Mutations in Planar Cell Polarity Gene SCRIB Are
Associated with Spina Bifida
Yunping Lei1, Huiping Zhu1, Cody Duhon1, Wei Yang3, M. Elizabeth Ross4, Gary M. Shaw3, Richard H.
1 Dell Pediatric Research Institute, Department of Nutritional Sciences, the University of Texas at Austin, Austin, Texas, United States of America, 2 Department
of Chemistry and Biochemistry, College of Natural Sciences, the University of Texas at Austin, Austin, Texas, United States of America, 3 Department of
Pediatrics, Division of Neonatology, Stanford University School of Medicine, Stanford, California, United States of America, 4 Center for Neurogenetics, Brain
and Mind Research Institute, Weill Cornell Medical College, New York, New York, United States of America
Neural tube defects (NTDs) (OMIM #182940) including anencephaly, spina bifida and craniorachischisis, are severe
congenital malformations that affect 0.5–1 in 1,000 live births in the United States, with varying prevalence around
the world. Mutations in planar cell polarity (PCP) genes are believed to cause a variety of NTDs in both mice and
humans. SCRIB is a PCP-associated gene. Mice that are homozygous for the Scrib p.I285K and circletail (Crc)
mutations, present with the most severe form of NTDs, namely craniorachischisis. A recent study reported that
mutations in SCRIB were associated with craniorachischisis in humans, but whether SCRIB mutations contribute to
increased spina bifida risk is still unknown. We sequenced the SCRIB gene in 192 infants with spina bifida and 190
healthy controls. Among the spina bifida patients, we identified five novel missense mutations that were predicted-to-
be-deleterious by the PolyPhen software. Of these five mutations, three of them (p.P1043L, p.P1332L, p.L1520R)
significantly affected the subcellular localization of SCRIB. In addition, we demonstrated that the craniorachischisis
mouse line-90 mutation I285K, also affected SCRIB subcellular localization. In contrast, only one novel missense
mutation (p.A1257T) was detected in control samples, and it was predicted to be benign. This study demonstrated
that rare deleterious mutations of SCRIB may contribute to the multifactorial risk for human spina bifida.
Citation: Lei Y, Zhu H, Duhon C, Yang W, Ross ME, et al. (2013) Mutations in Planar Cell Polarity Gene SCRIB Are Associated with Spina Bifida. PLoS
ONE 8(7): e69262. doi:10.1371/journal.pone.0069262
Editor: Michael Jay Brownstein, Pisces Therapeutics, United States of America
Received April 16, 2013; Accepted June 6, 2013; Published July 26, 2013
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise usd by
anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This research was supported by National Institute of Health [grant numbers R01-NS050249, NS076465, and HD067244]; and the Centers for
Disease Control and Prevention, Center of Excellence Award [grant number U01/DD000491]. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Neural tube defects (NTDs) are a class of human birth
defects that result from a failure of embryonic neural tube
closure. Failure to complete low spinal closure causes spina
bifida, incomplete cranial closure results in anencephaly, while
the failure of closure of the entire neural tube is a defect
referred to as craniorachischisis. Worldwide, NTDs affect 0.5-2
per 1,000 live born infants, with varying prevalence across
populations. Spina bifida and anencephaly are the two most
common forms of NTDs, occurring in 0.5-1 per 1,000
pregnancies in the United States . Many infants with spina
bifida can survive, but may endure a greatly diminished quality
Although genetic factors are believed to contribute in part, to
the etiology of spina bifida, the elucidation of such factors has
remained elusive. This is likely due to the complex inheritance
pattern and the contribution of a range of environmental factors
including folic acid . Indeed, more than 250 genes were
causally linked to NTDs in mice . Interestingly, all known
planar cell polarity (PCP) genes are involved in the process of
neural tube closure . Homozygous PCP mutations, such as
Vangl2 D255E and S464N [5,6], Celsr1 D1040G and N1110K
, produced a craniorachischisis phenotype in mice. When
heterozygous PCP gene mutations such as Vangl2 D255E are
combined with non-PCP mutations in mice, they produce
embryos with spina bifida or exencephaly . In humans,
mutations in PCP core genes including VANGL2, FZD6,
CELSR1, PRICKLE and DISHEVELLED, are associated with
several kind of NTDs. including spina bifida, anencephaly and
SCRIB is a PCP-associated gene in mammals . It is a
member of the LAP (leucine rich repeats and PSD-95/Discs
Large/ ZO-1) protein family [9,10]. The LRR region and PDZ
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regions are important for SCRIB localization and stabilization at
the plasma membrane [11,12]. The SCRIB PDZ domain also
plays an important role in physical interaction with other
proteins, including the core PCP gene Vangl2, which has a
PDZ binding domain . In Drosophila, homozygous Scrib
mutations result in loss of apicobasal cell polarity and
neoplastic tissue overgrowth . In mice, homozygous Scrib
mutations, such as circletail (3182-3183insC)  and line-90
(p.I285K) , cause the most severe type of NTD,
craniorachischisis. In humans,
associated with craniorachischisis  and several kinds of
cancer . It remains uncertain whether it is associated with
non-craniorachischisis NTDs in human, such as spina bifida.
