AGACCCAGAAGGCTGACAAGC-3’;
R: 50 -GGCACTGGCACAAAGTTCATAG-3’.
hMDX4: F:50 -CATCAAGAAACTGGAGGAGCAGG-30 , R:50 -TCCGT-
GGAGACAGCAGAGCC-3’.
b-Actin: F: 50 -TGACGTGGACATCCGCAAAG-3’;
R: 50 -CTGGAAGGTGGACAGCGAGG-3’.
2.7. Bioinformatics and statistics
For label-free protein quantification, we used MaxQuant LFQ
algorithm to quantitate the MS signals, and the proteins intensities
were represented in iBAQ. For normalization, protein's intensity in
one sample was first divided by the total intensity of all identified
proteins of this sample and then multiplied by the median intensity
of summed proteins' intensities of all samples. After normalization,
proteome data was filtered to remove outlier samples until the
standard deviation of protein intensities for triplicate biological
replicates was &1.5 for each time point. Proteins identified in at
least two biological replicates were used for subsequent analysis.
Proteins in each time point was compared to proteins at 0 h. Pro-
teins with fold change j2j and p value < 0.05 were defined as
significant proteins. Biological function annotation was conducted
by GO enrichment through String platform or Ingenuity Pathway
Analysis platform (QIAGEN Redwood City). Protein-protein inter-
action network was conducted in String platform and high
 S. Li et al. / Biochemical and Biophysical Research Communications 511 (2019) 855e861
confidence (0.7) was set as requited interaction score. Pearson
correlation, PLS-DA, unsupervised hierarchical clustering, and
bubble plot analysis were handled in R package. A two-sample,
two-side student t-test was applied to compare the difference be-
tween the mean values of two groups.
3. Results
3.1. Overview of identification and quantification of TFs proteome
To systematically depict the dynamic features of TFs during adi-
pogenesis of hADSCs, we performed quantitative proteomics of
endogenous TFs across a high-density temporal course (0, 0.5 h, 2 h,
8 h, 1D, 3D, and 7D), which covered both early and later stages of
adipogenesis [5,11]. A synthetic DNA containing a concatenated
tandem array of the consensus TFREs (catTFRE) was used as an af-
finity reagent to permit efficient enrichment of TFs (Fig. 1A).
Consequently, aided by annotation in TcoF-DB v2 database (https://
tools.sschmeier.com/tcof/home/) [12], we could assign 472 of iden-
tified proteins as TFs, 285 out of which (60.38%) were found matched
TF family information in DBD database (http://dbd.mrclmb.cam.ac.
uk/DBD) and almost all TFs families were recovered [13]. TF fam-
ilies of ZF-C2H2, Homeobox, and HLH accounted for the most of
identified TFs (Fig. 1B). Our results showed good quality of triplicate
replicates with a correlation value of R ¼ 0.93 on average (Fig. 1C)
and an average of 71% overlap of protein identification in each group
(Fig. 1D). Notably, the degree of Pearson correlation and overlap of
TFs identification were decreased during the later process of adipo-
genesis, indicating some special features underlying.
3.2. TF's proteome characterized different stages of adipogenesis
We first examined the temporal features of adipogenesis via TFs
Fig. 1. Overview of TFs proteome during adipogenesis of hADSCs. (A) Illustration of hADSCs adipogenesis and workflow of catTFRE enrichment, MS-based quantitative proteomic
and bioinformatic analyses. (B) Family information for identified TFs. (C) Pearson correlation analysis on quality of biological replicates. (C) Overlap of identified TFs of triplicate
biological replicates.
