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Characterization of two novel small molecules targeting melanocyte development in zebrafish embryogenesisLu Chen, Xi Ren, Fang Liang, Song Li,Hanbing Zhong and Shuo Lin

Submit your next paper to PCMR online at http://mc.manuscriptcentral.com/pcmr

DOI: 10.1111/j.1755-148X.2012.01007.xVolume 25, Issue 4, Pages 446-453

Characterization of two novel small moleculestargeting melanocyte development in zebrafishembryogenesisLu Chen1, Xi Ren1, Fang Liang1, Song Li1, Hanbing Zhong1 and Shuo Lin1,2

1 Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology, Peking University ShenzhenGraduate School, Shenzhen, China 2 Department of Molecular, Cell and Developmental Biology, University ofCalifornia, Los Angeles, Los Angeles, CA, USA

CORRESPONDENCE Shuo Lin, e-mail: shuolin@ucla.edu and Hanbing Zhong, e-mail: zhong@pkusz.edu.cn

KEYWORDS melanocyte ⁄ zebrafish ⁄ chemicalscreen ⁄ melanoma ⁄ drug candidate

PUBLICATION DATA Received 20 January 2012,revised and accepted for publication 19 April 2012,pulished online 1 June 2012

doi: 10.1111/j.1755-148X.2012.01007.x

Summary

Melanocytes are pigment cells that are closely associated with many skin disorders, such as vitiligo, piebal-

dism, Waardenburg syndrome, and the deadliest skin cancer, melanoma. Through studies of model organ-

isms, the genetic regulatory network of melanocyte development during embryogenesis has been well

established. This network also seems to be shared with adult melanocyte regeneration and melanoma forma-

tion. To identify chemical regulators of melanocyte development and homeostasis, we screened a small-mole-

cule library of 6000 compounds using zebrafish embryos and identified five novel compounds that inhibited

pigmentation. Here we report characterization of two compounds, 12G9 and 36E9, which disrupted melano-

cyte development. TUNEL assay indicated that these two compounds induced apoptosis of melanocytes. Fur-

thermore, compound 12G9 specifically inhibited the viability of mammalian melanoma cells in vitro. These

two compounds should be useful as chemical biology tools to study melanocytes and could serve as drug

candidates against melanocyte-related diseases.

Introduction

Melanocytes are melanin-producing pigment cells that

mainly distribute underneath the skin and are responsi-

ble for skin and hair pigmentation. Many human skin

disorders, such as vitiligo, piebaldism, Waardenburg

syndrome, and the deadliest skin cancer, melanoma

(Miller and Mihm, 2006), are closely associated with

melanocytes (Nordlund et al., 1998; Rawls et al., 2001).

Approximately, 160 000 new cases of melanoma are

diagnosed every year worldwide (Parkin et al., 2005),

and melanoma-related deaths reach about 48 000 per

year (Lucas et al., 2006). Given the important role of

melanocytes in skin-related diseases, enormous effort

has been employed to study the mechanisms underly-

ing its differentiation and proliferation. Through studies

of model organisms, including rodents, birds, amphibi-

ans, and fish, the framework of melanocyte develop-

ment is well established. Skin melanocytes and other

types of pigment cells are derived from the neural crest

through hierarchical gene regulation of transcription fac-

tors and signaling molecules.

The neural crest is a multipotent and highly migratory

cell population. In embryogenesis, neural crest cells

migrate away from the dorsal neural tube along defined

routes to specific destinations and give rise to a variety

Significance

Two novel small molecules that specifically inhibit zebrafish melanocyte development have been identi-

fied in this screen. Compound 12G9 also specifically inhibits mammalian melanoma cell viability in vitro.

Further investigation of these two compounds may lead to a discovery of new reagents to study melano-

cytes and drugs against melanocyte-related diseases.

