Abstract
Classical swine fever virus (CSFV) replicates in macrophages and causes
persistent infection. Despite its role in disastrous economic losses in
swine industries, the molecular mechanisms underlying its pathogenesis
are poorly understood. The virus evades the neutralizing immune
response, subverting the immune system to ensure its own survival and
persistence. Our genome-wide analysis of porcine alveolar macrophage
transcriptional responses to CSFV Shimen infection using the
Solexa/Illumina digital gene expression system revealed that p53
pathway components and cell cycle molecules were differentially
regulated during infection compared to controls. Further, we
investigated the molecular changes in macrophages infected with CSFV
Shimen, focusing on the genes involved in the p53 pathway. CSFV Shimen
infection led to phosphorylation and accumulation of p53 in a
time-dependent manner. Furthermore, CSFV Shimen infection upregulated
cyclin-dependent kinase inhibitor 1A (p21) mRNA and protein. In
addition, CSFV Shimen infection induced cell cycle arrest at the G1
phase, as well as downregulation of cyclin E1 and cyclin-dependent
kinase 2 (CDK2). The expression of genes in the p53 pathway did not
change significantly after p53 knockdown by pifithrin-α during CSFV
Shimen infection. Our data suggest that CSFV Shimen infection increases
expression of host p53 and p21, and inhibits expression of cyclin E1
and CDK2, leading to cell cycle arrest at the G1 phase. CSFV may
utilize this strategy to subvert the innate immune response and
proliferate in host cells.
Keywords: CSFV Shimen, p53 pathway, p21, cell cycle dysregulation,
macrophages, Pathology Section
INTRODUCTION
Macrophages play an important role in both innate and acquired immune
responses [[44]1]. Initially, macrophages are required to kill
microorganisms such as viruses when they enter the body [[45]2].
However, viruses are often able to survive through co-evolution with
their hosts. Macrophages are often attacked during viral infection and
become carriers of the virus, leading to chronic infection [[46]3].
Classical swine fever (CSF), caused by classical swine fever virus
(CSFV), is a highly contagious disease affecting swine and wild boar;
it is listed as a notifiable disease by the World Animal Health
Organization [[47]4]. Acute CSF, caused by a virulent strain of CSFV,
presents with a typical pathology, including hemorrhagic lymphadenitis
and diffuse hemorrhage in the skin, kidneys, and other organs [[48]5].
Over the past few decades, the epidemic of acute CSF has been
effectively controlled by administration of the CSFV C strain vaccine
and culling. However, the incidence of chronic and atypical CSF is
increasing, which makes prevention and control difficult owing to the
inability to distinguish this disease from other pig diseases [[49]6].
A cellular microenvironment that facilitates virus replication is
essential for the establishment of CSFV infection [[50]7]. Although
macrophages play a crucial role in protecting the host from viral
infection, their protective activities are subverted by CSFV [[51]8],
which could cause persistent infection in pigs.
Despite progress in CSFV pathogenesis research, the key mechanism by
which CSFV subverts the immune system is still poorly understood. The
characteristics of the cellular microenvironment are likely to
determine the outcome of the infection. To investigate the mechanism of
CSFV pathogenesis, we performed digital gene expression (DGE) profiling
to identify key genes that regulate signaling pathways in macrophages
during infection. Furthermore, we investigated the effects of viral
infection on the genes involved in transformation related protein
(Trp53 or p53) signaling and cell cycle progression in macrophages.
RESULTS
p53 signaling pathway as the most enriched pathway in CSFV Shimen-infected
macrophages
To determine the extent of proliferation of the CSFV Shimen and C
strains after infection, we assessed total viral RNA content in
infected porcine macrophages (cell line CRL-2843) by real-time
polymerase chain reaction (qPCR) (Figure [52]1A-1B). Both strains
underwent exponential growth in macrophages in the 48-h period after
infection. Given that the CRL-2843 cells maintained their morphology 48
h after infection, we selected this time point for the DGE study. As
the CSFV Shimen and C strains have similar gene sequences and encoded
proteins, we performed high resolution melt analysis to definitively
identify the virus in each sample. As shown in Figure [53]1C, the high
resolution melt results were consistent with those of the qPCR assay
and confirmed infection with either CSFV Shimenor C with no
cross-contamination between the samples.