857
proteomic profiles. First, supervised PLS-DA analysis was per-
formed to group samples according to their quantitative protein
profiles. Our results revealed two main and four sub-phases during
adipogenesis, in which 0 he8 h was grouped as one main phase,
and 1D to 7D belong to the other phase. We further defined 0 h as
the pre-early stage and 7D as the post-late stage, which are the
demarcation points for sub-phases (Fig. 2A). The number of iden-
tified proteins also provided similar stratified features, in which the
number of identified proteins jumped at 0.5 h, and persisted to 8 h,
while straightly dropped down from 1D to 7D (Fig. 2B). The pattern
of significant proteins showed that 8 h could be the critical point to
switch the stage of adipogenesis, in which the number of down-
regulated proteins started to increase, and reached the most at
7D (Fig. 2C). All these results indicated that proteomic profiles of
TFs could well characterize and provide more clues about the dy-
namic process of adipogenesis.
3.3. Hierarchical clustering and network analysis revealed featured
biological responses during different stages of adipogenesis
Since dynamic proteomic profiles of TFs revealed different
stages during adipogenesis, we next investigated what biological
responses were involved. Unsupervised hierarchical clustering was
conducted on significant proteins in at least one time points to
depict biological clusters. Consequently, two main clusters and four
sub-clusters were obtained (Fig. 3A). Proteins in cluster 1 pre-
dominantly kept unchanged at 0.5 h and 2 h, while increased from
1D to 7D. Proteins in cluster 2 highly expressed at 0.5 and 2 h, while
markedly decreased from 1D to 7D. Distinct patterns of 8 h in either
cluster 1 or cluster 2 further revealed that it could be the turning
point that switch the process of adipogenesis to the next stage.
Cluster 1 mainly involved in process of response to lipid and fat cell
differentiation (Fig. 3B). Network analysis of proteins in cluster 1
858 S. Li et al. / Biochemical and Biophysical Research Communications 511 (2019) 855e861

Fig. 2. TF's proteome characterized different stages of adipogenesis. (A) Partial least squares discrimination analysis (PLS-DA) of temporal proteomic data. (B) Distribution of
identified TFs in each time points. (C) Distribution of significant proteins in each time point. Each time point was compared with 0 h.

Fig. 3. Hierarchical clustering and network analysis TF's proteome. (A) Hierarchical clustering analysis of significant proteins. 221 proteins identified across all time points were
used and 160 of which were significant proteins in at least one time points (compared with 0 h). (B) GO analysis of proteins in cluster 1. (C) Protein-protein interaction network
analysis of proteins in cluster 1. (D) GO analysis of proteins in cluster 2. (E) Network analysis of proteins in cluster 2. (F) Fold changes of protein of component of SWI/SNF complex.
(G) Number of unique proteins. (H) GO analysis of unique proteins on 7D.

showed three major sub-networks, in which PPARg, CREB1 and
MYC were the centroid nodes, regulating lipid response, fat cell
proliferation and Notch signaling pathway (Fig. 3C). Proteins in
Cluster 2 mainly regulate stem cell proliferation and differentiation
(Fig. 3D). Network analysis showed that proteins, including
SMARCA1-5, SMARCC1-2, SMARCD1-3, and SMARCE1, which are
components of SWI/SNF chromatin remodeling complexes, were
the major nodes (Fig. 3E and F). These proteins regulate chromatin
remodeling, and mediate transition from proliferating stem/pro-
genitor cells to adult state. Previous evidences pointed out that
adipogenesis was composed of commitment phase and terminal
differentiation phase. During commitment phase, stem cells lose
pluripotency and become preadipocytes, and in terminal differen-
tiation phase, preadipocytes acquire some traits of mature adipo-
cytes [11,14,15]. Hence, we speculated that 0 he8 h matched the
commitment phase, which is responsible for stem cell proliferation
and differentiation, and chromatin assembly or disassembly,
accompanying the acquisition of lineage-committed cellular
phenotype [11,15]. 1D to7D matched the terminal differentiation,
which response to lipid and regulate fat cell differentiation, facili-
tating to get the characteristics of mature adipocyte.