We hypothesized that SCRIB mutations were associated with
non-craniorachischisis NTDs, and investigated this hypothesis
among infants born in California with spina bifida.
SCRIB mutations are
Materials and Methods
Data were obtained from a population-based case–control
study conducted by the California Birth Defects Monitoring
Program (CBDMP). The CBDMP is an active, population-based
surveillance system for collecting information on infants and
fetuses with congenital malformations, which has been
described elsewhere . Included for study were 192
singleton infants with spina bifida (cases) and 190 non-
malformed infants (controls). Cases were randomly selected
from all live born cases and a random sample of non-
malformed control infants ascertained by the CBDMP
corresponding to birth years 1994–1998. The case and control
infants were linked to their newborn bloodspot. All samples
were obtained with approval from the State of California Health
Table 1. Novel Rare Variants detected in NTDs in the CDS
of SCRIB Gene in Spina Bifida.
c.1096 G>Ap.A366T LRR 14 No
c.1655 C>Tp.T552MN/A No
c.3128 C>T p.P1043L3rd PDZYes
c.3943 G>Ap.A1315TN/A NobenignTolerant
c.3995 C>T p.P1332LN/AYes
c.4559 T>GP.L1520RN/A Yes
a Nucleotide numbering reflects cDNA numbering with + 1 corresponding to the A
of the ATG translation initiation codon 1 in the reference sequence. NCBI
Reference sequence number for SCRIB: NM_015356.
N/A: Not Available
and Welfare Agency Committee for the Protection of Human
DNA for genotyping for this study derived from anonymous
newborn bloodspots. Bloodspots are collected on all newborns
in California for genetic testing purposes by the State of
California. The State retains the residual, unused, portion of the
bloodspot and makes these bloodspots available to approved
researchers. The approval process includes detailed review by
the State of California Committee for the Protection of Human
Subjects (the primary IRB).
The original collection of bloodspots for newborn testing
includes an information form but it is not an official informed
consent form. The purpose of the form is to disseminate
information to the parents as to what occurs with their babies’
bloodspots and provides them with the instructions so that they
can opt out and request destruction by writing to the State of
California. Therefore this process is similar to the newborn
genetic screening tests - "informed dissent". For the use of
anonymous bloodspots for research, an "opt out" policy is
applied. In other words, parents are given written materials
which explain that if they do not want their child’s specimen
used in research studies, they can write to the State and the
State will destroy the sample. Thus, no bloodspots were used
in this research project for anyone whose parents had "opted
Genomic DNA was extracted from newborn screening
bloodspots using the Puregene DNA Extraction Kit (Qiagen,
Valencia, CA) and amplified using the GenomiPhi Kit (GE
Healthcare). Coding exons and flanking exon-intron regions of
the human SCRIB gene (NM_015356) were amplified by
polymerase chain reactions (PCR) from the whole genome
amplification (WGA) product. Primer sequences are available
upon request. The PCR products were sequenced using the
Prism Bigdye Terminator Kit (v3) on an ABI 3730XL DNA
analyzer (Life Technologies, Carlsbad, CA). Both case and
control samples were sequenced with either a specific forward
or reverse primer. Sequencing results were analyzed using the
Mutation Surveyor software V 4.0.7 (Softgenetics, Stage
College, PA). The detected mutations were subsequently
confirmed by a second round of whole genome amplification,
PCR and sequencing analysis.
