NR5A1 prevents centriole splitting by inhibiting centrosomal DNA-PK activation and ?-catenin accumulation
Cell Communication and Signaling
NR5A1 prevents centriole splitting by inhibiting centrosomal DNA-PK activation and -catenin accumulation
Chia-Yih Wang 0 1 2
Pao-Yen Lai 0
Ting-Yu Chen 2
Bon-chu Chung 0
0 Institute of Molecular Biology, Academia Sinica , Taipei 115 , Taiwan
1 Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University , Tainan 701 , Taiwan
2 Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University , Tainan 701 , Taiwan
Background: Adrenogonadal cell growth and differentiation are controlled by nuclear receptor NR5A1 (Ad4BP/SF-1) that regulates the expression of adrenal and gonadal genes. In addition, SF-1 also resides in the centrosome and controls centrosome homeostasis by restricting the activity of centrosomal DNA-PK and CDK2/cyclin A. Results: Here we show that SF-1 depletion resulted in centriole splitting and amplification due to aberrant activation of DNA-PK in the centrosome of mouse adrenocortical Y1 cells. In the absence of SF-1, GSK3 was aberrantly phosphorylated during G1 phase and -catenin was accumulated in the centrosome, but not in the nucleus. DNA-PK inhibitor vanillin reversed these phenomena. SF-1 overexpression led to inhibition of centrosomal DNA-PK activation caused by SF-1 depletion. Both full-length SF-1 and truncated SF-1 devoid of its DNA-binding domain rescued the multiple centrosome phenotype caused by SF-1 depletion, indicating that the effect of SF-1 in the centrosome is not contributed by its DNA-binding domain. Furthermore, SF-1 interacted with cyclin A in the centrosome, but not in the nucleus. Depletion of SF-1 also resulted in centriole splitting, genomic instability and reduced growth of mouse testicular Leydig MA10 cells. Conclusion: Centrosomal DNA-PK signaling triggers the accumulation of -catenin, leading to centrosome over-duplication and centriole splitting. This cascade of centrosomal events results in genomic instability and reduced cell numbers.
NR5A1; Centriole splitting; DNA-PK; GSK3; -catenin; Cyclin A
Steroidogenic factor 1 (SF-1, NR5A1, Ad4BP) is a
tissuespecific transcription factor expressed mainly in the adrenal
glands and gonads. It belongs to the nuclear receptor
superfamily that binds to its cognate DNA sequence to
activate the expression of its target genes [1,2]. SF-1
regulates genes important for energy metabolism,
steroidogenesis and reproduction. SF-1 also maintains adrenogonadal
cell growth and differentiation; SF-1 knockout mice are
sex reversed and lack adrenals and gonads . Being a
transcription factor, SF-1 is located in the nucleus.
However, SF-1 also resides in the centrosome and its
centrosomal residency is required for the maintenance of
centrosome homeostasis .
Centrosomes consist of a pair of centrioles and the
surrounding pericentriolar materials (PCM). During each
cell cycle, centrosomes duplicate only once in a tightly
controlled manner [5,6]. The pair of centrioles are usually
configured perpendicularly, but they lose this
perpendicular relationship (disengage) at late mitosis/early G1 phase.
This process relieves the physical constraint of centrioles
to permit their duplication. The disengaged centrioles are
maintained at a distance of 2 m or less . During S
phase, both centrioles serve as a platform for the growth
of new centrioles . The duplicated centrioles are
separated and form mitotic spindle poles for proper
segregation of replicated chromosomes.
The distance between two disengaged centrioles are
regulated by centrosomal -catenin . Increased
abundance of -catenin in the centrosome induces centrosome
separation during mitosis. Upon entering mitosis,
duplicated centrosomes go to the opposite sites of the nucleus
forming spindle poles. Centrosome separation requires
Nek2 (NIMA-related protein kinase 2), which
phosphorylates and stabilizes the -catenin in the centrosome during
mitosis. Aberrant accumulation of -catenin in the
centrosome during G1/S phase causes centriole splitting to a
distance of more than 2 m between two centrioles; it also
causes centriole over-duplication [7,9]. Thus the precise
control of centrosomal -catenin is important to maintain
centriole configuration and copy numbers.
