Cell culture and treatment
Human promyelocytic leukemia cell line HL60 was purchased from Procell (Wu Han, China). Cells were maintained in Iscove’s Modified Dulbecco Medium (IMDM) supplemented with 20% fetal bovine serum and 1% penicillin-streptomycin solution under 5% CO2 at 37 ℃. Except for CCK8 experiment, cells were treated with erastin (15 µM, #HY-15763, MedChemExpress, USA) with or without Fer-1 (15 µM, #HY-100579, MedChemExpress, USA), Hemin (20 µM, #GC14591, Good Laboratory Practice Bioscience, USA) and Znpp (10µM, HY-101193, MedChemExpress, USA) for a duration of 24 h at 37°C.
Cell counting kit-8 (CCK-8) assay
Cells were seeded at a density of 5×104 cells/well in 96-well plates and exposed to varying concentrations of erastin (5, 10, 20, 30 and 40 µM) and Fer-1 (15 µM) for 24 h. Following this incubation, 10 µL of CCK-8 solution(K1018, Apexbio, USA)was added to each well and incubated for 2 h at 37℃. Using a microplate reader (#EL808, Biotek, USA), the viability of cells was determined by measuring the absorbance at a wavelength of 450 nm.
Transmission electron microscopy (TEM)
The cell precipitate was collected and fixed sequentially with 2.5% glutaraldehyde and 1% osmium. Following fixation, the cells were dehydrated using ethanol, embedded in pure acetone, and subsequently stained with lead citrate and uranyl acetate. Finally, the samples were observed and photographed using a Tecnai Spirit electron microscop.
Measurement of malondialdehyde (MDA), glutathione (GSH) and ROS
The level of MDA (Beyotime, China) and GSH (Solarbio, China) were determined according to the manufacturers’ instructions. For ROS assessments, cells treated with the aforementioned compounds were harvested, centrifugation and precipitation (no less than 106 cells). These cells were then suspended in IMDM medium containing DCFH-DA (Beyotime, China) at a working concentration of 10 µM. After incubation at 37℃ for 20 min, the cells were washed with phosphate buffered saline (PBS) for three times to eliminate any residual DCFH-DA, and the labeled ROS was subsequently detected via flow cytometry. To assess fluorescence, a fluorimeter(FACS CANTO II, BD Bioscience, USA)was used to excite the sample at 488 nm and measure emissions at 535 nm.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using Trizol reagent (Solarbio, China) and subsequently reverse-transcribed using the PrimeScript™ RT kit (Takara, Japan). The cDNA was then amplified with TB Green™ Premix Ex TaqTM Ⅱ (Takara, Japan) in a Thermo Fisher instrument. Using the ∆∆Ct method, the relative expression of genes was calculated and normalized. The primer sequences were as follows:
ACSL4 (acyl-CoA synthetase long chain family member 4): F 5’-AGTCCATATCGCTCTGTCACA-3’ and R 5’-GCTGTCCTTCTTCCCAAACTT-3’
ALOX15 (arachidonate 15-lipoxygenase): F 5’-GCTGGAAGAGAGAAGGAAGTTG-3’ and R 5’-CACAGGGAGGTCATATAGTTTGG-3’
β-actin:F5’-AGTTGCGTTACACCCTTTCTTG-3’and R 5’-GCTGTCACCTTCACCGTTCC-3’
Western blot (WB) assay
Cells were lysed on ice with RIPA lysis buffer (#C5029, BIOSS) containing 1 nM phenylmethylsulfonyl fluoride (PMSF, #C05-01002, BIOSS), and protein concentration was determined using the BCA protein detection kit (#E-BC-K318-M, Elabscience). Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (MILLIPORE, USA) via membrane transfer apparatus. All membranes were sealed with Quickblock solution (#P0256, Beyotime) and incubated overnight with primary antibody at 4℃. Following three washes with TBST, the membranes were incubated with secondary antibody at room temperature for 1 h, and protein bands were ultimately detected using an ECL kit (#180–501, Tanon) and Tanon imaging system (Tanon). Image J software was utilized to calculate gray values of resulting images, with β-actin serving as a contro. Primary and secondary antibodies are as follows: anti-glutathione peroxidase 4 (GPX4, #ER1803-15), anti-HO-1 (#ER1802-73), anti-mitogen-activated protein kinase 14 (MAPK14, #R1308-3), anti-calreticulin (CALR, #EM1701-60), anti-apolipoprotein E (APOE, #0809-6), anti-ferritin heavy chain (FTH1, #ET1705-55) and light chain (FTL, #ET1705-54), anti-tranferrin receptor (TfR, CD71, #ET1702-06) were purchased from HUABIO (China). Anti-apolipoprotein C3 (APOC3, #A2209), anti-β-actin (#AC026) and anti-rabbit secondary antibody (1:5000 dilution, #AC026) were purchased from ABclonal (USA).
Label free proteomic analysis
HL60 cells were subjected to 24 h incubation with erastin and control group was not treated. Proteomic data were subsequently acquired via Label-free technique and a Q Exactive™ HF-X mass spectrometer (MS, Thermo Fisher). The peptides digested by trypsin were analyzed by high-resolution MS, which produced both primary ions and secondary fragments of the peptides. All resulting data were then analyzed through the UniProt database.
Liquid chromatograph (LC)-MS/MS Analysis
Mobile phase A (100% water, 0.1% formic acid) and B solution (80% acetonitrile, 0.1% formic acid) were prepared. Separated peptides were analyzed by Q Exactive™ HF-X MS, which utilized a Nanospray Flex (ESI) ion source with a spray voltage of 2.1 kV and ion transport capillary temperature of 320℃. Full scan ranged from m/z 350 to 1500 with a resolution of 60000 (at m/z 200), automatic gain control (AGC) target value was set at 3×106 and a maximum ion injection time was 20 ms.
Statistical analysis
Graphpad Prism 8 software was used for graph drawing and data analysis in this study, t-test was used for comparison between two groups and one-way ANOVA test was used for comparison between more than two groups. P < 0.05 was considered to be statistically significant.