3.4. Time restricted biological response revealed by TFs proteome
To examine the time restricted biological responses during
adipogenesis, we first investigated the distribution of unique TFs
numbers. Proteins that were not identified in all triple replicates or
only identified in one of triple replicates, while identified in at least
two replicates of all other time points were defined as unique
proteins of this time point. Our results showed that 7D had the
most heterogeneity with the greatest number of unique proteins,
indicating its specific role during adipogenesis (Fig. 3G). GO
 S. Li et al. / Biochemical and Biophysical Research Communications 511 (2019) 855e861
enrichment showed that these unique TFs was mainly focused on
response to oxygen and cell death (Fig. 3H), which is necessary for
terminal differentiation of adipogenesis and adipocyte maturation
[16,17], indicating that 7D may be the critical point to enter
adipocyte maturation.
3.5. Three TFs as novel regulators responsible for adipogenesis
We also aimed to discover new potential regulators of adipo-
genesis. As TF's function is strongly dose-dependent, it could be
considered that drastic changes in expression are often accompa-
nied by drastic functional changes [18]. Hence, we selected three
TFs, that is, MAFF, BATF3 and MXD4, which presented declining
tendency and dramatically changed during adipogenesis in our
proteomics results (Fig. 4A). Due to the robustness and redundancy
of TFs network, inactivation of any one of target genes probably
would not affect the function of TFs network, while over-expression
could be an alternative way to examine their intrinsic functions
[19]. We respectively over-expressed MAFF, BATF3 and MXD4 in
hADSCs. EGFP fluorescence from target genes showed good trans-
fection efficiency (Fig. 4B). RT-PCR showed 30e10000-fold of over-
expression for target genes (Fig. 4C). Each overrepresented hADSCs
were then induced to differentiate. Since 7D could be the point that
adipocytes begin to enter maturation, we examined the efficiency
of adipogenesis of transfected cells on 7D. We found significantly
inhibited Oil Red O accumulation, especially in MXD4 over-
expressed hADSCs (Fig. 4DeF). These results showed that over-
expression of MAFF, BATF3 and MXD4 could inhibit adipogenesis
of hADSCs and MXD4 showed the most efficiency.
4. Discussion
Our study presented the first dynamic TFs profiles about adi-
pogenesis of hADSCs at proteome level, which covered high-
density temporal courses, ranging from stem cell stage (0 h) to
later stage of adipogenesis(7D). This systematic TFs proteomics
study benefited from the recently well-developed enrichment
Fig. 4. MAFF, BATF3 or MXD4 over-expression inhibits adipogenesis of hADSCs. (A) Time course expression of MAFF, BATF3and MXD4 in proteomics data. (B) EGFP expression in
hADSCs infected for 48 h. Scale bar, 50 mm. (C) mRNA over-expression of targeted genes in hADSCs infected for 48 h. (DeE) Oil Red-O staining and microscopic images. Scale bar,
50 mm.
859
technology of catTFRE [10], which could provide high-quality of TFs
data to build more precise models of TFs network. Our data could
also serve as a valuable public resource for adipogenesis study.
Increasing evidences have showed that differentiation may be a
stochastic process, in which an increase in cell-cell variability have
been observed in many mammalian systems [20]. Notably, some
scholars believe that this stochastic process is caused by the
random response of TFs to external signals [21]. Our results also
observed similar phenomena that Pearson correlation and over-
lapped number of proteins among three biological replicates
decreased in later stages of adipogenesis, indicating the increased
variability of TFs network and suggesting that changes of TFs
network during adipogenesis is also a stochastic process.