446 ª 2012 John Wiley & Sons A/S

Pigment Cell Melanoma Res. 25; 446–453 ORIGINAL ARTICLE

of tissues and organs, including the craniofacial skele-

ton, peripheral nerves, smooth muscles, adrenal and

thyroid glands, and pigment cells (Betancur et al., 2010;

Sauka-Spengler and Bronner-Fraser, 2008). Genetic

work in mouse remarkably helped to understand the

process of pigment cell development and lead to the

discovery of pivotal genes in melanocyte development.

Sox10 encodes a transcription factor with a DNA-bind-

ing HMG domain and a C-terminal transcriptional activa-

tion domain and is critical for the formation of neural

crest cells. In strong mouse sox10 mutant alleles, many

neural crest derivatives are reduced or absent (Sout-

hard-Smith et al., 1998). Mitf is a basic helix-loop-helix

leucine zipper transcription factor, which is expressed in

melanocyte precursors shortly after neural crest cell

migration from the neural tube. Mouse mitf mutants dis-

play pigmentation defects ranging from minor functional

disturbances to a complete loss of mature melanocytes

(Opdecamp et al., 1997). Therefore, mitf is considered

to be a master regulator in melanocyte specification

from neural crest cells (Levy et al., 2006) (reviewed in

(Sommer, 2011)). Dct encodes the enzyme dopachrome

tautomerase, which is needed in the synthesis of eu-

melanin (Jackson et al., 1992). Both mitf and dct are

early melanocytic markers.

Because of the transparency of embryos, fast exter-

nal development, and amenability to forward genetic

analysis, zebrafish has been established as an excellent

vertebrate model to study melanocyte development

(Kelsh et al., 1996). The development of melanocytes is

mostly conserved between mammal and fish with some

minor variation. There is one type of pigment cell (mela-

nocyte) in mammals, whereas there are three primary

types of pigment cells in zebrafish: the black-brown

melanocyte (also referred to as melanophore), the yel-

low xanthophore, and the reflective iridophore (Kelsh,

2004; Rawls et al., 2001). Melanocytes start to produce

melanin and become visible at about 24 hours post-fer-

tilization (hpf). At this time, the pigmented melanocytes

possess a dendritic morphology. Afterward, the melano-

cytes change to a flat polygonal shape, and pigment is

accumulated. The embryonic pigment pattern is largely

formed by 48 hpf (Kelsh et al., 2000). All of the key reg-

ulatory genes found in mouse have homologs in zebra-

fish and play conserved roles. Zebrafish sox10 mutant

colorless has severe defects in most neural crest deriva-

tives, including pigment cells, peripheral neurons, and

glia (Dutton et al., 2001; Kelsh and Eisen, 2000). Zebra-

fish mitfa ⁄ nacre mutants lack melanocytes, but have

increased numbers of iridophores (Lister et al., 1999).

Zebrafish also has a dct homolog that is expressed in

melanoblasts, melanocytes, and retinal pigment epithe-

lium (RPE) (Kelsh et al., 2000).

Besides the above intrinsic factors, the development

of melanocytes is also regulated by extrinsic signals.

The generation of neural crest cells requires the integra-

tion of the BMP and Wnt pathways. Wnt signaling is

also needed in the subsequent specification of melano-

cytes as well (Raible, 2006). The Kit pathway regulates

proliferation, survival, and migration of melanocytes.

The endothelin pathway modulates the migration and

melanization of melanocytes (Sommer, 2011; Thomas

and Erickson, 2008).

In adults, the melanocytes underneath the skin regen-

erate every day to continually supply melanin to prevent

damage caused by UV light. Malignant proliferation of

melanocytes results in melanoma. Recent studies show

that embryonic melanocyte development, adult melano-

cyte regeneration, and melanoma formation seem to

share some common cellular and genetic events (White

and Zon, 2008). Therefore, the knowledge gained from

investigation of embryonic melanocyte development

should help in understanding the pathogenesis of adult

skin disorders.