In steroidogenic cells, SF-1 functions as a centrosomal
guardian to maintain centrosome homeostasis. SF-1
maintains centrosome copy numbers by controlling the
activity of DNA-dependent protein kinase (DNA-PK) in
the centrosome . Centrosomal SF-1 interacts with
and sequesters Ku70/80, the subunits of DNA-PK, from
the catalytic subunit of DNA-PK (DNA-PKcs) to prevent
the activation of centrosomal DNA-PK. Once SF-1 is
depleted, DNA-PKcs is recruited to the centrosome
forming an active complex with Ku subunits to
phosphorylate downstream Akt; this signaling cascade induces
centriole over-duplication. The activation of DNA-PK in
steroidogenic cells is not due to nuclear DNA damage
response, but caused by SF-1 depletion .
In this study we have investigated in more detail the
mechanism by which SF-1 controls centrosome
homeostasis. We showed that centrosomal SF-1 also maintained
centriole configuration by controlling centrosomal GSK3
and -catenin signaling. We found that SF-1 depletion led
to the activation of centrosomal DNA-PK/Akt signaling
pathway which further phosphorylated GSK3, resulting
in the accumulation of -catenin and centriole splitting.
SF-1 maintains genomic integrity and proper cell growth
SF-1 is important for genomic stability and proper growth
of Y1 cells . Here we tested whether the role of SF-1
can be extended to other cell types such as mouse Leydig
MA-10 cells. When SF-1 was depleted by shsf1#3 shRNA
treatment for eight days, MA-10 cells contained both
enlarged nuclei and micro-nuclei (Figure 1A).
Counting the numbers of these nuclei, we found that most
of the control shluc cells contained normal nuclei that
were less than 150 m2 in size, whereas a higher
proportion of shsf1#3 cells contained nuclei larger than
150 m2 (Figure 1B). The proportions of shsf1#3 cells
with micro-nuclei that scattered around the enlarged
nuclei were also increased (Figure 1C). A different shRNA
sequence, shsf1#2, also induced the formation of bigger
nuclei and micronuclei. This result indicates that MA-10
genomes were unstable when SF-1 was depleted. In
addition, MA-10 cell numbers were reduced when SF-1
was depleted (Figure 1D). Thus SF-1 is important for
the maintenance of genomic integrity and proper MA-10
Since SF-1 depletion affected cell growth, we examined
cell-cycle profiles by flow cytometry after SF-1 depletion
for 2-days or 8-days. Two days after SF-1 depletion, the
proportion of cells in different cell-cycle stages was
similar to that of control cells infected by shluc lentivirus
(Figure 2A). However, when these shsf1#3 cells were
cultured for eight days, the proportions of subG1 (apoptotic
cells) and polyploid cells (>4 N) were increased, whereas
cells in G1 phase were reduced. These data indicated
that short-term depletion of SF-1 did not disturb cell
cycle progression, however, long-term depletion of SF-1
led to cell death and genomic instability.
SF-1 restricts aberrant centriole splitting via DNA-PK
We have previously shown that SF-1 depletion in Y1
cells leads to centrosome amplification . The
configuration of these centrioles was here examined in more
detail. During G1 phase, each PCM spot stained by
tubulin was associated with two centrioles with a distance
of less than 2 m. However, in many shsf1#3 cells that
contained normal numbers of centrioles at G1 phase, the
distance between centrioles was increased to more than
2 m (Figure 3A), a situation defined as centriole splitting
. Quantitation confirmed that shsf1#3 increased
centriole splitting both in Y1 (Figure 3B) and in MA-10 cells
(Figure 3C). Thus, SF-1 depletion resulted in centriole
To investigate the mechanism by which SF-1 regulates
centriole splitting, we tested the involvement of
DNAPK, which triggers centrosome amplification in response
to SF-1 depletion . The effect of DNA-PK inhibitor
vanillin on the distance between two centrioles in a cell
was examined. Increased centriole splitting was observed
in cells depleted of SF-1 by shsf1#3, but this number was
reduced after vanillin treatment (Figure 3D). This result
indicated that DNA-PK facilitated centriole splitting
induced by SF-1 depletion in Y1 cells.