Figure 1. qPCR and high resolution melt analysis of CSFV Shimen and C strain
proliferation in macrophages.
Figure 1
[54]Open in a new tab
A. qPCR analysis of the proliferation of CSFV Shimenin macrophages. B.
qPCR analysis of the proliferation of CSFV C strain in macrophages. C.
High resolution melt analysis of the proliferation of CSFV Shimenand C
strains in macrophages in the absence of cross-infection. The results
are representative of three independent experiments, and data is
expressed as mean ± SEM.
To under stand the global molecular changes caused by infection with
CSFV Shimen and C strains, we used the DGE system to explore the
transcriptomes of macrophages infected with the two strains and an
uninfected control. We constructed cDNA libraries for uninfected and
CSFV (Shimen and C)-infected macrophages and analyzed their
characteristics by high-throughput Illumina sequencing
([55]Supplementary Table 1). All tags were aligned to the reference
sequences in the NCBI UniGene Susscrofa database, and gene products
with high homology to Susscrofa were identified by BLAST
([56]Supplementary Table 2) [[57]9].
To identify genes associated with CSFV Shimen infection, we analyzed
genes that were differentially expressed among the 3 samples. As shown
in [58]Supplementary Figure 1, we observed differential expression of
1,676, 3,074, and 2,525 genes in the three comparisons (Shimen vs.
control, C vs. control, and Shimen vs. C, respectively) (false
discovery rate ≤ 0.001 and |log2Ratio| ≥ 1). We performed pathway
analysis on the differentially expressed genes,using the Kyoto
Encyclopedia of Genes and Genomes (KEGG) database,to gain insight into
their functions. We found that the most significantly enriched pathway
in the CSFV Shimen-infected sample compared to that in the control was
the p53 signaling pathway (Figure [59]2). Interestingly, we did not
detect a significant difference in the p53 signaling pathway when
comparing CSFV C-infected and control samples ([60]Supplementary Figure
2). The cell cycle pathway was also differentially regulated in the
CSFV Shimen-infected and control samples. These results suggested that
upregulation of the p53 signaling pathway by CSFV Shimen in macrophages
may mediate their response to infection by modulating the cell cycle.
Figure 2. Pathway enrichment analysis in CSFV Shimen-infected versus control
macrophage samples.
[61]Figure 2
[62]Open in a new tab
CSFV Shimen promotes phosphorylation and expression of p53 in macrophages
We next examined whether CSFV Shimen infection influenced p53
expression in macrophages. We compared protein expression in both CSFV
Shimen- and mock-infected macrophages. We observed a marked
time-dependent increase in CSFV Shimen E2 protein expression in whole
cell lysates of CSFV Shimen-infected cells (Figure [63]3A), which was
absent in mock-infected cells, consistent with the qPCR analysis
(Figure [64]1). Next, we investigated the levels and activity of p53 in
infected macrophages. We found that Shimen infection promoted the
accumulation of total cellular p53 3 h post-inoculation, and the levels
remained elevated up to 12 h post-inoculation. Importantly, an increase
in the level of phosphorylation of p53 at serine 15was observed in CSFV
Shimen-infected macrophages at 0, 3, 6, 12, 24, or 48 hpost-infection
(Figure [65]3A). We did not detect phospho-p53(Ser15) in the negative
control at any of the time points tested (Figure [66]3A).
Figure 3. CSFV Shimen promoted the phosphorylation and expression of p53 in
macrophages.
[67]Figure 3
[68]Open in a new tab
A. Macrophages were infected with CSFV Shimen for the indicated time.
At the end of the infection, the expression of CSFV E2, p53,
phospho-p53(ser15), and β-actin (loading control) were analyzed by
immunoblotting with specific antibodies. The levels of the proteins at
0 h were considered 1-fold. Control group, mock-infected cells. B.
