Biochemical and Biophysical Research Communications 509 (2019) 435e440

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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

The induction of RANKL molecule clustering could stimulate early
osteoblast differentiation
Eri Sone a, b, Daisuke Noshiro c, 1, Yuki Ikebuchi d, Mami Nakagawa e, Masud Khan b,
Yukihiko Tamura f, Masaomi Ikeda g, Meiko Oki b, Ramachandran Murali h,Toshihiko Fujimori e, Tetsuya Yoda a, Masashi Honma d, Hiroshi Suzuki d, Toshio Ando c,
Kazuhiro Aoki b, *a

Department of Oral Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima,
Bunkyo-ku, Tokyo, 113-8549, Japanb
Department of Basic Oral Health Engineering, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima,
Bunkyo-ku, Tokyo, 113-8549, Japanc
Nano Life Science Institute (WPI NanoLSI), Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Japand
Department of Pharmacy, The University of Tokyo Hospital, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo, 113-8655, Japane
Division of Embryology, National Institute for Basic Biology, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi, 444-8787, Japanf
Department of Bio-Matrix (Pharmacology), Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima,
Bunkyo-ku, Tokyo, 113-8549, Japang
Department of Oral Prosthetic Engineering, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima,
Bunkyo-ku, Tokyo, 113-8549, JapanhResearch Division of Immunology, Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USAa r t i c l e i n f o

a b s t r a c t

Article history:
Received 30 November 2018
Accepted 13 December 2018
Available online 26 December 2018

We recently found that the membrane-bound receptor activator of NF-kB ligand (RANKL) on osteoblasts
works as a receptor to stimulate osteoblast differentiation, however, the reason why the RANKL-binding
molecules stimulate osteoblast differentiation has not been well clariﬁed. Since the induction of cellsurface receptor clustering is known to lead to cell activation, we hypothesized that the induction of
membrane-RANKL clustering on osteoblasts might stimulate osteoblast differentiation. Immunoblotting
showed that the amount of RANKL on the membrane was increased by the RANKL-binding peptide OP34, but not by osteoprotegerin (OPG), the other RANKL-binding molecule, in Gfp-Rankl-transfected ST2
cells. Observation under a high-speed atomic force microscope (HS-AFM) revealed that RANKL molecules
have the ability to form clusters. The induction of membrane-RANKL-OPG-Fc complex clustering by theaddition of IgM in Gfp-Rankl-transfected ST2 cells could enhance the expression of early markers ofosteoblast differentiation to the same extent as OP3-4, while OPG-Fc alone could not. These results
suggest that the clustering-formation of membrane-RANKL on osteoblasts could stimulate early osteoblast differentiation.
© 2018 Elsevier Inc. All rights reserved.

Keywords:
Osteoblast differentiationRANKL clustering
RANKL-binding peptide
OPG
HS-AFM

1. Introduction
Receptor activator of NF-kB ligand (RANKL) is known to activate
osteoclast formation and bone resorption activity through RANKL
and the receptor activator of NF-kB (RANK) interactions [1,2]. We* Corresponding author.
E-mail address: kazu.hpha@tmd.ac.jp (K. Aoki).
1
Present afﬁliation: Laboratory of Structural Biology, Institute of Microbial
Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141e0021, Japan.
https://doi.org/10.1016/j.bbrc.2018.12.093
0006-291X/© 2018 Elsevier Inc. All rights reserved.

recently showed that the RANKL expressed on osteoblasts works asa receptor of RANKL-binding molecules, which activate osteoblast
differentiation. This stimulatory signal in osteoblasts was designated “RANKL-reverse signaling”, since the ligand works as a receptor [3]. However, osteoprotegerin (OPG), which is known to be a
RANKL-binding molecule [2,4], does not have the anabolic effects
on bone formation since the deletion of OPG does not reduce boneformation [5]. Furthermore, OPG-Fc, which like OPG has been used
as a RANKL-binding molecule [6e8], did not stimulate the RANKLreverse signaling or osteoblast differentiation in ST2 cells (a murine