Compared with genetic analysis, chemical biology

studies with small molecules offer better temporal con-

trol of gene function by allowing the addition and

removal of certain compounds at preselected time

points. For instance, Yang and Johnson, (2006) used a

small molecule, MoTP, to ablate zebrafish melanocytes

and let the embryo recover, leading to the discovery of

a small number of melanocyte precursors or stem cells

that were able to reconstitute the larval melanocyte

population. To identify more novel chemicals regulating

melanocyte development, we carried out a small-mole-

cule chemical screen using zebrafish embryos and

obtained five compounds that can specifically disrupt

pigmentation. Here, we report the characterization of

two compounds, 12G9 and 36E9, out of these five com-

pounds for their functions in melanocyte lineages in ze-

brafish embryos, and their ability to inhibit mammalian

melanoma cell viability.

Results and discussion

Identification of two compounds disrupting

pigmentation in zebrafish embryos

Zebrafish embryos were placed into 96-well plates, and

chemical compounds were added to embryos at 6 hpf,

when embryos are at the gastrulation stage prior to neu-

ral crest specification. Pigmentation was examined and

recorded at 48 hpf, when the embryonic pigment pat-

tern is formed (Figure 1A). Two hundred micromolar

PTU (1-phenyl 2-thiourea, a well-known pigment inhibi-

tor by blocking tyrosinase activity) was used as a posi-

tive control (Karlsson et al., 2001), and 1% DMSO was

used as a vehicle control. Through screening 6000 com-

pounds of the DIVERSet chemical library, which was

built to cover the broadest biologically relevant pharma-

cophore diversity, five compounds were found to inhibit

pigment formation in zebrafish embryos. Here, we

report two compounds (12G9 and 36E9, Figure 1B,C)

based on their specificity to inhibit melanocyte develop-

ment. Literature searches with SciFinder Scholar

Two compounds disrupting melanocyte development

ª 2012 John Wiley & Sons A/S 447

revealed that these two compounds have never been

subjected to biological activity assessment and there-

fore represent novel small molecules. After optimiza-

tion, the working concentration of these two

compounds was determined to be 20 lM. At this con-

centration, the treated embryos appeared normal,

although their development was slightly delayed, and

the compounds did not cause permanent abnormalities.

12G9 and 36E9 inhibited pigmentation of both skin and

RPE (Figure 2E,G).

The absence of skin pigmentation could be caused by

either blocking melanin synthesis or disrupting melano-

cyte development. We employed RNA whole-mount in

situ hybridization with melanocytic marker dct to

address this issue. To obtain clear staining, embryos

were bleached with H2O2 after fixation with paraformal-

dehyde. The result showed that the number of dct posi-

tive cells decreased substantially in embryos treated

with compounds 12G9 or 36E9 (Figure 2F,H). To com-

pare all embryos at the same developmental stage,

12G9 and 36 E9 treated embryos were allowed to grow

an extra 6 h to 54 hpf to compensate the developmen-

tal delay. At 54 hpf, the body length and brain morphol-

ogy of treated embryos were the same as 48 hpf

control embryos, indicating that they were at the same

developmental stage. This finding implied that 12G9 and

36E9 disrupted melanization as well as the development

of melanocytes and RPE. To further investigate which

major stage of melanocyte development was disrupted,

three more time windows were assessed. First,

embryos were treated with 12G9 and 36E9 from 6 to

18 hpf followed by gene expression analysis using

sox10 for neural crest cells. The expression pattern and

level of sox10 did not significantly change (Figure S1),

suggesting that 12G9 and 36E9 did not interrupt the

generation of neural crest cells. Second, embryos were

treated with 12G9 and 36E9 from 6 to 24 hpf followed

by gene expression analysis of mitfa for melanoblast

and dct for both melanoblasts and melanocytes. The

expression patterns of mitfa and dct were normal, but

the number of mitfa and dct positive cells slightly

A

B C

Figure 1. (A) Horizontal arrows indicate four time windows in

which embryos are treated with given small molecules. (B and C)

The molecular structures of compounds 12G9 and 36E9.