Co-localization of CSFV E2 protein with phospho-p53(ser15) in
macrophages infected with CSFV Shimen. After 24 h, macrophages were
fixed with methanol and stained with phospho-p53(ser15) and CSFV E2
antibodies. Green, CSFV E2 fluorescein isothiocyanate-labeled antibody
staining; red, phospho-p53(ser15)
tetramethylrhodamineisothiocyanate-labeled antibody staining. The
position of the nucleus is indicated by DAPI staining in blue. Merged
images show significantly enhanced fluorescence representing
phospho-p53(ser15) in macrophages corresponding to time post-infection
with CSFV Shimen. The images are representative of 3 experiments with
similar results. Bar = 10 μm, except for the enlarged figures.
To assess the relationship between activated p53 and CSFV Shimen E2
protein, we stained macrophages at various time points post-infection
with fluorescently labeled antibodies against CSFV Shimen E2 and
phospho-p53(Ser15) and tracked their cellular localization by confocal
microscopy. We observed co-localization of CSFV Shimen E2 protein and
phospho-p53(Ser15), and the expression of both molecules
significantlyincreased over the course of CSFV Shimen infection (Figure
[69]3B). Our results suggested that CSFV Shimen infection activated the
p53 signaling pathway.
CSFV Shimen infection induces p21 expression
Given that CSFV Shimen infection led to p53 accumulation and
phospho-p53(Ser15) activation, we next examined which downstream
molecules in the p53 signaling pathway were upregulated by infection.
The DGE data revealed 3-fold higher expression of cyclin-dependent
kinase inhibitor 1A (Cdkn1a or p21) in CSFV Shimen-infected macrophages
than in controls 48 h post-infection (Figure [70]4A), suggesting
increased p21 biosynthesis in virus-infected macrophages compared with
that in controls. Increased transcription of p21, a downstream target
of p53 [[71]10], likely follows activation of p53 during CSFV Shimen
infection. We analyzed the expression of p21 mRNA and protein in
macrophages after infection with CSFV Shimen. As shown in Figure
[72]4B, p21 transcript levels were significantly higher in CSFV
Shimen-infected cells than in controls (up to 4.63 ± 0.20-fold). We
also determined p21 protein expression levels in macrophages after
infection with CSFV Shimen. Importantly, p21 accumulated in
virus-infected macrophages (Figure [73]4C). We did not observe
significant changes in p21 accumulation in the control cells.
Figure 4. CSFV Shimen infection increased the expression of p21 mRNA and
protein in macrophages.
[74]Figure 4
[75]Open in a new tab
A. DGE analysis of macrophages revealed that p21 correlated with CSFV
Shimen infection when compared to CSFV C infection and negative
controls (mock-infected cells). B. Analysis of p21 mRNA levels by qPCR
on macrophages infected with CSFV Shimen and controls. β-actin was
probed as the loading control. Control group, mock-infected cells. C.
Effects of CSFV Shimen infection on p21 protein expression. Macrophages
were infected with CSFV Shimen for 0, 12, 24, and 48 h. At the end of
the infection, the expression of CSFV E2, p21, and β-actin (loading
control) were analyzed by immunoblotting with specific antibodies. The
levels of indicated proteins at 0 h were considered 1-fold. Control
group, mock-infected cells.
CSFV Shimen decreases cyclin E and CDK2 expression, resulting in cell cycle
dysregulation
The cell cycle pathway is tightly regulated by a p53
transcription-dependent mechanism. As the DGE pathway analysis showed
that CSFV Shimen infection activated the cell cycle pathway of
macrophages (Figure [76]2), we wanted to investigate whetherdownstream
cell cycle molecules were suppressed in infected cells. We found that
the increase in E2 protein correlated with a decrease in cyclin E
levels, as measured by western blot, in a time-dependent manner (Figure
[77]5A). The expression of cyclin E was not altered in the control
cells. In addition, we observed a dramatic reduction in CDK2 expression
in CSFV Shimen-infected cells (Figure [78]5A). The G1-to-S phase cell
cycle transition is controlled by specific interactions between cyclin
E and CDK2. To assess the effects of CSFV Shimen infection on this
transition, we synchronized cells in pseudo-G0 phase by serum
withdrawal [[79]11] before viral infection. Macrophages were collected
at 48 h post-infection, with uninfected macrophages serving as
controls. Flow cytometric analysis revealed that CSFV Shimen infection
suppressed the cell cycle in macrophages and retained cells in the G1
phase (Figure [80]5B).