Mutations were annotated according to the HGVS
nomenclature (http://www.hgvs.org/mutnomen/). Nucleotide
numbering reflects cDNA numbering with +1 corresponding to
the A of the ATG translation initiation codon 1 in the reference
sequence. A variant was designated as novel if it was not
found in dbSNP Build 136 or in 1000 genome data (www.
1000genome.org) or the Exome Variant Server (NHLBI GO
Exome Sequencing Project (ESP), Seattle, WA (URL: http://
evs.gs.washington.edu/EVS/) [November 2012 accessed]).
Rare mutations were defined as having an allele frequency of
less than 1%. The potential pathogenic effect of the missense
mutations on protein function was predicted using two online
programs: PolyPhen (Polymorphism Phenotyping) (http://
SCRIB Gene Mutation and Spina Bifida
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genetics.bwh.harvard.edu/pph/) and SIFT (http://sift.jcvi.org/).
CLUSTAL W program with built in Mega software (V 5.1)
(http://www.megasoftware.net/) was used to analyze the
conservation of SCRIB amino acids that were changed by the
mutations. Residues were considered to be highly conserved if
there was no variation in amino acid properties observed
across the compared seven orthologous proteins, included five
mammalian orthologus plus zebrafish and Drosophila.
Human SCRIB full-length cDNA was cloned into pEGFP-C1
plasmid (GFP-SCRIB) and was graciously provided for our use
by Dr. Patrick Humbert (Peter MacCallum Cancer Centre,
Melbourne, Australia). GFP-SCRIB I285K mutant construct
was acquired from Dr. Philip Stanier (UCL Institute of Child
Health, London, UK). Human influenza hemagglutinin (HA)
tagged VANLG2 (HA-VANGL2) plasmid was obtained from Dr.
Hongyan Wang (Fudan University, Shanghai, China). SCRIB
missense changes were introduced into GFP-SCRIB by
Technologies, Carlsbad, CA).
Site-Directed Mutagenesis System (Life
MDCK II cell line was chosen for use in SCRIB mutation
subcellular localization studies
characteristic and being commonly used in epithelial cell
surface polarity studies. MDCK II cells were purchased from
Sigma-Aldrich and cultured according to the manufacturer’s
protocols. One day before transfection, cells were seeded in 4
chamber 35 mm glass bottom dishes (4x104/chamber) (In Vitro
Scientific, Sunnyvale, CA). Transfection was performed using
Lipofectamine 2000 regent (Life Technologies, Carlsbad, CA)
according to the manufacturer’s manual. Forty-eight hours
later, cells were washed twice with PBS and incubated 5
minutes with Hochest 3342 (1µg/ml) (Invitrogen) and
CellMask™ Plasma Membrane Stains (Life Technologies,
Carlsbad, CA) (2µg/ml) solution. The cells were then washed 3
times with PBS and fixed in 4% PFA (paraformaldehyde in
phosphate-buffered saline) for 10 minutes at 37°C, followed by
due to its epithelial
Figure 1. NTD specific SCRIB substitutions location and conservation analysis. (A) Location of missense changes and
mouse mutations. (B) A partial alignment of SCRIB amino acid sequence between human and other vertebrates. The SCRIB
variants found in NTDs affect conserved and less conserved residues. National Center for Biotechnology Information accession
numbers are NP_874365.2 for human SCRIB, XP_003431867.1 for dog SCRIB, XP_003586927.1 for cattle SCRIB, NP_598850.1
for mouse Scrib, NP_001178808.1 for rat Scrib, NP_001007176.1 for zebrafish scrib and NP_001163747.1 for Drosophila.
SCRIB Gene Mutation and Spina Bifida
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3 more PBS washes. Cells were examined and photographed
by a laser scanning confocal microscope (LSM710, Leica). The
transfection efficiency of GFP-SCRIB wild type and its mutant
constructs was measured and calculated on the Operetta High
Content Screening System (PerkinElmer).
