Materials and Methods
Animals. Generation of Plscr1 Knockout mice were
generated using the clustered regularly interspaced short palindromic
repeat (CRISPR)-associated Cas9 nuclease (CRISPR/Cas9) genome editing
technique as previously described(Yang , 2013). Briefly, C57BL/6 female
mice (7–8 weeks old) were superovulated by intraperitoneally injecting
pregnant mare serum gonadotropin (PMSG) and human chorionic
gonadotrophin (hCG) and then mated to C57BL/6 male mice. The fertilized
embryos (zygotes) were collected from the oviducts, and mixed Cas9 mRNA
(50 ng/µl) and small guide RNA (sgRNA; 25 ng/µl) were injected into the
cytoplasm of zygotes with visible pronuclei in Chatot-Ziomek-Bavister
(CZB) medium. The injected zygotes were then cultured in Quinn’s
Advantage cleavage medium (in vitro Fertilization, Inc.) for 24 h, at
which time 18–20 2-cell–stage embryos were transferred into the
oviduct of a pseudopregnant ICR female mouse at 0.5 day post coitus
(dpc). The accession numbers of the Plscr1 cDNAs used to design sgRNA is
NM_011636.2.
To determine the nucleotide sequence of mutated alleles, genomic DNA of
F0 mice was amplified using the following primers: forward,
5, -GGTGATCTCGATTCAGGGGT-3, ,
reverse, 5, - GGGGTTACTCGACCCTAAAA
-3,. DNA sequencing was then performed after TA
cloning into plasmid pMD19T. In order to obtain F1 knockout mice, F0
mice were crossed with C57BL/6 mice and newborns were examined by Sanger
sequencing .All animal experiments were approved by the Shandong Academy
of Agricultural Science and conducted accordingly. All methods were
performed in accordance with the relevant guidelines and regulations.
Virus. SIV(QD-2018 strain) was a H1N1 strain that isolated and
obtained from the diseased pigs in Shandong province of China, which can
be used as one of representative strains for the analysis of variant
strains. The virus was continuously passaged on MDCK cells and virus
titer was as high as 104.5 TCID50/mL.
Cell culture, transfection. HEK 293T ,COS7 and A549 cells were
maintained with 10% FBS DMEM medium at 37°C under 5%
CO2, and transfected with expression vectors or siRNAs
using
Lipofectamine
2000 Transfection Reagent (thermofisher) according to the
manufacturer’s instructions.
Plasmid construction. The cDNAs encoding the mouse Ildr1and Plscr1 were cloned into pmCherry-N1, pEGFP-C2, and pMyc-C2
(modified pEGFP-C2 with EGFP-coding sequence replaced by Myc-coding
sequence) as we have reported. All the constructs were verified by
Sanger sequencing.
Western blot. Cultured cells were transfected with expression
vectors as described above or siRNAs synthesized by the Sigma-Aldrich
company, then protein was resuspended with RIPA cell lysis containing 1
mM PMSF (Beyotime; Shanghai, China). After centrifuging at 4℃ for 20
min, the supernatant was analyzed by western blot. Protein samples were
resolved by 10% SDS-PAGE, then transferred to a PVDF membrane. After
blocking with 5% BSA buffer for 1 h, the membrane was incubated with
Rabbit Polyclonal-PLSCR1 Polyclonal Antibody (Proteintech,
Cat#11582-1-AP,1:1000 diluted), Rabbit Polyclonal-Anti-ILDR1 antibody
(Abcam, Cat#ab89847,1:1000 diluted), Rabbit Polyclonal-Anti-H1N1
Influenza A virus Nucleocapsid protein
antibody(Abcam,Cat#ab104870,1:1500 diluted), rabbit anti-GAPDH
antibody(Abcam,Cat.#ab181602, 1:5000 diluted), rabbit anti-LaminB1
antibody(Abways,Cat.#AB0054, 1:3000 diluted) at 4℃ over night, followed
by incubation with goat anti-mouse secondary antibody or goat
anti-rabbit secondary (Cell Signaling Technology, Danvers, MA) at room
tempreture for two hours. The signals were detected with the ECL system
(Cell Signaling Technology, Danvers, MA).