Figure 5. CSFV Shimen decreased cyclin E and CDK2 expression, which promoted
cell cycle arrest.
[81]Figure 5
[82]Open in a new tab
A. Macrophages were infected with CSFV Shimen for 0, 12, 24, and 48 h.
At the end of the infection, the expression of CSFV E2, cyclin E, CDK2,
and β-actin (loading control) were analyzed by immunoblotting with
specific antibodies. The levels of the proteins at 0 h were considered
1-fold. Control group, mock-infected cells. B. G1 arrest induced by the
infection of CSFV Shimen in macrophages. Cell cycle status was
determined by flow cytometry, as described in Materials and Methods.
The results are representative of three independent experiments.
CSFV Shimen induces cell cycle arrest via p53
Our results suggested that CSFV Shimen infection induced
phosphorylation of p53(Ser15) and expression of p21. We treated cells
with pifithrin-α (PFT-α), a specific inhibitor that blocks
transcription of p53-responsive genes, to investigate the role of p53
in CSFV Shimen-induced cell cycle arrest [[83]12]. We examined the
levels of p53 and phospho-p53(Ser15) in macrophages after PFT-α
treatment. We found, by western blot, that treatment diminished p53
protein expression (Figure [84]6, left panel). We also observed that
pre-treatment of macrophages with PFT-α blocked the CSFV Shimen-induced
phosphorylation of p53 and expression of p21 (Figure [85]6, middle
panel). We did not detect alterations to the levels of cyclin E and
CDK2 in PFT-α treated cells (Figure [86]6, middle panel) compared to
the levels in infected cells without PFT-α treatment (Figure [87]6,
right panel).
Figure 6. Activation of p53 in CSFV Shimen-infected macrophages is required
for cell cycle arrest.
[88]Figure 6
[89]Open in a new tab
To determine the effect of p53 in CSFV Shimen-induced cell cycle
arrest, p53 was depleted with the inhibitor PFT-α in CSFV
Shimen-infected and mock-infected macrophages. A significant reduction
in p53 protein synthesis was detected in the presence of PFT-α (Lane 1
in the left panel and the middle panel). In the middle panel,
pre-treatment of macrophages with PFT-α blocked CSFV Shimen-induced
phosphorylation of p53 and blocked the upregulation of p21. The levels
of cyclin E and CDK2 were not reduced and no significant changes were
observed in PFT-α-treated cells (middle panel), compared to CSFV
Shimen-infected cells without PFT-a treatment (the right panel).
Taken together, our findings demonstrated that CSFV Shimen increased
p53 signaling, leading to upregulation of p21 and concomitant
downregulation of cyclin E1 and CDK2 in a time-dependent manner,
resulting in cell cycle arrest at the G1 phase.
DISCUSSION
CSF is one of the most severe diseases that affect pigs worldwide, with
massive economic consequences [[90]13]. Macrophages are the immune cell
type primarily responsible for the phagocytosis of pathogens and
activation of the immune system. However, macrophages are also target
cells for CSFV, and the migration and diffusion of macrophages in vivo
is an important mechanism of infection for CSFV [[91]14]. Thus,
identification of the mechanisms by which viruses subvert the immune
system could provide insight into virus infection strategies.
We investigated the mechanism of CSFV infection of macrophages by DGE.
We compared macrophages infected with the CSFV Shimen strain to cells
infected with the CSFV C strain and uninfected controls to elucidate
the mechanism of persistent CSFV infection. CSFV Shimen is a virulent
strain, which causes the typical clinical symptoms of swine fever
[[92]15]. CSFV C is an attenuated strain that does not cause
pathological symptoms [[93]16]. Comparison of the differences in gene
regulation between CSFV Shimen infection and C strain or mock infection
by DGE analysis facilitates an understanding of the pathogenesis of the
infectious strain.
