Recombinant LukS-PV production and purification
Recombinant LukS-PV purification and purification were described previously by Ma et al .
AML patient samples and cell lines
THP-1, NB4, HL60 and OCL-AML3 cell lines were purchased from the Shanghai Institute for Biological Sciences (SIBS, shanghai, PR, China). Twenty untreated and newly diagnosed adult AML patients were recruited from January 2015 through January 2020. BM specimens were obtained before chemotherapy. The cells were separated by using the lymphocyte separation medium (TBD, China), and then purified by anti-CD33 and anti-CD34 magnetic-activated cell sorting (MACS) separation columns (Miltenyi Biotec, Bisley, United Kingdom). Cells with >95% viability detected by trypan blue staining were used.
Antibodies and reagents
LC3B (D11, # 3868S), HOXA9 (# ab140631), anti-HOXA9/AF 350 (# bs-6667R-AF350), 3-Methyladenin (3-MA, #M9281) and Bafilomycin A1 (Baf-A1, #S1413) were obtained from Cell Signaling Technology, Abcam, Bioss, Sigma and Selleck, respectively.
RNA extraction, real-time PCR and HOXA9 mRNA expression analysis
Total RNA from cell lines and human BM specimens were extracted using the Trizol (Invitrogen). Reverse transcription and quantitative real-time PCR for determination of HOXA9 and GAPDH mRNA expression were performed using the SYBR Green PCR Kit (GenePharma, shanghai, PR China). The primer sequences of HOXA9 was as follows: HOXA9 forward: 5′-CACCAGACGAACAGTGAGGA-3′; reverse (5’-3’): TGGTCAGTAGGCCTTGAGGT. Analysis of relative mRNA expression was using 2−△△CT method.
mRFP-GFP-LC3 transfection and immunofluorescence staining
All of the cells were transfected with an adenovirus encoding both mRFP-GFP and LC3 proteins. The cells were first seeded into 96‐well plates at a final density of 5×104 cells per well, followed by transfection in different conditions.
Proteins were resolved using 10% SDS-polyacrylamide gels and electro-transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA).. After blocked in Tris-buffered saline 0.1% Tween 20% to 10% milk, membranes were probed with an appropriate amount of primary antibodies and horseradish peroxidase-conjugated secondary antibodies, and then visualized with an enhanced chemoluminescence detection system.
Cell proliferation was assessed by 5-ethynyl-2’-deoxyuridine (EdU) assay (KeyGen BioTECH, JiangSu, China) according to the manufacturers' instructions. Cells were analyzed by fluorescence activated cell sorter Calibur flow cytometer (BD Pharmingen, San Diego, CA).
Cell counting kit‐8 assay
Cell viability was detected by cell counting kit‐8 (CCK‐8, Dojindo) following the manufacturers' instructions. Cells were first grown in 96‐well plates at a final density of 5000 cells (200μL) per well, and then incubated with different reagents in different conditions. Afterward, 20ml of CCK‐8 solution was added to each well to culture the cells for 1-2 hours, and the absorbance at 450 nm (OD450nm) in a microplate reader was recorded subsequently. All of the CCK-8 data were performed in triplicate.
Data of different groups were analyzed by the unpaired two-tailed Student’s t test with the help of SPSS (version 24), R language, and GraphPad Prism (version 7) software. A log-rank test was used for survival analysis and Kaplan-Meier survival curve was applied to the assessment of overall survival (OS) in AML patients.