To further confirm the role of SF-1 in regulating
DNAPK in the centrosome, we purified centrosomes from Y1
cells by sucrose gradient fractionation. The centrosome
was enriched in fraction number 5, as shown by the
presence of -tubulin (Figure 4A). This fraction was devoid of
nuclear or cytoplasmic contaminations, as shown by the
absence of mitochondrial complex II and nuclear hnRNP
A1 (Figure 4A). In Y1 cells that over-expressed control
EYFP, the amounts of DNA-PKcs phosphorylation was low
(Figure 4B), and this amount was increased after SF-1
depletion by shsf1#3 (shluc: shsf1#3 = 1: 2.4 0.5). SF-1
depletion also led to increased phosphorylation of Akt
(shluc: shsf1#3 = 1: 1.5 0.2, Figure 4B). Overexpression of
SF-1, however, blocked the phosphorylation of
DNAPKcs (shluc: shsf1#3 = 1.0 0. 8: 1.2 0.7) and Akt (shluc:
shsf1#3 = 0.9 0.2: 0.9 0.3) in shsf1#3 cells. Thus SF-1
prevented aberrant DNA-PK activation in the centrosome.
Figure 1 SF-1 depletion causes genomic instability and reduced MA-10 cell growth. (A-C) SF-1 depletion causes MA-10 genomic instability.
(A) Staining of MA-10 nuclei with DAPI after MA-10 cells were depleted of SF-1 by the infection of shsf1#3 lentivirus. The inset is a higher
magnification showing micro-nuclei stained by DAPI. Enlarged nucleus (asterisk) and micro-nuclei (arrow) were observed. The scale bar is 5 m.
(B-C) Quantitation of nuclear areas (B) and the population of cells with micro-nuclei (C). The areas of nuclei from at least 100 cells were counted
and compared in three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. At least 100 cells were counted in three independent
experiments and the meanS.D. is shown. (D) SF-1 depletion inhibits MA-10 cell growth. Growth curve of MA-10 cells after infection with
shRNA-encoding lentivirus against luc (shluc) or two different sequences of SF-1 (sh#2, sh#3). *P < 0.05. Inset shows the Western blot analysis of
SF-1 expression after infection of lentivirus for shluc, shsf1#2 (#2) and shsf1#3 (#3). Hsc70 was an internal control.
We also generated SF-1 that lacks the N-terminal
DNAbinding domain (D70) to examine the requirement of SF-1
domains in centrosome homeostasis. The transcriptional
activity of this truncated D70-SF-1 was tested after
transfection with a CYP11A1-luc reporter gene. Wildtype SF-1,
but not D70, activated reporter gene expression efficiently
(Figure 4C), indicating that D70 lost transcriptional activity.
We also depleted SF-1 from Y1 cells that expressed EYFP
or D70 stably. A big population of EYFP-expressing cells
contained multiple centrosomes when depleted of
endogenous SF-1 (Figure 4D). This multiple centrosome
phenotype was reversed by the re-introduction of SF-1.
D70 also inhibited multiple centrosomes in SF-1-depleted
Y1 cells. Thus, truncation of the DNA-binding domain
abolished the transactivation function of SF-1, but did not
affect its ability to maintain centrosome homeostasis.
When SF-1 is depleted, cyclin A, but not cyclin E, is
accumulated in the centrosome . We therefore
checked whether SF-1 overexpression would affect cyclin
A and cyclin E accumulation. SF-1 overexpression did
not affect the amount of cyclin A and E in the
centrosome, but SF-1 co-immunoprecipitated with
centrosomal cyclin A (Figure 5A). In the nucleus, SF-1 did
not co-immunoprecipitate with cyclin A, and only did
so slightly with cyclin E (Figure 5B). The amount of cyclin
A and cyclin E in the nucleus were also not affected by
SF-1 overexpression. SF-1 interacted with cyclin A in the
centrosome but not in the nucleus, implying that nuclear
Figure 2 Long-term, but not short term, SF-1 depletion disturbs
cell cycle profiles. Quantification of different cell cycle stages of Y1
cells infected with control shluc or shsf1#3 lentivirus that express
shRNA to deplete SF-1 for two days (A) or eight days (B) using
Aberrant accumulation of -catenin in the centrosome
upon SF-1 depletion
Centriole splitting depends on the accumulation of
catenin in the centrosome at G1 phase . To find
out whether centriole splitting induced by SF-1
involves -catenin, we examined the level of -catenin
after SF-1 depletion and found that it was accumulated in
the centrosome (shluc: shsf1#3 = 1: 2.7 0.4, Figure 6A).