A B

C D

E F

G H

Figure 2. The two compounds 12G9 and 36E9 inhibit pigmentation. The development of 12G9 and 36E9 treated embryos was slightly

delayed. To analyze treated embryos at the same stage as control groups, these embryos were collected at 54 hpf. Lateral view, anterior to

the left and dorsal to the top. A, C, E, and G show the live embryos; B, D, F, and H show in situ hybridization with dct probe. (A and B)

DMSO control group. Treated embryos had stellate melanocytes with corresponding dct expression. (C and D) Embryos were treated with

PTU. PTU blocked melanin synthesis and caused unpigmented embryos. However, the expression of dct showed that melanocytes were still

present. (E and F) Embryos treated with 12G9. (G and H) Embryos treated with 36E9. Both 12G9 and 36E9 prevented pigmentation, and the

number of dct positive cells in the trunk and RPE are much less than that of control group.

Chen et al.

448 ª 2012 John Wiley & Sons A/S

decreased (Figure S2), implying that 12G9 and 36E9

likely impaired the proliferation or differentiation of mela-

noblasts, but not their migration. These findings suggest

that the time window after 24 hpf is critical for the bio-

logical action of 12G9 and 36E9.

12G9 and 36E9 are cytotoxic to differentiated

melanocytes

We next added 12G9 and 36E9 to the embryos at

30 hpf and examined pigmentation at 48 hpf. At 30 hpf,

melanocytes have already produced melanin, and the

preliminary embryonic pigment pattern is established

(Figure 3A). The results showed that 12G9 and 36E9

were able to reduce pigmentation maintenance. In trea-

ted embryos, pigmented melanocytes condensed as

small dots and the RPE was impaired as well (Fig-

ure 3G,I). In situ hybridization with dct confirmed that

the number of dct positive cells was indeed decreased

(Figure 3H,J), which was consistent with the result of 6

to 48 hpf treatment (Figure 2F,H), indicating that 12G9

and 36E9 disrupted both melanization and survival of

melanocytes. To closely examine this morphological

change of the melanocyte, time-lapse microscopy was

used to observe the treated embryos. Soon after adding

the compounds, the pre-existing melanocytes started to

lose their normal stellate shape and finally condensed

as small black dots (Figure 4). Many of these shrunk

pigmented cells also lost their expression of dct, as

shown by analyzing overlapping images of fluorescent

in situ hybridization with dct and melanin signals (Fig-

ure 5). Meanwhile in the DMSO control group, dct is

always expressed in pigmented melanocytes.

Cell shrinkage is one of the main features of apoptosis

and apoptotic melanocytes become round (Cooper and

Raible, 2009). To determine whether these dot-like mela-

nocytes were undergoing apoptosis, a TUNEL assay was

performed. The overlap of black melanin and red TUNEL

signals indicated that apoptosis indeed was detectable in

many melanocytes in the compound-treated embryos

(Figure 6). In the 12G9 group, most of the TUNEL signal

was restricted to melanocytes (Figure 6C,E), while in the

36E9 group, TUNEL signal was also found in other types

of skin cells (Figure 6D,F), indicating that 36E9 was less

specific. In conclusion, compounds 12G9 and 36E9 were

able to disrupt pigmentation mainly by inducing apopto-

sis in melanocytes. Yang and Johnson (2006) reported

that the melanocytotoxicity of small-molecule MoTP

depended on tyrosinase activity. Therefore, PTU, the

tyrosinase inhibitor, could relieve the toxicity of MoTP.

We also treated embryos with PTU and 12G9 or 36E9

together and performed TUNEL assay, but did not see

any anti-apoptotic protection of melanocytes, which

implied that the functions of 12G9 and 36E9 were not

tyrosinase dependent (data not shown).