RNA extraction, RT-PCR and Quantitative real-time PCR. Total
RNA was isolated from virus-infected cells or mouse tissues and cDNA was
carried out by reverse transcription (RT) following the kit instructions
(TaKaRa Bio Inc., Dalian, China). The expression of gene or virus was
analyzed by quantitative real-time PCR (SYBR® Premix Ex TaqTM system,
Takara). The primers used were as follows: Ildr1 forward primer,
CCGGCGGCTGATGAAGAAAGACTC, reverse primer, AGGGCAGCAACAGCGGGTAGGA;Plscr1 forward primer, GTGGGGCGTC TAGACCTTTC, reverse primer,
CCAGGCATCACAGGTGAGTT; H1N1 forward primer, ACAGAAGTTATAAGAATGA, reverse
primer, TGTCTCCGAAGAAAT AAGA;β-actin forward primer,
ACGGCCAGGTCATCACTATTG, reverse primer, AGGGGCCGGACTCATCGTA. PCR reaction
system and procedures were refered to Premix Taq kit instructions
(Takara). PCR reaction sets were adjusted between 24 and 36 cycles , and
annealing temperatures were adjusted between 55 and 60 °C. The PCR
products were separated by electrophoresis on agarose gel.
Quantitative real-time PCR was carried out using SYBR® Premix Ex TaqTM
system (Perfect Real Time, Takara). The primers and template were the
same as that used in RT-PCR. Amplifiation and detection were run in a
Roche 480 Sequence Detection System with an initial cycle of 95 °C for
10 s followed by 40 cycles of 95 °C for 5 s, 62 °C for 10 s and 72 °C
for 5 s. All PCR reactions were performed in triplicate. Fold change in
gene expression level was calculated using the 2-ΔΔctmethod and all PCR reactions were performed in triplicate.
Immunofluorescence assay. Infected cells or transfected cells
with GFP- or mCherry-tagged proteins growing on Gelatin-coated glass
cover slips were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min
and blocked with PBT1 buffer (0.1%Triton X-100, 1% BSA, 5%
heat-inactivated goat serum in PBS, pH 7.3) for 30 min. Cells were
incubated overnight at 4°C with corresponding diluted in PBT1 then
washed twice with PBS for 10 min. And cells were incubated with
FITC-conjugated secondary antibody diluted in PBT2 (0.1% Triton X-100,
0.1% BSA in PBS) for 1 h, followed by washing with PBS three times for
10 min. For nuclei staining, cells were incubated with DAPI (Solarbio
Life Sciences) for 15 min, followed by three 10 min PBS washes, then
mounted in 50% glycerol/PBS. The cells were imaged with an inverted
fluorescence microscope(TE200, Nikon).
Immunohistochemistry. The tissues were fixed in 10% formalin
solution, embedded in paraffin, sectioned to 4 μm thicknesses and
dewaxed the paraffin sections to water. Then the sections were placed in
a retrieval box filled with EDTA antigen retrieval buffer (PH8.0) in a
microwave oven for antigen retrieval. After that, the sections were
blocked with 5% BSA for 30 min at room temperature. The section were
incubated with anti-PLSCR1antibody (1:50) overnight at 4°C. After a
brief wash , the secondary antibody were used for detection at room
temperature for 50 min and DAPI dye solution was added for 10 min in the
dark . The images were taken using a light microscopy (Nikon Eclipse
C1). For the controls, no antibody was added to the samples.
Cell fractionation. The PLSCR1-overexpressing or empty
retrovirus-transduced control A549 cells grown in 6-well plates were
infected with WSN virus at an MOI of 5. At 6 h p.i., the cells were
separated into nuclear and cytoplasmic fractions by using Minute Nuclear
and Cytoplasmic Extraction Reagents (SC-003, Invent) according to the
manufacturer’s procedure. The amount of NP, ILDR1 and PLSCR1 in each
fraction were determined by western blotting with a rabbit anti-NP pAb,
a rabbit anti-ILDR1 pAb and a rabbit anti-PLSCR1 pAb, respectively.
LaminB1 and GAPDH, nuclear and cytoplasmic fraction markers,
respectively, were detected by western blotting with a rabbit anti-GAPDH
pAb and a rabbit anti-LaminB1 pAb, respectively.
Co-immunoprecipitation (co-IP). HEK293T cells were transfected
with expression vectors as described above, then washed twice with PBS
24 h after transfection and resuspended in ice-cold lysis buffer
containing 150 mM NaCl, 50 mM Tris at pH 7.5, 0.1% Triton X-100, and
1×cocktail (Roche, Basel, Switzerland). After centrifuging at 4°C for 20
min, the supernatant was collected and incubated with immobilized
anti-Myc or anti-EGFP antibody at 4°C overnight. Immunoprecipitated
proteins were washed three times with 300 mM lysis buffer and then
analyzed by western blot.
Statistical analyses. All data were expressed as means ±
standard deviation (SD), and an independent-sample t -test was
used to evaluate data using Graph Prism software. *p<0.05,
**p<0.01, ***p<0.001.