GSK3 phosphorylation in the centrosome was also
increased (shluc: shsf1#3 = 1: 2.6 0.4), while total amounts
of GSK3 was not changed (Figure 6A). The global levels
of GSK3 and its phosphorylation in the whole cell lysate,
however, were not changed (Figure 6B). -Catenin was
also not accumulated in the whole cell lysate following
SF-1 depletion. This result indicates that SF-1 depletion
induced phosphorylation of GSK3 and accumulation of
-catenin only in the centrosome.
To investigate whether -catenin accumulation in
shsf1#3 Y1 cells is regulated by DNA-PK, cells were
Figure 3 SF-1 depletion induces centriole splitting during G1
phase. (A) Immuno-staining of shluc or shsf1#3 (sh#3) lentivirus
infected Y1 cells with antibodies against -tubulin (-tub) and
deploymerized acetylated -tubulin (Ace-tub). Scale bars are 5 m.
(B and C) Quantification of cells with centriole distance larger than
2 m in shsf1#3 infected Y1 (B) and MA-10 (C) cells. (D) Vanillin
inhibits centriole splitting induced by SF-1 depletion. Quantification
of centriole splitting in shluc and shsf1#3 infected Y1 cells in the
presence or absence of DNA-PK inhibitor, vanillin. These results are
mean S.D. from three independent experiments; more than three
hundred cells were measured in each individual group.
treated with DNA-PK inhibitor, vanillin. Vanillin blocked
phosphorylation of GSK3 (control shluc: control shsf1#3:
vanillin shluc: vanillin shsf1#3 = 1: 1.7 0.4: 0.9 0.1: 1.0
0.2) as well as the accumulation of -catenin (control
shluc: control shsf1#3: vanillin shluc: vanillin shsf1#3 = 1:
1.7 0.2: 0.9 0.2: 0.9 0.3, Figure 6C). These data further
indicated the importance of DNA-PK in the activation of
GSK3 and the accumulation of -catenin.
In this study, we have uncovered a novel function of
a tissue-specific factor SF-1 that maintains centriole
configuration in adrenocortical Y1 and testicular Leydig
MA-10 cells. In the centrosome, we showed that SF-1
prevented aberrant activation of the DNA-PK/Akt
signaling required for centriole splitting. When SF-1 was
depleted, activated DNA-PK/Akt signaling led to increased
GSK3 phosphorylation followed by the accumulation of
-catenin in the centrosome, causing centriole splitting.
Figure 4 SF-1 controls centrosome homeostasis independent
of its DNA binding domain. (A) Fractionation of centrosome
from FLAG-tagged SF-1 transfected Y1 cells by sucrose gradient
ultracentrifugation. Immunoblots of proteins in fractionated cell
extracts are shown. Cyt: cytoplasmic fraction; Nu: nuclear fraction;
Sup: post-centrosomal supernatant; WCE: whole cell extract; -tub:
-tubulin; hnRNAP: hnRNP A1 (nuclear marker) and Mito II: mitochondria
complex II (cytoplasmic marker). (B) SF-1 overexpression inhibits the
activation of DNA-PK in the centrosome. Centrosomal extracts of EYFP
(control) or shRNA-resistant 3-FLAG-SF-1 (3 F-SF1) overexpressing
shluc (luc) or shsf1#3 (#3) infected Y1 cells were analyzed by
immunoblotting with antibodies against phosphorylated DNA-PKcs
(pPKcs), phosphorylated Akt (pAkt), Akt, SF-1, and Ku70. For the
immunoblot of SF-1, the upper band is exogenous 3 F-SF1; the lower
band is endogenous SF-1 (C) Transcriptional activities of EYFP (negative
control), wild-type SF-1 (positive control), or D70-SF-1 (D70) were
measured in Y1 cells using human CYP11A1 2.3 k promoter linking to
luciferase as a reporter. (D) Quantification of cells with multiple
centrosomes in shluc (luc) or shsf1#3 (#3) infected EYFP, SF-1 or D70
expressing Y1 cells. These results are mean S.D. from three
independent experiments; more than three hundred cells were
measured in each individual group.