In this study, macrophages with CSFV Shimen, CSFV C, or mock infection
were analyzed after 48 h by DGE. We identified differential responses
to CSFV Shimen in the host macrophages compared to that in the
[94]controls.Interestingly, pathway analysis suggested that CSFV Shimen
triggered the accumulation and activation of p53, which promoted cell
cycle arrest in macrophages.
The p53 signaling pathway plays an important role in DNA damage repair
[[95]17];when DNA is damaged or cell proliferation is abnormal, p53 is
activated, resulting in cell cycle arrest and DNA repair. Recent
studies have demonstrated that when a cell is infected by a virus, p53
is involved in determining the fate of the cell [[96]18]. Viruses
activate or interfere with the p53 signaling pathway at different
levels to achieve proliferation. Hepatitis B virus interferes with the
function of p53 by blocking its nuclear localization and binding with
DNA [[97]19]. The core protein of hepatitis C virus activates p53
during the infection process [[98]20]. Avian reovirus p17 protein
regulates cell cycle and autophagy by activating the p53/PTEN pathway
[[99]21]. Infectious bursal disease virus infection increases the
activity of chicken p53, which is targeted by gga-miR-2127 [[100]22]. A
recent study indicates CAV1-mediated endocytosis is advantageous for
productive CSFV Shimen infection in macrophages [[101]8]. As a
principal component of caveolae membranes in vivo, caveolin-1
expression negatively regulates cell cycle progression by inducing
G0/G1 arrest via a p53/p21WAF1/Cip1-dependent mechanism [[102]23]. The
reported researches are beneficial to a deep-going understanding to the
activation of p53 signaling pathway by CSFV Shimen.
Our observations were consistent with those in a previous report
[[103]24], which suggested that activation of p53 causes accumulation
of p21, a cyclin-dependent kinase inhibitor, and eventual induction of
downstream target genes. Both p21 and p53 cause cell cycle arrest at
the G1 checkpoint, which promotes DNA repair by allowing sufficient
time for damaged DNA to be repaired before it is passed to daughter
cells [[104]25, [105]26]. The mechanisms used by viruses to manipulate
p53 and induce p21 expression are complex and diverse. For example,
hepatitis B virus X protein can circumvent the need for p53 and induce
transcription of the gene for p21 [[106]27], while hepatitis C virus
NS2 protein cooperates with p53 to inhibit DNA damage in cells but
cannot induce the expression of p21 [[107]28].
The main target of p21 is the cyclin E-CDK2 complex, in addition to the
cyclin D-CDK4/6 complex [[108]29]. Cyclin E participates in the
regulation of cell cycle progression [[109]30]. If the same signaling
event causes the synthesis of cyclin E, the GI-to-S phase transition is
initiated [[110]31]. The Cyclin E-CDK2 complex is inhibited by p21
during the progression from G1 to S phase [[111]32]. Cells regulate the
p53 pathway, which controls the cell cycle, in order to maintain their
stability and modulate their differentiation [[112]33]. Researchers are
investigating the infection strategies that facilitate viral
proliferation by manipulating the cell cycle. However, research into
the role of p53 signaling pathway in CSFV infection is still in its
nascent stage. Pathway analysis of our DGE data indicated CSFV Shimen
infection induced significant differences in the p53 signaling pathway
and cell cycle response compared to the mock- and C strain-infection.
Bioinformatics analysis highlighted the importance of the p53 signaling
pathway in CSFV Shimen infection. We found that CSFV Shimeninfection in
macrophages promoted phosphorylation of p53(Ser15) and increased the
expression of p21. Thus, infection with CSFV Shimen activated p53,
resulting in upregulation of p21, inhibition of cyclin E-CDK2, and,
ultimately, cell cycle arrest, which could facilitate persistent
infection (Figure [113]7).
Figure 7. Proposed mechanism for upregulated p53 signaling to promote cell
cycle arrest in porcine alveolar macrophages induced by CSFV Shimen.
