Cell lines and cell culture
The mouse ESC lines used were E14 and A17-2loxP mESCs (gift from Dr. Thomas P. Zwaka). Mouse ESCs were maintained under feeder-free conditions, on culture dishes coated with 0.1% gelatin (Sigma-Aldrich). Cells were cultured in Knockout DMEM (Gibco) supplemented with 15% (v/v) fetal bovine serum (Hyclone, Australia), 0.1 mM β-mercaptoethanol (Sigma), 2 mM GlutaMAX, 0.1 mM minimum essential medium (MEM) non-essential amino acids (Sigma), 100 U/mL penicillin (Gibco), 100 μg/mL streptomycin (Gibco), 1000 U/mL LIF (Millipore), 1 μM PD0325901 (Stemgent), and 3 μM CHIR99021 (Stemgent). 293T cells were from ATCC company (America) and cultured in DMEM (Corning) supplemented with 10% (vol/vol) FBS (Excell, Australia), 100 U/mL penicillin (Gibco), and 100 μg/mL streptomycin (Gibco).
Stable cell line generation
Sequence encoding Cas9, murine Oct4, Sox2, Ddx56, and EGFP were cloned into pENTR. Ddx56 domain truncations were generated by overlap PCR on pENTR-Ddx56. Cas9, Oct4, Sox2, EGFP, Ddx56, and its domain truncations were ligased to pDEST27 or pBabe by gateway® LR clonase reaction (Thermo Fisher) for immunoprecipitation experiments. A17-2loxP mESCs have a doxycycline-inducible promoter and two loxP loci near HPRT gene. Cas9, Ddx56, and its domain truncations were ligated to p2loxP plasmid, then cotransfected with pSalk-Cre plasmid which codes cyclization recombination enzyme (Cre) into A17-2loxP mESCs by Lipofectamine 2000 (Invitrogen). Positive ESC clones were selected by G418 and detected by western blot after adding doxycycline for 48 h. A pair of gRNA oligonucleotides with 5′-CACC and 3′-AAAC overhang was synthesized, annealed, and ligased to vectors (px330, px458, plenti-gRNA1-BSD, and plenti-gRNA2-Hygro). To get inducible knockout Ddx56 cell lines, plenti-gRNA2-BSD and plenti-gRNA2-Hygro were transfected into A17-2loxP-Cas9 cells via retroviral transfection system. The Ddx56 gRNA sequences were as follows: gRNA1, GCCATTCCTCTGGCGCTGG; gRNA2, GTGGTCTGTGAGACAGAAG. Target sites of Ddx56 were PCR amplified using primers in Additional file 1: Table S1. The PCR products were then used in T7 endonuclease I (T7EI) cleavage assay.
E14 cells were transfected with siRNA oligos targeting Ddx56 using RNAi Max (Invitrogen) and harvested 48 h after transfection. The small interfering RNA (siRNA) oligos were purchased from Guangzhou IGE Biotechnology Ltd., and their sequences are listed below:
Quantitative real-time PCR (qRT-PCR)
Total RNA was isolated using phenol-chloroform. The following cDNA synthesis was performed with PrimeScript™ RT Reagent Kit (TaKaRa), and qRT-PCR using GoTaq® qPCR Master Mix (Promega) was performed using qPCR primers with ABI StepOnePlus Real-Time PCR System. The primers are listed in Additional file 1: Table S1.
Co-immunoprecipitation (co-IP) and western blotting
293T cells were transfected with various expression vectors and harvested 48 h after transfection. Cells were lysed in 1× NETN buffer (40 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.5% NP40, 1 mM EDTA, 10% glycerol, containing protease inhibitors). The supernatant was incubated with Glutathione Sepharose 4B for 4 h at 4 °C. The immunoprecipitates were then eluted by SDS loading buffer. Whole cell lysates were also obtained by direct lysis of cells in SDS loading buffer. All samples were resolved by SDS-PAGE and transferred onto nitrocellulose filter membrane (Millipore) for blotting with appropriate antibodies. Antibodies for western blotting are anti-Flag (mouse) (Abmart M20008M), anti-GAPDH (mouse) (Proteintech group 60004-1-Ig), anti-GST (Rabbit) (Homemade), anti-mouse 680 (LI-COR 926-32220), and anti-rabbit 800 (LI-COR 926-32211).
His-tagged recombinant protein purification and pulldown in vitro
Sox2-His plasmid was transformed into E. coli strain BL21, and the fusion protein expression was induced by adding isopropyl thio-β-d-galactosidase (IPTG) in 1 mM final concentration at 18 °C. After 18 h, cells were centrifuged for 10 min at 4000g and 4 °C. The cell pellets were re-suspended in lysis buffer (Tris 50 mM, 500 mM NaCl, 10% glycerol, 0.5% NP40, 1 mM DTT, 1 mM EDTA, 1 mM PMSF, and protease inhibitor cocktail) and lysed with a sonicator. Cell lysis was centrifuged at maximum speed in a microcentrifuge for 10 min at 4 °C, then the supernatant was transferred to Ni-NTA resin column with incubation for 30 min. The column was washed for three times, then the His-tagged Sox2 affinity beads were detected by SDS-PAGE. 293T cells were collected in 48 h after transfected with Ddx56 full length and ΔC-ter plasmids, and grayscale in western blot experiment was used to balance the quantity of protein. The same quality of protein was added into beads and incubated 4 h at 4 °C with gentle agitation. The beads were washed and used for western blotting. Antibodies for western blotting are anti-GST (rabbit) (Homemade), anti-Sox2 (mouse), anti-mouse 680 (LI-COR 926-32220), and anti-rabbit 800 (LI-COR 926-32211).