The subcellular localization of GFP expression was classified
to three different categories: (1) GFP present only at
membrane; (2) GFP distributed at the membrane and in the
cytoplasm; and (3) GFP exclusively in the cytoplasm. For each
transfection, at least 50 cells were counted and each construct
transfection was performed three times. Quantitative data
Table 2. Rare variants (<1%) in the open reading frame of
SCRIB that are detected in NTD patients and controls or
silent, or recorded in NCBI database.
c.18 G>Cnew p. P6P1/0 N/AN/A
c.468 C>Tnew p.A1488A0/1 N/A N/A
c.693 G > Anew p.R231R0/1 N/AN/A
c.732 G>Anew p.L244L0/1 N/AN/A
c.756 G>A rs75171224p.Q252Q3/3 N/AN/A
c.762 G>A rs144565607 p.L254L1/0 N/AN/A
c.1375 G>A rs138716612p.D459N1/0
c.1460 G>C rs143419869p. S487T 1/0benign TOLERATED
c.3444 C>Trs150206819p.D1148D 0/1N/A N/A
c.3555 C>T rs142247868p.T1185T0/1 N/A N/A
c.3769 G>Anewp.A1257T 1/0 benignTolerant
c.3816 C>Tnew p.A1272A0/1 N/AN/A
c.3942 C>Tnew p.P1314P1/0N/A N/A
c.3943 G>Anewp.A1315T 1/1 benign Tolerant
c.4437 G >
c.4536 C>Tnewp.D1512D 0/1N/A N/A
c.4662 C>Trs111739279p.L1554L3/2 N/AN/A
c.4803 C>T rs150660931p.P1601P0/1N/A N/A
MAF: Mino Allele Frequency; Ct: Control; SB: Spina Bifida. N/A: Not done
NCBI Reference sequence number for SCRIB: NM_015356
between wild type and mutants were evaluated by Chi-square
The HEK293T cell line was chosen in immunoprecipitation
assay because of its high transfectability. In this study, the
HEK293T cell line was kindly provided by Dr. Edward Marcotte
(Department of Chemistry & Biochemistry, University of Texas
at Austin) and was cultured in DMEM (Life Technologies,
Carlsbad, CA) supplemented with 10% FBS and 2mM
glutamine. One day before transfection, cells were seeded into
6-well plates (6X105cells/well). GFP-SCRIB (2µg) or its relative
mutant plasmids were co-transfected with HA-VANGL2 (2µg).
Twenty-four hour post-transfection, cells were washed twice
with ice-cold PBS and lysed with 200µl 1% NP-40 lysate buffer
containing 1X cocktail protease inhibitor (Roche). We removed
30µl lysate as inputs, examined the input by anti-GFP or anti-
HA immunoblotting. The remaining cell lysate was used for
GFP-SCRIB and HA-VANGL2 co-immunoprecipitation. The co-
immunoprecipation was performed using a Pierce HA Tag
IP/Co–IP Kit (Thermo Fisher Scientific Inc.), according to the
instructions. The co-precipitates were run on SDS-PAGE
followed by western blot detection, immunoblotting with anti-
GFP antibody for GFP-SCRIB detection and anti-HA antibody
for HA-VANGL2 detection.
Six rare missense mutations were identified in SCRIB
in spina bifida
We detected six SCRIB missense mutations among 192
spina bifida infants. They were: c.1096 G > A (p.A366T); c.
1655 C>T (p.T552M); c.3128 C>T (p.P1043L); c.3943G>A
(p.A1315T); c.3995C>T (p.P1332L)
(P.L1520R) (Table 1). None of these mutations was found in
the dbSNP database or 1000 genome data, or in the 190 infant
controls. p.P1332L was found in the ESP database, with a
minor allele frequency of 0.007 among European American
(EA) populations. Among of these six mutations, p.A366T was
localized to the LRR domain, p.1043L was within the third PDZ
domain, while the other four variants did not locate in these two
classical domains of SCRIB (Figure 1A, Table 1). Five of the
Table 3. Common Variants (MAF>=0.01) in the Coding Sequence of SCRIB Gene detected in this study.