Compound 12G9 selectively inhibits mammalian

melanoma cell viability in vitro

To determine whether 12G9 and 36E9 could selectively

inhibit the viability of mammalian melanoma cells by

causing apoptosis, an MTT assay with murine mela-

noma cell line B16, human pancreatic carcinoma cell line

MIA PaCa-2, and murine lung carcinoma cell line Lewis

cells were used. The results showed that 12G9 inhibited

the viability of B16, but had no effect on MIA PaCa-2

A B

C D

E F

G H

I J

Figure 3. 12G9 and 36E9 induced

morphological changes in differentiated

melanocytes. All embryos were treated

from 30 to 48 hpf except in A and B.

Lateral view, anterior to the left and dorsal

to the top. A, C, E, G, and I show the

pictures of live embryos; B, D, F, H, and J

show the dct in situ hybridization. (A and

B) 30 hpf embryos before treatment. (C

and D) Embryos treated with DMSO had

stellate melanocytes, and the pattern of

dct positive cells matched the

melanocytes. (E and F) Embryos treated

with PTU had less melanin, and the dct

expression pattern was normal. Embryos

treated with compound 12G9 (G and H) or

36E9 (I and J) had less pigmentation,

condensed melanocytes, and severely

reduced number of dct positive cells.

Two compounds disrupting melanocyte development

ª 2012 John Wiley & Sons A/S 449

and Lewis cells (Figure 7A). The IC50 of 12G9 against

B16 viability is 46 lM. Although this IC50 is modest, the

specificity of 12G9 is impressive as it does not inhibit

viability of the other two tumor cells at concentrations

as high as 125 lM. Flow cytometry assays confirmed

that 12G9 also caused apoptosis of B16 cells (Fig-

ure 7C). 36E9 did not inhibit the growth of these three

cell lines, nor cause apoptosis (Figure 7B). This result

therefore warrants further studies of 12G9, including

chemical modification to improve its potency, pharmaco-

kinetics, and safety to make it a better drug candidate

for treating melanoma.

Our studies suggest that the efficiency of identifying

pigmentation inhibitors through zebrafish screening is

reasonably high. A pilot screen of 6000 small-molecule

compounds resulted in the identification of five new

compounds capable of blocking pigmentation in both

melanocytes and RPE. Two compounds, 12G9 and

36E9, could disrupt the development of melanocytes

and RPE. The cell lineages of melanocytes and RPE are

completely different. Melanocytes originate from neural

crest, while RPE originates from the optic cup. There-

fore, we focused on melanocytes and did not investi-

gate RPE in this study. The biological functions of 12G9

and 36E9 were completely unknown before. Recently,

a chemical screen for melanocyte regulators was

carried out, and several small-molecule modulators were

isolated and characterized (Anastasaki et al., 2009;

Figure 4. Time-lapse microscopy imaging

revealed melanocytes shrank to

condensed dots when embryos were

treated with 12G9 or 36E9 from 36 to

48 hpf. DMSO and PTU were used as

control. All panels show dorsal stripes of

trunk region. Time points are indicated on

top of each panel. Red arrowheads point

to the melanocytes under transformation.

A B C

D E F

G H I

Figure 5. 12G9 and 36E9 treatment leads

to loss of dct expression in melanocytes.

All embryos were treated from 30 to

48 hpf. Lateral view, anterior to the left

and dorsal to the top. Dct expression is in

red. The right two columns are higher

magnifications of boxed areas in the most

left column. (A to C) In DMSO treated

embryos, melanin (black) and dct

expression (red) overlapped perfectly. (D

to I) In 12G9 or 36E9 treated embryos,

only a few melanocytes had overlapping

melanin and dct expression.

Chen et al.

450 ª 2012 John Wiley & Sons A/S

Hultman et al., 2008; Ishizaki et al., 2010). The molecu-

lar structures of 12G9 and 36E9 are different from those

compounds. Time course analysis revealed that 12G9

and 36E9 could impair melanization and reduce the

number of pigmented melanocytes, but not neural crest

cells and melanoblasts. The inhibitory effects of 12G9

and 36E9 are reversible. When 12G9 and 36E9 were

washed out with fresh fish water at 48 hpf, the melano-

cytes gradually recovered and partially formed the ste-

reotyped pigment pattern at 96 hpf (Figure S3G,H).