SF-1 maintains centriole configuration
SF-1 is an orphan nuclear receptor that regulates the
expression of genes involved in reproduction and
development. In addition to being in the nucleus, SF-1 is also
located in the centrosome controlling centrosome
homeostasis . Here we demonstrated that SF-1 depletion led
to the accumulation of -catenin followed by centriole
splitting. SF-1 restrains the activity of centrosomal
DNAPK/Akt signaling by sequestering Ku from the DNA-PKcs
. When SF-1 is removed from the centrosome,
DNAPKcs is recruited to the centrosome and forms active
complex with Ku. Following the activation of DNA-PK/
Akt signaling, we showed that GSK3 was phosphorylated
and inactivated leading to -catenin accumulation in the
centrosome and centriole splitting. Centriole splitting
is a key step in centriole amplification in response to
DNA damage ; here we also show that centriole
splitting in SF-1-deficient Y1 also contributed to
Our previous study shows that transcriptional activity
of SF-1 is dispensable for its centrosomal function .
In this study, we demonstrated that deletion of DNA
binding domain, SF-1-D70, also prevented centrosome
amplification. The DNA-binding domain of SF-1 binds
DNA, however, it is still unclear whether it also has other
functions in the centrosome. For example, this domain
may function as a docking site of other centrosomal
regulatory components. Here we showed that SF-1-D70
rescued centrosome amplification phenotype in SF-1
deficient cells, thus DNA binding domain is not involved in
the regulation of centrosome homeostasis.
SF-1 maintains adrenogonadal cell growth
SF-1 is a transcription factor that activates the
transcription of adrenal and gonadal genes. In addition, it also
regulates the growth of adrenogonadal cells. SF-1 appears to
regulate cell growth via multiple mechanisms. Our
previous publication shows that SF-1 maintains centrosome
homeostasis and consequently sustains genomic stability
and cell growth . In the nucleus, SF-1 activates genes
that promote cell proliferation . In addition, it also
activates genes involved in glucose metabolism as well as
production of ATP and NADPH, thus maintaining proper
cell growth . In the absence of SF-1, cells do not
produce enough energy; and cell proliferation is
compromised. Thus SF-1 uses multiple methods, including the
activation of genes involved in cell proliferation and
energy production, as well as the assurance of centrosome
homeostasis, to maintain proper cell growth. SF-1 is
located both in the nucleus and the centrosome; the
communication between the nucleus and the centrosome
appears tight. SF-1 can function both in the nucleus and
in the centrosome to maintain proper cell growth. Upon
sensing a change in the environment, SF-1 can act both in
Figure 5 SF-1 interacts with cyclin A and E in the centrosome. (A) Centrosomal and (B) nuclear fractions of Y1 cells transfected with either
empty vector (EV) or 3-FLAG-tagged SF-1 were either immunoprecipitated (IP) with anti-FLAG antibody followed by immunoblotting or directly
immunoblotted with antibodies against FLAG, SF-1, cyclin A (CyA) or cyclin E (CyE).
Figure 6 SF-1 depletion activates DNA-PK signaling and causes
accumulation of -catenin and GSK3 in the centrosome. (A, C)
Centrosomal or (B) whole cell extracts (WCE) of shluc or shsf1#3
lentivirus infected Y1 cells in the presence or absence of DNA-PK
inhibitor, vanillin, were analyzed by immunoblotting with antibodies
against -catenin (-cat), phosphorylated GSK3 (pGSK3), GSK3,
SF-1, -tubulin (-tub), phsophorylated Akt (pAkt), Actin and Hsc70.
the nucleus and in the centrosome to modulate cell
growth. This is probably the function of a master regulator
such as SF-1 in the balance of differentiation and growth
of adrenogonadal cells.
