The human AML cell lines HL-60, OCI-AML3, KG-1, and Molm-13 were acquired from American Type Culture Collection (Rockville, MD, USA). HL-60 and OCI-AML3 cells were maintained in DMEM (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 20% fetal bovine serum (FBS) (Gibco), 1% penicillin–streptomycin (Gibco) and 4 mM l-glutamine (Gibco). KG-1 and Molm-13 cells were cultured using RPMI-1640 medium (Gibco) supplemented with 10% FBS, 1% penicillin–streptomycin (Gibco) and 2 mM l-glutamine (Gibco). Cells were grown at 37 °C in 5% CO2. Every three days, half of the medium was replaced.
The Cell Counting Kit-8 (CCK-8) assay was employed to analyze the effects of PAL on cell proliferation. The CCK-8 assay was performed using 2 × 104 cells/well in 96-well plates. After the cells were cultured with 2.5, 5, 7.5 or 10 μg/mL PAL, CCK-8 (10 μL) was added for an additional 4-hour incubation at 37°C. Cell suspensions were then prepared and vortexed for 5 min, after which the absorbance was read at 450 nm, and the cell proliferation rate was determined.
Annexin V-FITC antibody immunofluorescence combined with PI/DNA binding was adopted for fluorescent analysis of apoptosis. Cells (1× 106) treated with 5 μg/mL PAL for 24 h were collected and subjected to quantitative flow cytometry analysis according to the instructions of the Annexin V-FITC kit (BioVision, USA).
Western blot analysis
Cells (1 × 106) were collected and washed three times with PBS. Total protein was extracted by using an Animal Tissue/cells/bacteria total protein isolation kit (DocSense, Chengdu, China) according to the manufacturer’s instructions. The protein concentration was then determined by using a BCA protein kit (Sigma–Aldrich, St. Louis, MO, USA). Twenty micrograms of total protein was separated by SDS–PAGE and electrophoretically transferred onto a PVDF membrane (Millipore, Billerica, MA, U.S.A.). The membrane was blocked with 5% nonfat milk in TBST buffer and incubated overnight at 4 °C with specific primary antibodies, including anti-cleaved caspase-3 (cat. No.: ab32042), anti-cleaved PARP1 (cat. No.: ab32064), anti-beta actin (cat.:: ab8226), anti-pro-caspase-1 (cat. No.: 2225T, CST), anti-cleaved caspase-1 (cat. No.: 24232S, CST), anti-pro-caspase-5 (cat. No.: ab40887, Abcam), anti-cleaved caspase-5 (cat. No. LS-C380468-50, LSBio), anti-GSDMD (cat. No.: ab219800, Abcam), anti-cleaved GSDMD (cat. No.: 10137, CST) antibodies. The membrane was washed with TBST buffer and incubated with the appropriate secondary antibody (horseradish peroxidase-conjugated goat-anti-mouse IgG). Analysis was performed using enhanced chemiluminescence kits (Millipore, Billerica, MA, USA).
Measurement of intracellular ROS.
Cells were exposed to PAL alone or in combination with NAC or VX765 for the indicated time points (6, 12, 18, 24 h), the supernatants were removed, and the cells were labeled with 2′,7′-dichlorofluorescin diacetate (DCFH-DA, Invitrogen, Paisley, UK) at 37°C for 30 min. The cells were washed twice with PBS and maintained in 1 ml serum-free medium. Cellular ROS levels were analyzed using a fluorescent enzyme labeling instrument (Spectra Max M5; Molecular Devices LLC).
The mitochondrial membrane potential was detected using a JC-1 mitochondrial membrane potential assay kit (cat. No.: C2006; Beyotime Institute of Biotechnology). Briefly, the cells were collected and washed three times with PBS and then incubated with JC-1 staining solution for 20 min at 37°C. The cells were then washed twice with 1x JC-1 buffer, and the fluorescence intensity was analyzed using flow cytometry.
Colony formation in soft agar
To assess tumor formation ability in vitro, soft agar clonogenic assays were performed. Each well of a 6-well plate contained 2 mL of 0.5% (w/v) low-melting agar (Sigma–Aldrich, St. Louis, MO, USA) in DMEM with 10% FBS. The cells were mixed equally, and 1×104 cells in 2 mL of 0.3% low-melting agar in 10% FBS were added above the polymerized base solution. The plates were incubated (37°C, 5% CO2) for 14 days before colony number and diameter were quantified microscopically.
Cell migration and invasion assay
Transwell migration assay experiments were performed using a 24-well Transwell chemotaxis chamber technique (Millipore, Billerica, MA, USA). Briefly, DMEM/F12 (500 μL) supplemented with 10% FBS was placed in the lower chamber. A total of 1× 104 cells in 200 μL medium were seeded into the upper chamber (pore size, 8 μm). The chamber was then incubated for 24 h at 37 °C in a humidified atmosphere with 5% CO2. The membrane (Millipore) was removed, and its upper surface was wiped away with a cotton swab to remove the unmigrated cells. The membrane was then merged in 4% paraformaldehyde for 5 min at room temperature and stained with 0.1% crystal violet for 10 min followed by 3 washes with ice-cold PBS. The number of cells that migrated to the lower surface of the membrane was counted in 10 random high-power fields (HPFs) under a light microscope (BL-AC10DS, Olympus, Tokyo, Japan). Each assay was performed in triplicate wells.
To analyze invasion, the underlying surface of the membrane was coated with matrix gel (0.01%) at 37°C for 2 h. The lower chamber was filled with 1 ml of DMEM supplemented with 10% FBS. A total of 1×104 cells in a volume of 0.2 ml were added to the upper chamber. After incubation at 37°C for 24 h, the cells on the upper surface of the Transwell membrane were removed. The cells attached to the lower surface of the membrane were stained with 1% crystal violet and imaged.
Statistics and presentation of data
The results are expressed as the mean±SD. Statistical analysis involving two groups was performed using Student’s t test. All data were processed with SPSS 19 version software.