[114]Figure 7
[115]Open in a new tab
In conclusion, the present DGE study showed that the p53 signaling
pathway was significantly altered in response to CSFV Shimen infection
in macrophages. We observed an increase in p53 and p21 levels in CSFV
Shimen-infected macrophages, an arrest of the cell cycle, and cyclin E
and CDK2 downregulation. Furthermore, we showed that the effects of
both p53 and genes involved in the p53 pathway could be inhibited by
treatment with PFT-a. We concluded that CSFV Shimen infection may be
responsible for enhanced activation of the p53 pathway and is involved
in G1 arrest in response to p21 activation. The p53 signaling pathway
appears to be the most important regulatory pathway for CSFV Shimen in
macrophages, and most likely represents the key mechanism used by the
virus to escape host immune clearance and establish persistent
infection.
MATERIALS AND METHODS
Culture of macrophages, CSFV infection, and RNA isolation
The porcine alveolar macrophage cell line 3D4/21 (CRL-2843) was
obtained from the American Type Culture Collection (Manassas, VA, USA)
[[116]8]. The CSFV Shimen and C strains were obtained from the National
Control Institute of Veterinary Bio products and Pharmaceuticals
(Beijing, China). Macrophages were cultured in 25-cm2 tissue culture
flasks at a density of 2 × 107 cells per flask, and the CSFV Shimen and
C strains were added to the cultures at a multiplicity of infection of
5 (TCID50) when macrophages were 70%-80% confluent [[117]8]. After 1 h
of incubation at 37°C in 5% CO2, the medium was aspirated, fresh medium
containing 2% fetal calf serum was added, and the cultures were
incubated for 48 h in 5% CO2. High resolution melt analysis was
conducted to confirm infection with CSFV Shimen and C strains, and qPCR
was carried out to detect the proliferation of CSFV [[118]34]. Total
RNA was isolated from CSFV-infected macrophages and control samples at
48 h post-infection, using TRIzol® reagent (Invitrogen, Carlsbad, CA,
USA), according to the manufacturer’s protocol. RNA yields were
determined by absorbance of samples at 260 nm using a NanoDrop
(ND-2000; NanoDrop Technologies, Wilmington, DE, USA). An Agilent 2100
Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) was used to
evaluate RNA integrity. CSFV Shimen-infected, CSFV C-infected, and
control macrophage samples were separately subjected to DGE profiling
based on Solexa sequencing.
Solexa sequencing and DGE tag profiling analysis
We created cDNA librariesby our previous report [[119]9], using the
Illumina Gene Expression Sample Prep kit (Illumina, San Diego, CA,
USA). Raw reads were filtered through the Illumina pipeline to obtain
clean tags, which were normalized to the number of transcripts per
million clean tags [[120]9, [121]35]. The following formula was used to
demonstrate the probability that one gene was equally expressed in two
samples [[122]36]:
[MATH: p(y|x)=(N2N
1)y(x+y)!x!y
!(1+N2N1)
(x+y+1)
:MATH]
wherex and y indicate the clean tags mapping to the gene. N1 and N2
represent the total number of clean tags in two compared libraries. The
p-value corresponds to differential gene expression. The false
discovery rate determines the threshold of the p-value in multiple
tests and analyses [[123]37]. In this study, the statistical
significance of differences in gene expression were determined using
the threshold false discovery rate ≤ 0.001 and the absolute value of
log2 ratio ≥ 0.
Pathway enrichment analyses identify significantly enriched metabolic
pathways or signal transduction pathways from differentially expressed
gene data by the following formula [[124]38, [125]39]:
[MATH: p=1−∑i=0m−1<
mrow>(Mi)(N−Mn−i)(Nn) :MATH]
whereN is the total number of genes with KEGG functional annotations,
and n is the number of differentially expressed genes in N. M is the
number of the genes with specific KEGG annotations, and m is the number
of differentially expressed genes in M.
Western blot analysis
Protein extraction and western blot were performed as previously
described [[126]40, [127]41]. Protein concentration was determined
using a BCA Protein Assay Kit (Cowin Biotech, Beijing, China).
