1. Cell culture and treatment of leukemia cells.
The acute myeloid leukemia cell lines, including HL-60, THP-1 and NB4, were purchased from the Cell Bank of Chinese Academy of Sciences. The cells were cultured in RPMI-1640 (coring, 10-040-CVRC) supplemented with 10% fetal bovine serum (GIBCO) in the cell incubator with 5% CO2 at 37℃.
1×106 cells of each group were pretreated with or without G-CSF (100ng/ml) for 24 hours, and after that cells were treated with or without ATO (2μM) for 48 hours. Then 100μl cells of each group with gradient concentrations (2×105/ml, 1×105/ml and 5×104/ml) were centrifuged onto glass slides at 500rpm for 5 minutes. The slides were stained by Wright-Giemsa staining solution (Beyotime, C0135), and then were observed or photographed under microscope.
2. Flow cytometry analysis of cell apoptosis.
To analyze the apoptosis levels of cells, THP-1 or HL-60 cells were pretreated with or without G-CSF (100ng/ml) for 24 hours, followed by the treatment with or without ATO (2μM) for 48 hours. 2×106 cells were collected and washed with PBS buffer once and with binding buffer (ThermoFisher) once again. Then the cells were resuspended by 100μl binding buffer. Each sample was stained by 5μl Annexin V (ThermoFisher) for 15 minutes at room temperature in the dark. Then the cell samples were washed with 2ml binding buffer and resuspended by 200μl binding buffer. The cell suspension was added with 5μl propidium iodide (Invitrogen) to stain for 10 minutes before analysis by flow cytometry (BD).
3. Western blotting.
To prepare protein samples, 2×106 cells of each sample were collected and washed by PBS once. Each sample was lysed by 1×SDS loading buffer (Beyotime, P0015) and boiled at 100℃. Then protein samples were separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane (0.22μm). The antibodies and the usage concentrations are as follows: anti-AQP9 antibody (Alpha Diagnostic International, 1:1000), anti-CEBPB antibody (Santa Cruz, 1:1000), anti-GAPDH antibody (Proteintech, 1:2000), HRP-linked anti-Rabbit IgG antibody (Santa Cruz, 1:2000).
For RT-qPCR analysis, 4×106 cells of each sample were collected and washed by PBS once. Each sample was lysed by TRIZOL (Sigma). RNA was extracted and reversely transcribed as protocol. The transcriptional levels of each gene were analyzed on 7500 Fast Real-Time PCR System. The primers of the target genes and the reference gene (GAPDH) are as follows:
5. Cell transfection.
The THP-1 or HL-60 cells in logarithmic growth phase were transfected with siRNAs or plasmids. The siRNA targets of AQP9 gene and CEBPB gene are as follows:
negative control, UUCUCCGAACGUGUCACGU.
6. The atomic fluorescence spectrometry.
THP-1 or HL-60 cells were pretreated with or without G-CSF (100ng/ml) for 24 hours, and then were treated with ATO (2μM) for 48 hours. After that, 5×106 cells were collected and broken up by ultrasonication. The cell debris was dissolve by test solution (66.7% HNO3 + 33.3% H2O2) and was analyzed for the concentrations of atomic arsenic by Agilent 7800 ICP-MS.
7. The cell line derived xenograft mouse model and drug administration.
Approximately 6-8 weeks old male BALB/c nude mice were used for the xenograft animal model. To establish the xenograft mouse model of AML cells, THP-1 cells (1×107 cells per mouse) were subcutaneously injected into nude mice under brief anesthesia by intraperitoneal injection of tribromoethyl alcohol (350mg/kg). And then those mice bearing THP-1 cells were administered with ATO alone (2mg/kg) or ATO/G-CSF (G-CSF 300μg/kg, ATO 2mg/kg) by peritoneal injection when the tumor volumes reached 100mm3. The tumor volumes were recorded every 3 days. The tumor volume was calculated by the following formula: (length×width2)/2. All the mice were euthanized by intraperitoneal injection of pentobarbital (200 mg/kg) at Day 17 after treatment, and the carefully separated tumors were photographed and weighted. All the animal experiments were approved and supervised by the Medical Ethics Committee of Ren Ji Hospital affiliated to Shanghai Jiao Tong University School of Medicine.
8. Immunohistochemistry and HE staining.
The procedures of immunohistochemistry are as follows. The tumor tissues were carefully separated, fixed in 4% paraformaldehyde and embedded in paraffin. The embedded tissues were sliced into sections. The sections went through deparaffinization, rehydration and antigen retrieval. Then the sections were blotted with the primary antibodies against AQP9 (Alpha Diagnostic International) or CEBPB (Santa Cruz). After wash steps by PBS, the sections were blotted by biotin-associated secondary antibody. The sections were developed with substrate mixture.
As for the analysis of mouse livers, the liver tissues were carefully separated from the nude mice receiving ATO or G-CSF/ATO treatment. The tissue sections were prepared as the immunohistochemistry procedures. The sections were stained by hematoxylin and eosin (HE) using the HE Staining Kit (Beyotime, C0105S). All the procedures were performed as protocol. All the finished sections were observed and photographed under microscope.
9. Statistical analysis.
The data in this study were all analyzed and presented using Graphpad Prism software (version 8) or Excel software. The apoptosis levels of cells, the concentrations of ATO or the weights of tumors from different groups were statistically analyzed by unpaired student t test. P<0.05 represents significant difference between the different groups. NS represents no significant difference.