A17-2loxP mESCs were cultured in 60-mm dish and have been ~ 80% confluent on the day of the experiments. Firstly, the cells were treated with cycloheximide at a final concentration of 100 μg/mL in culture media for 5 min at 37 °C and washed once with 5 mL of ice-cold 1× PBS containing 100 μg/mL cycloheximide. Secondly, the cells were lysed with lysis buffer (140 mM NaCl, 5 mM MgCl2, 10 mM Tris-HCl pH 8.0, 1% Triton X-100, 0.5% sodium deoxycholate, 0.4 U/μL RNase inhibitor, 20 mM DTT, 0.1 mg/mL cycloheximide, 10 mM RVC, 0.1% cocktail), and incubated on ice for 15 min. Then, cell lysate was centrifuged at maximum speed (> 13,000 rcf) at 4 °C for 5 min. At last, the lysate supernatant was carefully transferred to the linear 10 to 50% sucrose gradients and centrifuged at 36,000 rpm for 2 h at 4 °C using the SW41Ti rotor. The sample was analyzed with a fraction collector and UV detector.
Propidium iodide (PI) staining
After doxycycline treatment for 3 days, the cells were collected and seeded on a 15-mm microscope cover glasses for 12 h. The cells on glasses were fixed, washed twice with cold PBS, and were treated with PI and Hoechst for 15 min at dark place. Then, the cells on glasses were detected using a fluorescence microscope.
Annexin V-FITC and PI staining
Cells were treated with or without Dox for 3 days, collected by centrifugation at 1200 rpm for 5 min, and washed twice with cold 1× PBS. Annexin V (5 μL) and PI (5 μL) were added and mixed gently and incubated in room temperature and dark place for 15 min. Then, the cells were analyzed by fluorescence-activated cell sorting (FACS) (Kit: Invitrogen Catalog no. V13241).
Cell cycle assay
After doxycycline treatment for 6 days, the cells were collected by centrifugation at 1200 rpm for 3 min, and washed once with 1× PBS. The cells were resuspended with 70% ethanol and stored at 4 °C overnight. The cells were washed twice with 1× PBS in the second day, resuspended with 200 μL 1× PBS containing 2 μL RNase in 37 °C for 30 min, and incubated with PI at a final concentration of 10 μg/mL for 5 min on ice. Then, the cells were analyzed by fluorescence-activated cell sorting (FACS) to determine cell cycle stages.
Cells were seeded on 15-mm microscope cover glasses a day before the experiment. For immunostaining, cells were fixed with 4% paraformaldehyde, permeabilized (5% Triton-X, 20 mM HEPES, 3 mM MgCl2·6H2O, 300 mM sucrose), blocked (3% goat serum, New Zealand origin (16210072, Gibco, Thermo Fisher), 0.1% BSA in PBS), and incubated with primary antibodies overnight at 4 °C. Then, they were stained with Alexa Fluor-conjugated secondary antibodies goat anti-rabbit IgG (H+L) (Thermo Fisher) and goat anti-mouse IgG (H+L) (Thermo Fisher) at room temperature for 1 h. Nuclear staining was performed with DAPI. Antibodies for immunofluorescence are anti-Flag (rabbit) (GenScript A00170-40) and anti-Oct4 (mouse) (BD 611202).
A17-2loxP-Ddx56, Ddx56ΔC-ter, and GFP cells were cultured in a 24-well plate; each well contains 4000 cells. After 24 h, cells were treated with or without doxycycline. And cells were counted at 2, 4, and 6 days. Each type has three wells repeats.
RNA sequencing and analysis
A17-2loxP-Ddx56, Ddx56ΔC-ter, and GFP cells were cultured and treated with doxycycline for 48 h. The cells were harvested and dissolved in TRIzol for total RNA extraction and treated with DNase I (Ambion) to remove any potential contaminated DNA fraction. The following library generation and sequencing were conducted by Ruibo Biotechnology Co., Ltd. in Guangzhou. The RNA-seq reads were mapped to the UCSC mouse genes (mm10). Only the uniquely mapped reads were retained for analysis in our study. Fragment per kilobase per million mapped fragments (FPKM) and DE genes were calculated.
Significant differences between groups were calculated by performing a two-way ANOVA and were defined as *p < 0.05, **p < 0.01, and ***p < 0.001. The error bars represent standard error of the mean (SEM) of three independent experiments.