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E. Sone et al. / Biochemical and Biophysical Research Communications 509 (2019) 435e440

osteoblastic cell line) [3]. Thus, it became known that not all theRANKL-binding
molecules
alwaysenhanceosteoblast
differentiation.
RANKL-binding peptides, another type of RANKL-binding
molecule, have been shown to stimulate osteoblast differentiation
and bone formation [9e12]. Both W9 and OP3-4 are known as
RANKL-binding peptides [9,13e16], and they are also known to beable to activate mammalian target of rapamycin complex 1(mTORC1) activity in osteoblasts [12]. Since mTORC1 activation has
been demonstrated to occur downstream of RANKL signaling in
osteoblasts [3], both RANKL-binding peptides are suggested toactivate RANKL-reverse signaling. Furthermore, the activation of
osteoblast differentiation by RANKL-binding peptides is retarded
when RANKL-deﬁcient osteoblasts are used [3,9], suggesting that
the RANKL-binding peptides stimulate osteoblast differentiation
under a RANKL-dependent mechanism. However, how the RANKLbinding peptides enhance osteoblast differentiation has not beenclariﬁed.
On the other hand, the induction of membrane receptor clustering is known to enhance cellular activities, through the mechanisms such as the stimulation of proliferation and/or differentiation[17] of several types of hematopoietic lineage cells. For example,
Kosmides et al. showed that the magnetically-induced clustering of
membrane molecules stimulated T cell activation [18]. Furthermore,
small molecules that induce the dimerization of thrombopoietin
receptors activate thrombopoietin-speciﬁc signal transduction inplatelet precursors [19]. The induction of granulocyte-colony stimulating factor (GCSF)-receptor clustering activates granulopoiesis inhematopoietic lineage cells [19]. Furthermore, the induction oferythropoietin-receptor-dimerization also enhances the productionof erythrocytes [20]. Thus, we hypothesized that the difference inosteoblast differentiation between OP3-4 and OPG could be
explained by the different molecular characteristics following theformation of RANKL molecule clusters.
2. Materials and Methods
2.1. Culture conditions and the alkaline phosphatase (ALP) assay
ST2 (a murine osteoblastic cell line, RIKEN, Ibaragi, Japan) cells,
were seeded at a density of 1  104/well and cultured in an osteogenic medium as described elsewhere [3,12].
For performing the ALP assay, we used 96-well plates. The culture conditions are described above. The culture was stimulatedwith 100 or 200 mM OP3-4 (YCEIEFCYLIR; the RANKL-binding
peptide, which was designed based on the critical contact site on
OPG for RANKL [15] (Atlantic Peptides, Lewisburg, PA, USA)
(Fig. 1A)), 5 or 20 ng/ml OPG (Prospec-Tany Technogene, Rehovot,
Israel). The conditioned medium was changed on day 3. ALP
staining was performed on day 6 using a Lab Assay ALP (FUJIFILM
Wako Pure Chemical Corp.) in accordance with the manufacturer's
protocol. The ALP activity in each well was measured at 405 nmwith a microplate reader (iMark, Bio-Rad, Hercules, CA, USA).
2.2. Cell surface RANKL detection
The mouse Rankl gene was subcloned into pEGFP-C1 vector.
Plasmids encoded with green ﬂuorescent protein (GFP) at the Nterminal were constructed by site-directed mutagenesis against
pEGFP-based constructs [21].
For the preparations to detect the amount of surface GFPRANKL, we used 12-well plates cultured for 72 h under the culture conditions as described in section 2.1., excluding ascorbic acid,b-glycerophosphate, and dexamethasone. After the ﬁrst 24 h, Gfpmouse-Rankl was transfected using Lipofectamine 2000 (aFig. 1. OPG, a RANKL-binding molecule, did not enhance ALP activity, while OP3-4,
a RANKL-binding peptide, stimulated it.
A. Scheme of OPG (purple)-RANKL (yellow) complex with putative location of OP3-4
binding (shown in green). OP3-4 was designed from the contact site of RANKL on
OPG using structure-based approach.
B. OPG did not stimulate ALP activity. ST2 cells were cultured in osteogenic medium for
6 days in the presence of the indicated reagents. The ALP activity was measured asshown in 2.1. of the Materials and Methods section. Data are shown as the mean ± SD.
*, p < 0.01 vs vehicle-control.

