The chemicals dissolved in dimethylsulfoxide (DMSO) were: Neflamapimod (synonyms: VX745, Code: HY-10328, MedChemExpress, Monmouth Junction, NJ, USA), adenosine monophosphate (Synonyms: AMP, Code: HY-A0181, MedChemExpress, Monmouth Junction, NJ, USA), RSL3 (Code: S8155, Selleck Chemicals, Houston, TX, USA) and Lipeoxstatin-1 (Code: S7699, Selleck Chemicals, Houston, TX, USA).
Human lung adenocarcinoma cell lines (A549 and H1299) were purchased from the American Type Culture Collection (Manassas, VA, USA). The cell lines were verified by STR profiling before distribution. The cell lines were grown in RPMI 1640 medium (ThermoFisher Scientific ,Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco, Shanghai, China) in a humidified 5% CO2 atmosphere at 37 °C.
For knockdown of AMPD2, target shRNA sequences were subcloned into pLKO.1-puro (Agel/EcoRI) shRNA Lentivector (GENERAY). shAMPD2-1: 5’-GCACGUCUAUGGAUGGCAATTUUGCCAUCCAUAGACGUGCTT-3’,Sh-AMPD2-3:5’-CCAUCGCUUUGACAAGUUUTTAAACUUGUCAAAGCGAUGGTT- 3’. Cell viability assay
The cell viability tests were analyzed by the standard cell counting kit-8 (CCK-8) assay method. CCK-8 (K1018) was purchased from APEXBIO (Houston, TX, USA). A549 and H1299 cells were seeded into the 96-well plate with a density of 4×104 cells/well. After 12 h, the medium was replaced with 100 µL of fresh medium and incubated overnight. Then, 10 µL CCK-8 solution was added to each well and the cells were subsequently incubated at 37℃ for 1 h. Absorbance was measured at 450 nm using the microplate reader (Thermo, USA).
All cells were washed with PBS and the protein were extracted using RIPA Lysis buffer (Thermo, USA). The protein concentration was quantified with BCA Protein Assay Kit (Beyotime, China). The protein was separated by SDS-PAGE and then transferred to NC membranes. The protein levels were analyzed via western blotting using the corresponding antibodies and were normalized to GAPDH expression. The antibody to AMPD2 (ab137598), Glutathione Peroxidase 4 (ab125066) were obtained from Abcam (Cambridge, MA, USA). The antibody to phospho-p38 MAPK (4511) was purchased from CST (Danvers, MA, USA). The antibody to GAPDH (10494-1-AP) and p38MAPK (14064-1-AP) were obtained from proteintech (Wuhan, China).
RNA isolation and quantitative real-time PCR (RT-qPCR)
Total cellular RNA was isolated from cells using Trizol reagent (TaKaRa Bio, Dalian, China) and then reversely transcribed into cDNA using PrimeScriptRT Reagent Kit (TaKaRa Bio, Dalian, China) according to the manufacturer’s instructions. Real-time PCR was performed using QuantiTect SYBR Green PCRkit (TaKaRa Bio, Dalian, China) in Stratagene MX3000P (Agilent Technologies, Santa Clara, CA, USA). The sequences of the primers were as follows: GAPDH, forward 5′-GCACCGTCAAGGCTGAGAAC-3', reverse, 5′-ATGGTGGTGAAGACGCCA GT-3', AMPD2, forward, 5′-ATGATCCCTTGCAGTTCACTT-3', reverse, 5′-GGCTCTTTACCTTGTGCGAGAA-3'. The expression level was normalized to the internal control and determined by a 2-∆∆CT method.
The MDA amount can reflect the degree of lipid peroxidation in the body, which indirectly indicates the extent of cell damage. We detected the MDA content according to manufacturer's protocol (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The absorbance at 532 nm was detected by a microplate reader (Thermo, USA).
A DCFH-DA probe Reactive Oxygen Species Assay Kit (Beyotime, China) was used to determine intracellular ROS levels according to the manufacturer's instructions. Briefly, A549 and H1299 cells were attached to 6-well plates. Then, DCFH-DA in serum-free RPMI 1640 medium was loaded with 10 μM at 37℃ for 40 min. After washing three times with serum-free medium, the fluorescence was recorded with a confocal scanning laser microscope (DP72, Olympus, Japan) and quantified with Image J® software.
Tissue microarray and immunohistochemistry
NSCLC tissue microarrays (TMA), including 30 paired cancerous tissue and adjacent normal tissue were purchased from Shanghai Outdo Biotech (Shanghai, China). TMA were deparaffinized in xylene and rehydrated in series of ethanol solutions and then boiled in a citrate buffer (pH 6.0) in a microwave oven for antigen retrieval and incubated in 3% hydrogen peroxide (H2O2) to deactivate potential endogenous peroxide activity. The sections were subsequently incubated with 5% normal goat serum and then with a rabbit polyclonal AMPD2 antibody overnight at 4 °C in a wet box. Next day, the sections were washed with PBS thrice and incubated with pv-9000 kit (ZSGB-Bio, Beijing, China) and then subjected to color development using 3,3-diaminobenzidine (ZSGB-Bio, Beijing, China) and counterstained with hematoxylin. The percentage (%) of positively stained cells was classified as 0 (0%–25%), 1 (26%–50%), 2 (51%–75%), or 3 (76%–100%), while the staining intensity was classified as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). We then plus these two scores to reach a staining index.
Gene Correlation Analysis in GEPIA
Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/index.html), an online database was used to evaluate the correlation between AMPD2 expression and p38 MAPK(MAPK14) expression in LUAD, LUSC and paracancer tissues. GEPIA is a web server for analyzing the RNA sequencing expression data of 9736 tumors and 8587 normal samples from the TCGA and the GTEx projects, using a standard processing pipeline(Tang, et al. 2017).
All statistical calculations were completed using GraphPad Prism (version 9.0). All results were presented as the mean±standard deviation (SD). The differences between the two groups were performed by the Student's t-test.