A C E

B D F

Figure 6. 12G9 and 36E9 caused apoptosis of melanocytes. All embryos were treated from 30 to 48 hpf. Lateral view, anterior to the left

and dorsal to the top. Red signals show the apoptotic cells detected by TUNEL assay. (A) Positive TUNEL assay control group, fixed embryos

were digested with DNase I and then subjected to TUNEL assay. Many cells were stained with red signal in nuclei. (B) DMSO control group,

no red TUNEL signals were detected. Embryos treated with 12G9 (C) and 36E9 (D) had overlap of black and red signals, indicating some

melanocytes were undergoing apoptosis. (E) and (F) show higher magnification of boxed area in (C) and (D).

CA

B

Figure 7. 12G9 induced apoptosis in melanoma cells. (A and B) Murine melanoma cell line B16, human pancreatic carcinoma cell line MIA

PaCa-2, and murine lung carcinoma cell line Lewis were incubated with 12G9 and 36E9 followed by MTT assay. Data show the mean ± SE

from three independent experiments. 12G9 inhibited B16 viability with IC50 of 46 lM, but did not inhibit MIA PaCa-2 and Lewis cells. 36E9

did not inhibit the growth of these 3 cell lines. (C) B16 cells were cultured with 12G9 for 24 h, then were double stained with Annexin

V-Alexa Fluor� 488 and 7-AAD. In the flow cytometry result, non-apoptotic cells were in Q4 (negative for both dyes), early apoptotic cell were

in Q3 (Annexin+ ⁄ 7-AAD)), late apoptotic cells were in Q2 (Annexin+ ⁄ 7-AAD+), and necrotic cells were in Q1 (Annexin- ⁄ 7-AAD+). The

percentage of cells of each state is shown in each quadrant.

Two compounds disrupting melanocyte development

ª 2012 John Wiley & Sons A/S 451

Molecular and cellular biology studies suggest that

12G9 and 36E9 disrupt melanocyte maintenance by

causing apoptosis. This mechanism of action is particu-

larly attractive for treating melanoma, as suggested by

compound 12G9 studies with B16 cells. Our screen and

follow-up molecular and cellular biology studies

designed in this study can be easily scaled up. Our find-

ings therefore establish an attractive approach to iden-

tify many more candidates for developing melanoma

drug candidates.

Methods

Zebrafish maintenanceWild-type AB line was maintained in a circulating aquaculture sys-

tem according to standards described in The Zebrafish Book (Wes-

terfield, 2000). Embryos were incubated at 28.5�C and staged

according to the description by Kimmel et al., (1995).

Chemical compounds and embryo treatment12G9(2-bromo-1,4-phenylene di(2-furoate)) and 36E9(N-(4-chlor-

ophenyl)-1-methyl-4-nitro-1H-pyrazole-3-carboxamide) were from the

DIVERSet� chemical library and purchased from Chembridge (San

Diego, CA, USA). These molecules were dissolved in dimethyl

sulfoxide (DMSO) to make stock solutions and then diluted with

fresh fish water to 20 lM for all treatments. PTU (Sigma-Aldrich,

St Louis, MO, USA) was dissolved in water to make stock

solutions and then diluted with fresh fish water to 200 lM for all

treatments.

Whole-mount in situ hybridizationWhole-mount RNA in situ hybridizations were performed essentially

as described by Westerfield, (2000). Embryos were bleached with

H2O2 after fixation with paraformaldehyde, except in dct fast red in

situ hybridization. Digoxigenin-labeled antisense RNA probes were

generated in vitro by using the zebrafish sox10, mitfa, and dct cDNA

as templates with RNA polymerase (Promega, Madison, WI, USA).

The dct cDNA was kindly provided by Simon Hughes. The cDNA

template of sox10 was amplified by RT-PCR with primers 5¢-AACGCGTTCATGGTGTGGGC-3¢ and 5¢-TGAACCGCTCGCCGCTGT.