transfection reagent; Thermo Fisher Scientiﬁc, Waltham, MA, USA).
ST2 cells were maintained in 100 or 200 mM OP3-4, 100 or 200 mM
W9 (YCWSQYLCY; Atlantic Peptides) [13], or 20 ng/ml OPG for
another 6 or 24 h. The cell surface proteins were biotinylated using
EZ-Link sulfo-NHS-SS-biotin (Thermo Fisher Scientiﬁc) in accordance with the manufacturer's protocol. The cells were then lysedwith a buffer composed of the following reagents; PBS, pH 7.4; 1.0%
Triton X-100; protease inhibitors (cOmplete, EDTA-free Protease
Inhibitor Cocktail, Merck & Co., Rahway, NJ, USA, #5056489001).
After lysing, the cells were centrifuged at 15,000g for 10 min. The
protein concentration of each supernatant was measured using a
BCA Protein Assay Kit (Thermo Fisher Scientiﬁc). Biotinylated protein in the supernatants was collected with Manga Bind Streptavidin Beads (Thermo Fisher Scientiﬁc). The blots were probed andproteins were detected with anti-GFP rabbit polyclonal (Thermo
Fisher Scientiﬁc, #A-11122), anti-Naþ/Kþ-ATPase, rabbit polyclonal(SantaCruz, Dallas, TX, USA, #sc-28800) and anti-GAPDH, rabbit
monoclonal (Cell Signaling Technology, Danvers, MA, USA, #2118).
Immunolabeled proteins were detected using anti-rabbit IgG, HRPlinked whole Ab donkey (GE Healthcare, Buckinghamshire, UK,#NA934) as corresponding secondary antibodies, and ECL prime
reagents (GE Healthcare) were used for ﬁnal detection.
2.3. High-speed atomic force microscope (HS-AFM)
Observations were performed in tapping mode using a labmade HS-AFM apparatus [22] and short cantilevers (BL-AC10DS,
Olympus, Tokyo, Japan). For the observation of soluble RANKL(FUJIFILM Wako Pure Chemical Corp.) in the absence or presence ofOP3-4, 100 mM RANKL dissolved in solution A (100 mM NaCl,
20 mM Tris-HCl pH 8.0) was deposited on a freshly cleaved mica
surface. After 3 min of incubation, unattached RANKL was washedE. Sone et al. / Biochemical and Biophysical Research Communications 509 (2019) 435e440