Equivalent amounts of proteins were subjected to 12% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and transferred to
polyvinylidenedifluoride membranes (Millipore, Atlanta, GA, USA). The
membranes were blocked for 2 h at 25°C± 2°C in Tris-buffered saline
containing 0.05% Tween® 20 and 5% nonfat milk, and incubated with
specific primary antibodies raised against p53 (1C12; Cell Signaling
Technology, Beverly, MA, USA), phospho-p53(Ser15) (Cell Signaling
Technology), p21Waf1/Cip1 (12D1; Cell Signaling Technology), cyclin E1
(HE12; Cell Signaling Technology), CDK2 (M2; Santa CruzBiotechnology,
Santa Cruz, CA, USA), and β-actin (Tianjin Sungene Biotech, Tianjin,
China) at 4°C overnight, and with the corresponding secondary antibody
conjugated to horseradish peroxidase at appropriate dilutions for 1 h
at 37°C. Images were captured using a GeneGnome XRQ chemiluminescence
detector (Syngene, Cambridge, UK); the densities of the protein bands
were normalized to the β-actin signal and quantified using GeneSys
software (VilberLourmat, France). The abundance of the proteins of
interest in the various treatments was expressed relative to the
abundance under control conditions.
Confocal immunofluorescence microscopy
CSFV-infected cells were washed in phosphate-buffered saline (PBS) and
fixed with methanol/acetone (1:1) for 20 min at 25°C ± 2°C and
permeabilized with 1% Triton X-100 in PBS for 10 min. After three
washes in PBS, samples were incubated with mouse anti-E2 antibody
(MssBio, Guangzhou, China) and rabbit anti-phospho-p53(Ser15) (Cell
Signaling Technology) for 1 h at 25°C ± 2°C, followed by staining with
donkey anti-rabbit IgG conjugated to Alexa Fluor® 594 and donkey
anti-mouse IgG conjugated to Alexa Fluor® 488 (YEASEN, Shanghai, China)
at a 1:200 dilution for 1 h at 25°C ± 2°C. After incubation with 4′,
6-diamidino-2-phenylindole (DAPI), the samples were observed using a
laser-scanning confocal microscope (LSM 510 META; Carl Zeiss, Jena,
Germany).
Flow cytometry
After infection or mock infection, cells were harvested and fixed in
70% ethanol for 30 min on ice. Cells were washed, pelleted in PBS, and
resuspended in propidium iodide solution (50 mg/mL) containing 0.1
mg/mL RNase for 40 min at 37°C. Flow cytometry was performed to examine
cell cycle status, using a Coulter® EPICS® XL/MCL Flow Cytometer
(Beckman Coulter, Hialeah, FL, USA). We acquired 10,000 events for each
sample. Data were analyzed using Expo32 ADC software (Beckman Coulter,
Miami, FL, USA).
qPCR analysis
We performed qPCR on a Bio-Rad iQ™5 system (Bio-Rad, California, USA)
with SYBR® Premix Ex Taq™ II (TaKaRa, Dalian, China), on the same RNA
samples used for the DGE experiments. We synthesized cDNA, using the
Transcriptor First Strand cDNA Synthesis Kit (TaKaRa, Dalian, China),
according to the manufacturer’s instruction. Each cDNA sample was
analyzed in triplicate, and the average threshold cycle (Ct) was
calculated per sample. The 2-ΔΔCt method [[128]42] was applied to
calculate the relative expression levels among CSFV-infected and
control samples.
Statistical analysis
Statistical analyses were conducted by one-way or two-way analysis of
variance, using the Statistical Package for the Social Sciences (SPSS)
16.0 (SPSS, Chicago, IL, USA), and differences with a p-value < 0.05
were considered statistically significant. Data are shown as the mean ±
standard error of mean (SEM) of three independent experiments.
SUPPLEMENTARY MATERIALS FIGURES AND TABLES
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[130]oncotarget-08-55938-s002.xls^ (7.9MB, xls)
Acknowledgments