AT-3¢, and the mitfa was amplified by 5¢-TACAGTGATGACATT

CTTGGGTT-3¢ and 5¢-AGAGTGGTAGGACGGGACA-3¢, then cloned

into pGEM-T easy vector.

Time-lapse microscopyEmbryos were mounted in 1% low-melting-point agarose contain-

ing compounds. Images were taken every 20 min until the round

dot morphology of melanocytes had formed. The control and

compound-treated embryos were mounted together and observed

in parallel.

Confocal microscopyLabeled embryos were fixed in 1% low-melting-point agarose and

imaged with the Zeiss LSM510 Meta and Axiovert 200M confocal

system. All pictures were edited with Photoshop CS2 (Adobe Sys-

tems, San Jose, CA, USA).

TUNEL staining and imagingEmbryos were fixed in 4% paraformaldehyde in PBST overnight at

4�C, dehydrated using methanol, and stored overnight at )20�C, re-

hydrated using decreasing concentration of methanol in PBST.

Fixed and permeabilized embryos were treated for 15 min with

20 lg ⁄ ml proteinase K (Sigma-Aldrich), followed by several washes

in PBST and then a 1 h incubation at 37�C in a red fluorescent

TUNEL cell death detection reagent (In situ Cell Death Detection

Kit TMR Red; Roche Applied Science, Basel, Switzerland). After

reaction, embryos were washed 3 · 5 min in PBST at room tem-

perature and stored in PBST at 4�C.

Cell cultureB16 melanoma cells, MIA PaCa-2 pancreas cancer cells, and Lewis

lung cancer cells were cultured at 37�C in a 5% CO2 incubator in

Dulbecco’s modified Eagle’s medium (DMEM) supplemented with

10% fetal calf serum and penicillin-streptomycin. All of the cells

were maintained according to recommendations of ATCC.

MTT assayB16, MIA PaCa-2, and Lewis cells (5 · 103) were seeded into 96-well

plates. After being cultured for 24 h, the cells were treated with

12G9 and 36E9 for 48 h with a dose range from 6.25 to 125 lM (Fig-

ure 7A). DMSO concentrations did not exceed 0.01%. The cells were

then cultured with 100 ll fresh growth medium with 10% FBS and

10 ll MTT solution (Sigma-Aldrich) for 4 h. The medium was replaced

with 200 ll DMSO followed by measuring absorbance using a Micro-

plate reader (Bio-Rad, Hercules, CA, USA) at 570 nm after 10 min

incubation. The results are presented as percentage of cell viability.

Detection of apoptosis with flow cytometry12G9 was added to B16, MIA PaCa-2, and Lewis cells (2 · 105) for

24 h. After treatment, cells were collected and washed in cold

PBS. After two washes, cells were resuspended in 1· annexin-

binding buffer, and 5 ll Annexin V- Alexa Fluor� 488 (Molecular

Probes, Inc., Eugene, OR, USA) and 10 ll 7-AAD (Invitrogen, Carls-

band, CA, USA) were added to cell suspension. They were incu-

bated at room temperature for 15 min in the dark. The stained cells

were analyzed by flow cytometry, measuring the fluorescence

emission at 530 and 692 nm under excitation at 488 nm.

Acknowledgements

We thank Dr. Zahra Tehrani for editing the manuscript. This work

was supported by grants from Shenzhen Science and Technology

Program (ZYC201006170364A, JC201005270280A, and JC201-

104220257A to Hanbing Zhong), 973 Program from MOST of China

(2009CB941203 to Hanbing Zhong and Shuo Lin), and National Nat-

ural Science Foundation of China (31071281 to Hanbing Zhong).

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Supporting information

Additional Supporting Information may be found in the

online version of this article:

Figure S1. 12G9 and 36E9 did not impair neural crest

cell development.

Figure S2. The effects of 12G9 and 36E9 on the

development of melanocyte lineage.

Figure S3. Recovery of melanocyte after removal of

12G9 and 36E9 at 48 hpf.

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Two compounds disrupting melanocyte development

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