out with solution A. For the observation of RANKL in the presence ofOPG, a drop of 10 nM RANKL mixed with 10 nM OPG in solution A
was deposited on mica. After 3 min of incubation, unattachedRANKL and OPG was washed out with solution A. The HS-AFM
imaging conditions were as follows: scan size and pixel size,
80  80 nm2 (80  80 pixels), 100  100 nm2 (100  100 pixels), or
50  50 nm2 (80  80 pixels); imaging rate, 5.0e6.7 frames/sec(fps). Imaging was performed for RANKL alone in solution A, RANKL
plus OP3-4 (100 mM) in solution A, and RANKL plus OPG in 50 mM
Tris-HCl (pH 8.0).
2.4. mRNA quantiﬁcation in the crosslink culture
We used 6-well plates and cultured ST2 cells for 5 days under
the culture conditions described in section 2.1. and the cells weretransfected as described in section 2.2. The stimulants in this assay
were 0.09 nM OPG-Fc (BioLegend, San Diego, CA, USA), 0.45 nM
mouse anti-human IgG Fc CH3 secondary antibody (IgM; LifeSpan
BioSciences, Seattle, WA, USA), and OPG-Fc þ IgM. To induce RANKL
molecule clustering, we added IgM 1 h after adding OPG-Fc to thetransfected cells. To check the marker-gene-expressions of early
osteoblast differentiation, reverse transcription and a PCR were
performed using cDNA from ST2 cells, with GoTaq PCR mix(Promega, San Luis Obispo, CA, USA). mRNA was quantiﬁed by aquantitative real-time PCR with SYBR GreenER qPCR SuperMix
Universal (Life Technologies, Carlsbad, CA, USA) and an Eco RealTime PCR System (Illumina, San Diego, CA, USA). The software
program and primers are described as follows;
50 -GAGTCAGATTACAGATCCCA-30 and 50 -TGGCTCTTCTTACTGAGAGA-30 for mouse Runx2, 50 -CCCCAAGATGTCTATAAGCC-30 and 50 CGCTCTAGCTCCTGACAGTT-30 for mouse Osterix, 50 -CCCCAACCCTGGAAACAGAC-30 and 50 -GGTCACGTTCAGTTGGTCAAAGG-30 for mouse
Col1a1, 50 -GGGCGTCTCCACAGTAACCG-30 and 50 -ACTCCCACTGTGCC
CTCGTT-30 for mouse Alp, 50 -ATGTGTCCGTCGTGGATCTG-30 and
50 -TGAAGTCGCAGGAGACAACC-30 for mouse Gapdh.
2.5. Statistics
All of the data were expressed as the mean ± SD. A t-test with
Bonferroni correction was used to analyze multiple comparisons
between two groups. All statistical analyses were performed using
the SPSS software program (ver. 22.0, IBM, Chicago, IL, USA).
3. Results
3.1. RANKL-binding molecules stimulated osteoblast differentiation
First, we performed an osteoblast differentiation assay to
conﬁrm the previous evidence showing that OP3-4 stimulates
osteoblast differentiation but OPG does not. As shown in Fig. 1B,
OP3-4, a RANKL-binding peptide, stimulated ALP activity, a marker
of early osteoblast differentiation in the osteogenic culture condition, while OPG, which also binds to RANKL did not. The concentrations of OP3-4 and OPG used in Fig. 1B are known to enhanceosteoblast differentiation and to inhibit osteoclastogenesis,
respectively [12,23].
3.2. RANKL-binding peptide increased the amount of membraneRANKL, but OPG did not
Since we thought that a certain amount of membrane-RANKL isnecessary to induce the clustering of RANKL molecules on theosteoblast membrane, we tried to ﬁnd the difference in the amountof membrane-RANKL after stimulation of RANKL-binding molecules in Gfp-Rankl-transfected ST2 cells. As shown in Fig. 2, both437

OP3-4 and W9, another RANKL-binding peptide, increased theamount of membrane-GFP-RANKL, after 6 h of stimulation, while
OPG did not. The expression level of Naþ/Kþ-ATPase, a standard
cell-surface-marker protein, appeared to be similar in all conditions. After 24 h of stimulation with both peptides, the amount of
membrane-GFP-RANKL was still higher in comparison to that after
stimulation with OPG. On the other hand, the amount of GFPRANKL in the whole cell lysates was similar among all conditions(Fig. 2), suggesting that the RANKL localization shifted from theintracellular organelle to the cell membrane after the stimulation
with the RANKL-binding peptides. On the other hand, confocal laser
scanning microscope (CLSM) images obtained from the cells in thesame experimental conditions as shown in Fig. 2 did not show
apparent changes of localization of GFP-RANKL after 6 h of stimulations of the RANKL-binding peptides or OPG (Supplementary
Fig. 1), suggesting that the portion of the RANKL protein, whichshifted from the intracellular organelle to the plasma membrane,
was not a major portion of the RANKL protein compared to thewhole amount of RANKL protein in the cells even in the immunoblotting analyses, which showed the apparent increase of themembrane-RANKL by the RANKL-binding peptides.

3.3. HS-AFM revealed the ability of RANKL molecules to form
clusters and highly ﬂexible OPG clinging to RANKLTo reveal the molecular features of RANKL in clustering, we
observed RANKL molecules on mica in solution, by HS-AFM, whichallows for the direct imaging of protein molecules in dynamic action without disturbing their function [24]. As shown in Fig. 3A,
linear cluster-like structures were observed, as well as monomers
with a height of ~1.5 nm. While the clusters were sometimes disassembled into smaller parts or monomers, oligomers including
trimers or monomers sometimes made contact with other clusters,
resulting in the formation of larger clusters. This cluster formationof RANKL was not inhibited by the presence of OP3-4 (Fig. 3B). Notethat the HS-AFM apparatus could not image the OP3-4 molecules
due to their small size (11 amino acids) and/or very fast diffusion on
mica. The characteristic behaviors of RANKL molecules in thepresence or absence of OP3-4 are shown in Video S1A.
Supplementary video related to this article can be found athttps://doi.org/10.1016/j.bbrc.2018.12.093.
Next, we imaged RANKL in the presence of OPG. As shown in
Fig. 3C and Video S1B, one OPG molecule with high ﬂexibility clung
to and moved around to one RANKL molecule. This intermolecularinteraction would prevent RANKL molecules from forming large
clusters.