Centrosomal DNA-PK signaling regulates centrosomal
In this report we showed that SF-1 depletion led to
the induction of centriole splitting and the
accumulation of -catenin in the centrosome. -Catenin is
involved in the separation of mitotic spindle poles at
the onset of mitosis . During G2/M transition,
catenin is stabilized in the centrosome to promote
centrosome separation. Aberrant accumulation of -catenin
in the centrosome during G1/S causes centriole
splitting and centriole over-duplication [7,9]. In our study,
we found SF-1 depletion led to accumulation of
catenin in the centrosome. This -catenin accumulation
was not due to G2/M arrest as the cell-cycle profile was
Examining the cause of -catenin accumulation in
SF1-deficient cells, here we uncovered that GSK3 was
phosphorylated by DNA-PK/Akt signaling.
Phosphorylated GSK3 resulted in stabilization of -catenin ,
and -catenin accumulation was blocked by DNA-PK
inhibitor vanillin. Thus the accumulation of -catenin in
the centrosome was due to activated DNA-PK signaling.
DNA-PK is not the only DNA damage regulator that
regulates -catenin. In the nucleus, down-regulation of
Ku70 increases the physical interaction between
catenin and TCF4, therefore inducing the transactivation
of the downstream target genes . Here we show that
the cross-talk between DNA-PK and -catenin in the
centrosome is regulated by SF-1. Thus the function of
catenin in different subcellular compartment is regulated
by distinct mechanism.
SF-1 controls the abundance of cyclin A in the
We have previously shown that cyclin A participates in
centrosome amplification caused by SF-1 depletion .
SF-1 interacted with cyclin A in the centrosome; this
interaction appears to sequester cyclin A. In the absence
of SF-1, cyclin A is recruited to the centrosome and
centrosomal CDK2 activated . CDK2 activity is required
for centrosome duplication . The recruitment of
DNA-PKcs and cyclin A to the centrosome contributes
to centrosome amplification in SF-1 deficient cells. Thus
we propose that SF-1 controls centrosomal CDK2 activity
by preventing aberrant accumulation of cyclin A in the
centrosome (Figure 7).
We found that SF-1 interacted with cyclin A in the
centrosome. SF-1 also interacts with Ku70, which has
been identified as a binding partner of cyclin A in the
centrosome . Thus SF-1 may form a complex with
Ku70 and cyclin A in the centrosome (see our model in
Figure 7). This interaction appears to be independent of
its DNA binding domain, as the SF-1-D70 mutant devoid
of the DNA-binding domain still had normal function in
the centrosome. We have shown that SF-1 depletion
induces cyclin A accumulation and CDK2 activation in the
centrosome . Once SF-1 is depleted, the composition
of the complex would be changed, more cyclin A would
be recruited followed by activation of CDK2 in the
centrosome. This will lead to centrosome amplification. Thus
Figure 7 Proposed model of SF-1 action in the centrosome. In
the centrosome, SF-1 interacts with Ku and cyclin A to prevent the
DNA-PK/Akt signaling cascade. In the absence of SF-1, DNA-PKcs
and cyclin A are recruited to the centrosome, leading to the
activation of DNA-PK and Akt. This signaling cascade leads to GSK3
inactivation and -catenin accumulation in the centrosome, therefore
inducing centriole splitting. On the other hand, accumulation of cyclin
A leads to CDK2 activation, thus facilitating centriole amplification. Solid
arrows: proven activation step. Dotted arrows: indirect activation,
perpendicular lines: inhibitory step.
the role of SF-1 appears to depend on its interaction with
Ku70 and cyclin A so as to sequester them. In the absence
of SF-1, Ku70 will interact with and activate DNA-PKcs,
while cyclin A will recruit and activate CDK2 in the
centrosome. The increased activities of DNA-PK in the
centrosome will lead to centriole splitting followed by
Cell culture and drug treatment
Mouse adrenocortical Y1 and testicular Leydig MA-10 cell
lines were grown in Dulbeccos modified Eagle medium
(DMEM)-F12 medium supplemented with 10% fetal
bovine serum at 37C in a humidified atmosphere at 5%
CO2. Human embryonic kidney 293FT cells were grown
in Dulbeccos modified Eagle medium (DMEM) medium
supplemented with 10% fetal bovine serum at 37C in a
humidified atmosphere at 5% CO2. For drug treatment,
cells were incubated with or without 1 mM vanillin 48 h
before analysis. The performance of all experiments were
approved by Academia Sinica biosafety committee.
