Patient samples
This study was approved by the Ethics Committee of the First Affiliated Hospital of Soochow University following the Declaration of Helsinki. Informed consent was obtained from the patient according to the Declaration of Helsinki.
Cytogenetic analysis
At the time of diagnosis, bone marrow cells were cultured for 24 h and analyzed for standard cytogenetic R-banding. The karyotype was described according to the International System for Human Cytogenetic Nomenclature (ISCN 2013).
RNA sequencing
RNA-sequencing analysis was performed according to Roche instruction for users. The cDNA sample library was prepared by using the KAPA Stranded RNA-Seq Library Preparation Kits. The target regions were captured with SeqCap RNA choice probe. The library was sequenced by the Nextseq 550 (Illumina) for 151bp on each paired-end.
Reverse transcription and Sanger sequencing
An aliquot of the RNA extracted for RNA sequencing was also reverse transcribed using random hexamers and standard techniques (Invitrogen). The primers used to detect the CTCF-ETO2 transcript were CTCF-exon3: 5’-CGATTACGCCAGTGTAGAAGT-3′ and ETO2-exon4: 5′-TGATGGCTGTTGGTGAGTG-3′. The primers used to detect the reciprocal ETO2-CTCF transcript were ETO2-exon 1: 5’-ATGTCCCAGACGCACCCT-3′ and CTCF-exon 6: 5’-GCTGCTTTCGCAAGTGGA-3’. The length of the sequence amplified by the primers is 554bp and 109bp, respectively. Sanger sequencing was performed using the Big Dye Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA).
Cell lines and reagents
The 293T and Hela cells were maintained in Dulbecco’s Modified Eagle Medium (Hyclone, SH30243.01); 32D cells was maintained in RPMI medium (Hyclone, SH30809.01); and all were supplemented with 10% fetal bovine serum (Gibco, 10099141) and antibiotic-antimycotic (Gibco, 15240-062). 10μM mIL-3 (PeproTech, 9621313) are provided for 32D cells because they are IL-3 dependent.
Plasmid constructs
The DNA sequence encoding CTCF-ETO2 and reciprocal ETO2-CTCF were synthesized and inserted into both pcDNA3.1 expression vector (Genewiz) with Flag tag fused to their carboxy termini and VENUS-N-FLAG lentiviral vector (Genewiz). Wild-type CTCF and ETO2 were also synthesized and inserted into pcDNA3.1 expression vector with Flag tag fused to their carboxy termini. The empty vector pcDNA3.1 and VENUS-N-FLAG were used as control vectors. All of the plasmids were purchased from Genewiz.
Immunofluorescence
The Hela cells was transiently transfected with Flag-tagged pcDNA3.1 expression plasmid by JetPEI transfection kit (Polyplus, 26032C1F). Then the cells were permeabilized and sequentially incubated with primary anti-Flag antibodies (CST, 14793), secondary antibodies (DyLight 549 goat anti-mouse IgG antibody; Abbkine, A23310), and 4-6-diamidino-2-phenylindole (DAPI; Beyotime, C1005). Images were obtained with a laser scanning confocal microscope (Leica SP2).
Lentivirus production and transduction
The 293T cells were transfected with lentiviral vector constructs and pPACK packaging plasmids mix by calcium phosphate precipitation method. Cells were then infected with prepared lentivirus and sorted by BD FACS Aria II System for green fluorescent protein (GFP)-positive cells.
Flow cytometry analysis
Transduced 32D cells were washed with PBS and stained with APC-conjugated mCD71 (Biolegend, 113820) antibody. For apoptosis, VENUS, CTCF-ETO2 and ETO2-CTCF transduced 32D cells were treated with either 10ng/ml or 0 mIL-3 for 24 hours, and then the cells were stained with Annexin-V (BD Pharmingen, 550474) and 7-AAD (BD Pharmingen, 559925) according to the manufacturer's protocol. Antibody staining was monitored with a Novocyte flow cytometer. Data analysis was carried out using FlowJo software.
Q-PCR
RNAs were extracted from cells using total RNA extraction (Trizol, Invitrogrn, 15596026). 0.5μg–1μg RNA was used to make cDNA library using Primescript RT master mix (Takara, RR036B). The resulted cDNA library was diluted five-fold and used for SYBR green based qPCR reactions (Takara, RR420B) on an ABI 7500 Real-Time PCR system. The following primers were used for mouse ζ-globinqF: 5’GAAGCCTGG GACAAGTTCAT-3’; ζ-globinqR: 5’GGGTTCAATAAAGGGGAGGA-3’; mβ1-globin qF: 5’GCTCTTGCCTGTGAACAATG-3’; β1-globinqR: 5’ GTCAGAAGACAGATTT TCAAATG-3’; mβh1-globinqF: 5’ TTGCCAAGGAATTCACCCCA-3’; βh1-globinqR: 5’CTCAATGCAGTCCCCATGGA-3’; mε-globinqF: 5’GTTTTGGCTAGTCACTTCG G-3’; ε-globinqR: 5’CAAGGAACAGCTCAGTATTC-3’; GAPDH was used as the internal control: mGAPDHqF: 5’CATCACTGCCACCCAGAAGACTG-3’; mGAPDH qR: 5’ATGCCAGTGAGCTTCCCGTTCAG-3’. Gene expression levels were quantified with the 2−ΔΔCt method.
CCK8 assay
Transduced 32D cells were rinsed with PBS for 3 times and treated with either 10ng/ml or 0 mIL-3 in a 96-well cell culture plate. Approximately 15000 cells were seeded in each well. Three replicates were made for each measurement. Then, cells were incubated at 37 °C in a humidified atmosphere with 5% CO2 for 24 h, 48 h and 72h. Finally, 10 μL of the CCK-8 reagent (Donjindo, KR675) was added into each well, and OD at 450 nm was measured using a multifunction microplate reader (Infinite M200 Pro, Tecan) after incubation for 3 h at 37 °C. The fold each concentration accounted for of the control was presented as relative cell proliferation.
Western blot
Cell lysates were separated by SDS–PAGE gel and transferred to PVDF membrane (Millipore). The membrane was probed with primary antibody and then with secondary antibody. Antibody binding was revealed by using an enhanced chemiluminescence reagent (GE Healthcare Biosciences). ImageJ was used to quantify the density and size of the blots.
C-myc antibody was purchased from Active Motif (61075). The following antibodies were obtained from Cell Signaling Technology: p-STAT3 (9145), STAT3(12640), p-STAT5(9359), STAT5(9363), p-AKT(4060), AKT(4691), p-Erk(4370), Erk(4695), p-JNK(9255), JNK(9252), BCL2(15071), P53(2527), CDK9(81464), Actin(4967). Other commercial antibodies: HOXA9(Abcam, ab140631), CyclinT1(Abcam, ab264326). Secondary antibodies for western blotting were from Beyotime: HRP goat anti-rabbit IgG (A0208), HRP goat anti-mouse IgG (A0216), HRP goat anti-rat IgG (A0192).
Statistical analysis
Data were shown as mean ± standard deviation (SD). The significance of differences between different groups was determined by ANOVA. Data analyses were performed using Graphpad Prism v6.0. Statistical significance threshold was set at 0.05; asterisks indicate significant differences (*P < .05;**P< .01; and ***P < .001).