Nucleotide changers ID Amino Acid change PolyPhen PredictionSIFT PredictionMAF Ct/NTDp Value
c.474 C>T rs56748182p.L158LNANA 0.0421/0.0520.504
c.1276 G > A rs148555909p.D426N possibly damaging TOLERATED0.0211/0.01 0.233
c.1512 G > Ars144626855p. S504S NA NA0.0316/0.0340.857
c.1651 G > A rs118022661p.A551T benignTOLERATED 0.0421/0.0490.617
c.3921 G > Ars11784217p.P1307PNA NA 0.0211/0.0130.387
c.4098 C>T rs72693351p.R1366RNA NA 0.0263/0.0420.291
c.4663 G > Ars117338714p. G1555S benign TOLERATED0.05/0.0470.836
c.4751 C>Trs146664605p.P1584LbenignTOLERATED 0.0158/0.013 0.714
MAF: Mino Allele Frequency; Ct: Control; SB: Spina Bifida. NA: Not Done
NCBI Reference sequence number for SCRIB: NM_015356
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six mutations were predicted-to-be-deleterious by PolyPhen,
while three of them were predicted-to-be-deleterious by SIFT.
PolyPhen and SIFT each has its own advantages and
disadvantages; therefore, combining the results from both
programs was thought to increase the prediction sensitivity
. Using this strategy, five mutations were determined as
“predicted-to-be-deleterious” (Table 1). During amino acid
conservation analysis, three (p.P1043L, p.P1332L and
p.L1520R) mutations located at highly conserved nucleotides,
and the others (p.A366T, p.T552M and p.A1315T) involved
less conserved nucleotides (Figure 1B). p.P1043L was
identified in a case with an open defect in the thoracic area,
others were found among cases with defects in the
lumbosacral area. We also detected six novel synonymous
mutations in case infants, but not control infants (Table 2).
Eight common SNPs were identified in both cases and
controls, and there were no significant differences between the
groups (Table 3).
Figure 2. Effect of SCRIB mutations on protein subcellular localization. A: Protein localization in MDCK cells, green indicated
GFP-SCRIB and its mutant, blue indicated cell nucleus. B: Quantitative analysis of protein localization for GFP-SCRIB. Cells were
scored as cytoplasmic only, plasma membrane (PM) and cytoplasmic combined, or PM only. **Statistically significant differences of
percentage cytoplasmic and percentage PM compared with wild-type (P < 0.001) were determined by Chi-square analysis.
SCRIB Gene Mutation and Spina Bifida
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Previous studies demonstrated that SCRIB must localize to
the cell membrane in order to function normally [13,17]. The
SCRIB mutation (p.I285K) which caused craniorachischisis in
mice adversely affected SCRIB membrane localization .
We compared the SCRIB membrane localization of the six
case specific missense mutations with the wild type localization
serving as a positive control, and the mouse mutation p.I285K
serving as a negative control.
mutations(p.P1043L, p.P1332L and p.L1520R), all of which
change a highly conservative amino acid (Figure 1B),
significantly increased the proportion of cells with cytosolic
localization, like the mouse pathogenic line-90 mutant
(p.I285K), while the other three mutations (p.A366T, p.T552M
and p.A1315T) maintained the same subcellular localization
pattern as wild type (Figure 2). No significant difference in
transfect efficiency was detected between GFP-SCRIB wild
type and its mutant constructs (Supplementary Figures S1, S2,
and S3 in File S1).
Three of the six
Effect of mutant on physical interaction with VANGL2
VANGL2 is a core PCP gene that when mutated, causes
craniorachischis in mice [5,6] as well as different kinds of NTDs
in humans [21,22]. Of the six mutations identified, one of them
(p.P1043L) was located in the third PDZ domain (PDZ3) of
SCRIB, which could affect the physical interaction with PZD
binding motif in VANGL2.
immunoprecipitation of SCRIB and its mutants with VANGL2.
We found that none of them lost the physical interaction
between SCRIB and VANGL2 (Figure 3).