Our TFs proteome results revealed two-main phases during the
first 7 days of adipogenesis. The first phase (commitment phase,
0 h-8h) could be characterized by stem cell proliferation and dif-
ferentiation, in which proteins of components of SWI/SNF chro-
matin remodeling complexes, that dramatically increased at this
period, could play key roles in this process (Fig. 3F). Chromatin-
remodeling complexes is an important step in cell differentiation
to facilitate the access of multiple transcription factors. SWI/SNF
complex is involved in the differentiation of a variety of mamma-
lian tissues, including adipocytes [22]. It has been proven to be
necessary for transcription of PPARg2, which is the most important
regulator of adipogenesis [23]. The first phase can be further
divided into two sub-phases (0 h; 0.5e8 h). The number of identi-
fied TFs rapidly reached maximum at 0.5 h, and about 10% of the TFs
changed dramatically, suggesting that TFs network at this time
point responded to differentiation stimulus rapidly and dramati-
cally. The second phase (determination phase, 1De7D)was char-
acterized by response to lipid and fat cell differentiation. In this
phase, PPARg, CREB1 and MYC are the key nodes. PPARg is the most
important regulator in adipogenesis. CREB1 and MYC has also been
proven to promote adipogenesis in 3T3-L1 and ADSCs, respectively
[24,25]. The second phase could also be further divided into two
sub-phases (1D-3D; 7D) and 7D is the special time, in which the
most unique TFs were found, and these time-restricted proteins
860 S. Li et al. / Biochemical and Biophysical Research Communications 511 (2019) 855e861
were responsible for response to oxygen and regulation of cell
death, which may involve in adipocyte maturation. Interestingly,
our time window of sub-phases is not the same as that depicted by
transcriptome data. Transcriptome data indicates three distinct
sub-phases (0 he3h; 6 he1D; 2De4D) within the first 4 days of
differentiation [15], suggesting that our dynamic proteome of TFs
network could provide more information about the process of
adipogenesis.
Our study also found and validated three TFs (MAFF, BATF3 and
MXD4) as novel regulators of adipogenesis. Among them, over-
expression of MXD4 has the strongest inhibitory effect on adipo-
genesis. MXD4 is a member of MAD family. Pulverer has reported
that ectopic expression of MXD1(also the member of MAD family)
in 3T3-L1 cells resulted in inhibition of adipogenesis by affecting
the balance of Myc/Max/Mad network [26]. We speculated that
MXD4 could affect adipogenesis through similar mechanisms and
this should be investigated in the future. Several reports have
revealed that MAF family was involved in maintaining the char-
acteristics of stem cells, cell differentiation and disease develop-
ment [27e29]. Our results showed that over-expression of MAFF, a
member of MAF family, inhibited adipogenesis of hADSCs. How-
ever, Maeda found that adipogenesis of 3T3L cells was inhibited
through down-regulation of MAFA (also belongs to MAF family)
[30]. This contradiction indicated that regulation of adipogenesis
through MAF family was diverse and intricate. Furthermore, a
recent research that summarized 72 genetic association studies for
obesity-related traits provided a comprehensive annotation of all
susceptibility loci. This study pointed out that a series of SNPs
linked to obesity-related traits could affect the binding of tran-
scription factor on the promoter of MAFF, or affect the binding of
MAFF to tissue/cells specific enhancers or disturb microRNA tar-
geting on MAFF [31]. Therefore, further study of MAFF in adipo-
genesis may deepen our understanding of obesity development.
Overall, our TFs proteome data provided a reference for studies
on adipogenesis. Mapping TFs on proteomics level provides a new
strategy to further understand the complex network of transcrip-
tion factors during adipogenesis and help advance our under-
standing of obesity.
Authorship
S.L and J.W conceived and designed the study; S.L, L.Z, H.F and
D.C performed experiments; S.L, T.X and S.O analyzed data; S.L
drafted manuscript; J.W and T.X revised manuscript critically for
important intellectual content; J.W approved manuscript for
submission.
Conflicts of interest
All authors declare no conflict of interest.
Acknowledgements
This work was supported by CAMS Central Public Welfare Sci-
entific Research Institute Basal Research Expenses (2016ZX310182-
3) and CAMS Initiative for Innovative Medicine (CAMS-I2M).
Transparency document
Transparency document related to this article can be found
online at https://doi.org/10.1016/j.bbrc.2019.02.134.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bbrc.2019.02.134.
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