Fig. 2. The RANKL-binding peptides OP3-4 and W9 increased the membraneRANKL, but OPG did not.
Gfp-mouse-Rankl was transfected to ST2 cells and the indicated reagents were added tothe transfected cells 2 days after the transfection. The cells were lysed 6 h and 24 hafter each condition, and biotinylated surface proteins were collected using streptavidin beads. Membrane-RANKL was detected using anti-GFP antibody. Naþ/Kþ-ATPasewas used as a representative cell surface protein. OP3-4 and W9: 100 or 200 mM, OPG:
20 ng/ml.

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E. Sone et al. / Biochemical and Biophysical Research Communications 509 (2019) 435e440

Fig. 3. HS-AFM imaging revealed the formation of clusters of RANKL molecules and highly ﬂexible OPG clinging to RANKL.
Representative HS-AFM images showing molecular interaction among RANKL molecules in the absence (A) and presence (B) of 100 mM OP3-4. (C) HS-AFM images showing
molecular interaction between RANKL and OPG. Scale bars, 20 nm. The detailed experimental conditions are shown in the Materials and Methods section. See also Videos S1A and B.

3.4. Cross-linker of the OPG-Fc-RANKL complex stimulated early
osteoblast differentiation
The mRNA expression of osteoblast differentiation markers wasinvestigated when IgM was used as a cross-linker of Fc-fusion
protein. First we found the enhanced expression of markers ofearly osteoblast differentiation (Runx2, Osterix, Col1a1 and Alkaline
phosphatase), in the OP3-4-treated Gfp-Rankl-transfected ST2 cells(Fig. 4A). These osteoblast differentiation markers were signiﬁcantly enhanced when IgM was added to the OPG-Fc culture, while
they were not enhanced in the presence of OPG-Fc or IgM alone(Fig. 4A). Since the amount of membrane-RANKL might be a key tostimulating osteoblast differentiation, we also performed immunoblotting to clarify the difference in the amount of membraneRANKL after using IgM, a cross linker of OPG-Fc fusion protein(Fig. 4B). As we expected, the amount of membrane-RANKL protein
appeared to show a similar tendency to the markers of osteoblast
differentiation (Fig. 4B).
4. Discussion
In this study, we attempted to reveal reason why RANKLbinding peptides simulate stimulate osteoblast differentiation,
but OPG does not. Both OPG and OP3-4 are RANKL-binding molecules, but the afﬁnity of OPG to RANKL is much higher than that ofOP3-4 [1,15,23], suggesting that we cannot explain the difference ofboth molecules on osteoblast differentiation by comparing thedifference in afﬁnity to RANKL. We mainly showed the difference
that both molecules made on the amount of RANKL on the osteoblast membrane at 6 and 24 h after stimulation. Immunoblotting
showed that OP3-4 enhanced the amount of membrane-RANKL on
the cell surface, but that OPG did not (Fig. 2). Based on the HS-AFM
data showing that RANKL molecules have an ability to cluster