We also performed co-
heterozygous mutations of two or more PCP genes are known
to cause spina bifida, exencephaly and craniorachischisis in
mice . SCRIB mutations have previously been identified in
craniorachischisis patients; however, it is not clear whether
SCRIB mutations are associated with non-craniorachischisis
types of NTDs in humans. We identified for the first time five
predicted-to-be-deleterious mutations of which three were
confirmed in functional analysis, in 192 spina bifida case
infants. All of these mutations save one (p.A1315T), was found
among infants born before 1998, the year when mandatory folic
acid fortification started in the US. No novel predicted-to-be-
deleterious mutations were found in control infants. Our data
indicate that SCRIB mutations may underlie the pathogenesis
of PCP mutations and compound
Figure 3. Effect of mutations on SCRIB physical interaction with VANGL2. Western blotting of the lysate with anti-GFP
demonstrates the presence of wild-type GFP-SCRIB protein and mutant proteins with different missense changes. Following HA
immunoprecipitation, blotting with anti-GFP confirms interaction between SCRIB and VANGL2 with all of the missense variants
SCRIB Gene Mutation and Spina Bifida
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of human spina bifida. The number of patients with spina bifida
carrying novel SCRIB mutations predict to be pathogenic in this
study (5 of 192; 2.6%) is comparable to the previous study (1
out of 36; 2.8%). The number of confirmed functional SCRIB
mutations identified in spina bifida in this study (3 of 192; 1.6%)
is less than that identified in a previous craniorachischisis study
(1 out of 36; 2.8%) .
In the subcellular localization assay, mutations p.P1043L,
p.P1332L and p.L1520R significantly altered the distribution of
SCRIB subcellular localization, as more GFP-SCRIB localized
to cytoplasmic instead of membrane domains. Previous
structure–function analysis of human SCRIB indicated that both
LRR and PDZ domains are required for correct localization
[24,25]. In our study, one of the three functional mutations
(p.P1043L) was located at the third PDZ domain. Prolines were
highly conserved in PDZ1 domain, and were less conserved in
PDZ3 and PDZ4 domain. They were not conserved in PDZ2
domain (Supplementary Figure S3 in File S1). Two of the three
functional mutations (p.P1332L and p.P1520R) were not
located in either the LRR or the PDZ domain. They were
located close to the C terminal of SCRIB, after the fourth PDZ
domain. This is similar to the previously published SCRIB
mutation p.R1535Q . Our data, when combined with
previously published results, suggested that in addition to the
LRR and PDZ domains, other regions of the SCRIB protein,
especially the C terminus of SCRIB, may play an important role
in human SCRIB subcellular localization.
We did not detect any obvious adverse effects of the
mutations on the physical interaction of SCRIB with VANGL2.
Although one of the conservative mutations (p.P1043L) was
located in the third PDZ domain, which is the direct interaction
partner with VANGL2 PDZ binding domain, it did not abolish
the interaction between SCRIB and VANGL2. SCRIB has four
PDZ domains, and both the second and third PDZ domain
strongly interact with VANGL2 . Although the p.P1043L
mutation may affect the third PZD domain, the second PZD
domain still could interact with VANGL2, hence compensating
for some third PZD domain variations. Due to the quantitative
limitation of western blot assay, there is the possibility that
these mutations may partly affect the interaction between
SCRIB and VANGL2 which could not be detected by Western
Spina bifida is a birth defect with a multi-factorial etiology.
SCRIB mutations may interact with mutations among other
non-PCP genes, or other genetic and environmental factors,
and contribute to the spina bifida phenotype observed here. In
the future, high-throughput
technology will allow us to perform a thorough search among
the whole exome or even the whole genome, to identify the
additional mutations that may interact with PCP mutations.
File S1. Supporting figures. Figure S1. Transfection
efficiency calculation: a. count total cells number (n) by
Operetta; b. selected and counted GFP positive cells number
(a). Transfection efficiency = (a/n) *100%. Figure S2. GFP-
SCRIB constructs transfection efficiency. No significant
difference was detected between SCRIB wild type and the
mutant. Figure S3. GFP-SCRIB constructs transfected GFP-
positive cells fluorescence intensity. a. individual GFP-positive
cell (green dot) green fluorescence intensity, red dot indicate
GFP-negative cells. b. Different GFP-SCRIB constructs GFP-
positive cells average fluorescence intensity.
We thank Dr. Yue Li for her excellent assistance with the
confocal microscopy. We thank the California Department of
Public Health Maternal Child and Adolescent Health Division
for providing data for these analyses. The findings and
conclusions in this report are those of the authors and do not
necessarily represent the views of the NIH, CDC or California
Department of Public Health.
Conceived and designed the experiments: YL HZ MER GMS
RHF. Performed the experiments: YL CD. Analyzed the data:
YL WY HZ. Contributed reagents/materials/analysis tools: HZ
RHF. Wrote the manuscript: YL HZ.
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