Protein depletion or over-expression
A lentiviral system for gene silencing was obtained from
the National RNAi Core Facility (Institute of Molecular
Biology, Academia Sinica, Taipei, Taiwan). Short hairpin
RNA (shRNA)-encoding pLKO.1 vectors were as follows:
A lentiviral system was also used for
tetracyclineinducible protein expression. The pTrip-aOn plasmid
was constructed by inserting the coding sequence of the
Tet-On advanced transcriptional activator into
pTripIRES-neo downstream from the elongation factor 1 alpha
promoter. The plasmids for overexpression of EYFP,
EYFP-D70-SF-1 and 3-FLAG-SF-1 were constructed by
inserting the coding sequence of enhanced yellow
fluorescent protein (EYFP), or wild-type or N terminal truncated
(D70) SF-1 into pAS4w.1.Pbsd, which contains seven
copies of modified tetO sequence.
293FT cells were cotransfected with packaging vectors
pCMVdelR8.91 and pMD.G as well as transfer vectors,
which were either pLKO.1-derived plasmids encoding
shRNA or pAS4w.1.Pbsdbased protein overexpression
plasmids according to the protocols provided by the
Taiwan National RNAi Core Facility. 16 hours after
cotransfection, culture media were removed and fresh
media were added and incubated for an additional 24 h.
The medium were collected and new medium were added
for further 24 h, then the new media were collected and
pooled with previously collected one. The pooled viral
harvests were purified by removing cell debris after
centrifugation at 1250 rpm for 5 min to remove cell debris and
stored at -80C for future use.
For the generation of tetracycline-inducible SF-1
overexpression cells, Y1 cells were infected with pTrip-aOn
lentivirus at a multiplicity of 3 and incubated for 24 h
before G418 (500 g/ml) selection. Infected cells were
selected by G418 for more than one week until no further
cell death was observed, indicating all the un-infected cells
were removed. After selection, pooled G418-resistant cells
were infected again by AS4w based, EYFP-, EYFP-SF1- or
EYFP-SF1-D70-expression lentivirus at a multiplicity of 3
and incubated for 24 h before selection with 10 g/ml
blasticidin. The infected cells were selected again by
blasticidin for more than one week until no further cell death
was observed. The surviving cells were pooled, amplified,
and treated with 1 g/ml doxycycline for 48 h to induce
transgene expression. To eliminate endogenous SF-1
expression, doxycycline and shRNA-encoding shsf1#3
lentivirus were added to the cells simultaneously.
Y1 cells were transiently transfected with
pcDNA5-3FLAG-SF-1 using Lipofectamine and Plus Reagents
(Invitrogen, Carlsbad, CA) according to the manufacturers
instructions. Five microgram pcDNA5-3-FLAG-SF-1 
and 15 l Plus Reagent were first mixed in 500 l
Opti-MEM medium (Life Technologies, Grand Island,
NY) for 15 min, then mixed with pre-mixed 15 l
Lipofectamine in 500 l Opti-MEM medium and incubated at
room temperature for 20 min before the mixture was
layered onto cells in 1 ml DMEM/F12 medium.
To measure SF-1 transcriptional activity, wild-type or
D70 truncated SF-1 stable cells were co-transfected with
a CYP11A1:luciferase reporter plasmid and pRLuc that
encodes Renilla luciferase as an internal control.
Luciferase activities were measured 24 h after transfection. The
firefly luciferase activities were normalized with Renilla
The following antibodies were obtained commercially:
anti--tubulin, polyclonal anti-FLAG, anti-Cyclin A,
monoclonal anti-FLAG M2, anti--tubulin and
anti-acetylated-tubulin (all from Sigma, St. Louis, MO), anti-Cyclin E,
anti-CDK2 phospho-Thr160, anti-Akt and anti-Akt
phospho-Thr308 (Cell Signaling, Beverly, MA), anti-centrin
20H5 (Millipore, Billerica, MA), polyclonal anti--catenin
and anti-GSK3 (Abcam, Cambridge, UK), anti-Ku70
(Genetex, Trvine, CA), anti-DNA-PKcs, and
anti-DNAPKcs phospho-Thr 2609 (Santa Cruz Biotech, Santa Cruz,
CA). The immune sera against SF-1 have been described
Cells were grown on glass coverslips at 37C before
fixation with ice-cold methanol at -20C for 6 min.