together (Fig. 3A), it is considered that the higher amount of RANKL
molecules on the cell membrane induced by OP3-4 means that
more clusters can form on the cell membrane at a higher rate incomparison to when a smaller amount of RANKL is induced by OPG.
The osteoblast differentiation stimulated upon OPG-Fc crosslinking
by IgM is possibly accounted for by the increased RANKL clusteringon the cell membrane through the crosslinked OPG (Fig. 4A).
Although the signaling to increase the amount of RANKL by the
induction of the crosslinking is not clariﬁed yet, the accumulationof RANKL on the cell membrane might be taken place in the similar
way as shown in the previous study using the RANK-coated beads[21,25]. On the other hand, a single OPG molecule was observed tocling to a RANKL molecule (Fig. 3C). This sticky intermolecular association is very likely to prevent RANKL clustering, which could bea key to clarify why OPG has no stimulatory effect on osteoblast
differentiation.
As mentioned earlier, we have found that RANKL-reverse
signaling stimulated osteoblast differentiation [3]. In this study,
OP3-4 probably worked like an agonist, binding to the membraneRANKL to enhance osteoblast differentiation (Figs. 1B and 4A). The
amount of membrane-RANKL after 6 h of OP3-4 stimulation was
higher in comparison to the vehicle-control (Fig. 2), suggesting thatsome unknown signaling might be initiated to the osteoblastic cells
by OP3-4. The induction of RANKL clustering was probably took
place on the osteoblastic cell membrane. Kariya et al. showed thatRANKL was predominantly expressed in the intracellular compartments of ST2 with an almost undetectable level of RANKL in theplasma membrane [21], suggesting almost no endogenous RANKL
protein was expressed on the ST2 plasma membrane in this study,
either. Both the accumulation of RANKL protein and the formationof RANKL clusters might be an induction-switch of both theenhancement of osteoblast differentiation and the accumulation of
RANKL molecules on the cell membrane. Further studies are

E. Sone et al. / Biochemical and Biophysical Research Communications 509 (2019) 435e440439

Fig. 4. OPG-Fc increased markers of early osteoblast differentiation when RANKL molecules were oligomerized by crosslinking OPG-Fc
A. Comparison of the mRNA expression levels of early osteoblast differentiation markers in ST2 cells after 5 days of stimulation with OP3-4, OPG-Fc, IgM, or OPG-Fc þ IgM. Data areshown as the means ± SD. *, p < 0.05 vs vehicle-control. y, 0.05 < p < 0.1 vs vehicle-control.
B. Comparison of the RANKL expression on ST2 cells stimulated with OPG, OPG-Fc, IgM, or OPG-Fc þ IgM.

needed to clarify the details of this stimulatory signaling andwhether the signaling is the same as the reverse signaling as shownin our previous study [3,12].
In conclusion, we showed that after OP3-4 stimulation, theamount of RANKL on the plasma membrane of Gfp-Rankl-transfected ST2 cells became higher in comparison to after OPG stimulation. HS-AFM revealed the characteristic ability of RANKL
molecules to form clusters, and highly ﬂexible OPG clinging to
RANKL prevented productive clustering. Since the induction of theclustering of RANKL molecules on the plasma membrane stimulated early osteoblast differentiation, the induction of RANKLclustering on the membrane could be a molecular switch that induces osteoblast differentiation.
Conﬂicts of interest
The authors declare no conﬂicts of interest in association withthe present study.

and wrote the manuscript. D.N. performed HS-AFM analyses. Y.I.
performed immunoblot analyses. M.N. and T.F. obtained the CLSM
images. M.K. supervised for cell culture. Y.T. taught the skills for cell
culture. M.I. assisted statistical analysis. M.O. advised and edited
the manuscript. R.M., T.Y., M.H. and H.S. provided critical advices tothe manuscript. T.A. supervised the AFM experiments and edited
the manuscript. K.A. designed and supervised the study and edited
the manuscript.
Funding
This work was supported by Grants-in-Aid for ScientiﬁcResearch (KAKENHI) from the Japan Society for the Promotion of
Science (grant numbers: 17F17124, 16K15656, and JP16H06280),
and a Grant-in-Aid for Scientiﬁc Research on Innovative AreasPlatforms for Advanced Technologies and Research Resources“Advanced Bioimaging Support.”
Acknowledgements

Author contributions
E.S. designed the study, performed experiments, analyzed data

The authors thank Mariko Takahashi and Dr. Hitoyata Shimokawa for their support in this experiment, and the laboratory

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E. Sone et al. / Biochemical and Biophysical Research Communications 509 (2019) 435e440

members of the department of Materials Science and Engineeringin Tokyo Institute of Technology, especially Takashi Nyu, Tatsuhiro
Maekawa and Dr. Tomohiro Hayashi.
Appendix A. Supplementary data
Supplementary data to this article can be found online athttps://doi.org/10.1016/j.bbrc.2018.12.093.
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