To visualize centriolar -tubulin staining, cells were
treated with 30 M nocodazole on ice for 1 h to
depolymerize microtubule networks, followed by brief
extraction with saponin (20 ng/ml) for 2 min and
fixation with ice-cold methanol for 5 min. After
blocking with 5% BSA for 1 h, cells were incubated with
antibodies for 24 h at 4C, washed extensively with
phosphate-buffered saline (PBS), and incubated with
fluorescein isothiocyanate-conjugated and Cy3-conjugated
secondary antibodies (Invitrogen, Carlsbad, CA) and
4, 6-diamino-2-phenylindole (DAPI, 0.1 g/ml) for 1 h in
the dark. After extensive washing, the coverslips were
mounted in 50% glycerol on glass slides. Fluorescent
cells were examined with an AxioImager Z1
fluorescence microscope or an LSM 510 confocal
microscope (both from Zeiss, Oberkochen, Germany). The
numbers of centrosomes and centrioles from more
than 100 cells were counted under the microscope in
three independent experiments and shown as mean
standard deviation. Students t test was performed to
analyze the difference between different groups as
Subcellular fraction and crude centrosomes were
prepared by modifying a published procedure . Briefly,
4 109 cells were treated with nocodazole (10 g/ml)
and cytochalasin B (5 g/ml) for 60 min, followed by
sequential wash with cold PBS, 8% (w/w) sucrose in
0.1 PBS, and 10 mM TrisHCl, pH 8.0. Cells were
then lysed in lysis buffer (10 mM TrisHCl, pH 8.0,
0.5% NP-40 and 0.1% -mercaptoethanol), and the cell
lysate was centrifuged at 2,000 g. The nuclear pellet was
further lysed with lysis buffer containing 0.5% NP-40,
300 mM NaCl, 1 mM EDTA, and the protease inhibitor
cocktail (Roche, Mannhein, Germany).
The cytoplasmic fraction was centrifuged again at
10,000 g for 1 h on a 50% sucrose (w/w) cushion layer.
Supernatant was removed and the resulting sucrose
cushion containing the concentrated centrosome was
centrifuged over a discontinuous 75%, 50% and 40% (w/w)
sucrose gradient at 35,000 g. Samples (300 l/fraction)
were collected from the bottom of the tube and the crude
centrosomes were enriched in sucrose fraction #5 at about
Protein extracts were incubated with specific antibodies
for 1 h at 4C before further incubation with protein-G
beads for 1 h at 4C. The beads were washed with buffer
containing 10 mM TrisHCl, pH 8.0, 120 mM NaCl,
1 mM EDTA, 1 mM PMSF, and the protease and
phosphatase inhibitor cocktail. The samples were eluted with
3-FLAG peptide or sample buffer.
Quantification of immunblots
The images of Western blot analysis on the X-ray film
were scanned and saved as the Tagged Image File (TIF)
format. The intensity of each protein band was quantified
by the Image J software (NIH, Bethesda, MD). The
intensities of protein bands were normalized against those of
the internal controls and shown as relative units.
SF-1: Steroidogenic factor 1; DNA-PK: DNA-dependent protein kinase;
DNA-PKcs: Catalytic subunit of DNA-PK; PCM: Pericentriolar material;
D70-SF-1: SF-1 that lacks the N-terminal DNA-binding domain; Nek2:
NIMA-related protein kinase 2; DMEM: Dulbeccos modified Eagle medium;
DAPI: 4, 6-diamino-2-phenylindole; EYFP: Enhanced yellow fluorescent protein.
C-YW: designed and performed experiments; drafted manuscript. P-YL:
generated Y1 derivatives that stably express shRNA-resistant SF-1. T-YC:
counted proportions of MA-10 cells with split centrioles. BC: designed
experiments and interpreted data; drafted manuscript. All authors read and
approved the final manuscript.
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