Paraformaldehyde solution

To measure the cell invasion ability, transwell chambers (8-μm pore size) were coated with Matrigel (50 μL/well with a solution containing four volumes of serum-free medium to one volume of Matrigel solution) in 24-well plates. Cell migration assay was performed with transwell chambers (8-μm pore size) coated with serum-free medium in 24-well plates. Cells at a density of approximately 1.5 × 10⁵ cells/well were seeded into the upper chamber of the insert. After incubation for 48 h at 37 °C with 5% CO₂, any cells that had invaded the lower cavity were fixed by 4% paraformaldehyde solution (Beyotime, Cat no: P0099) for 10 min and then stained with 5% crystal violet solution (Beyotime, Cat no: C0121) for 10 min. The cells were counted in three randomly selected visual fields using an inverted microscope (IX71, Olympus). The quantification analysis was carried on using the Image J software (Fiji Software). Cell numbers were counted at random three views, the average number of migratory and invasive cells were shown as histogram, respectively. All the experiments, including the quantification analysis, were performed in triplicate.

Dopamine hydrochloride (purity = 99%), SN38 (98%), dimethyl sulfoxide (DMSO), ammonia (28–30%), 3H-Indolium,5-[[[4-(chloromethyl)benzoyl]amino]methyl]-2-[3-(1,3-dihydro-3,3-dimethyl-1-octadecyl-2H-indol-2-ylidene)-1-propenyl]-3,3-dimethyl-1-octadecyl-,chloride (CM-Dil), fluorescein isothiocyanate (FITC), phenylmethylsulfonyl fluoride (PMSF), deionized water (pH 7.0) and absolute ethanol (99.8%) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) with high glucose, Minimum Essential Medium α (MEM α), and fetal bovine serum (FBS) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). RIPA lysis buffer, a bicinchoninic acid (BCA) protein kit, enzyme-linked immunosorbent assay (ELISA) kit for tumor necrosis factor (TNF)-α, 4% paraformaldehyde solution, 4′,6-diamidino-2-phenylindole (DAPI), Triton X-100, and Coomassie Brilliant Blue were purchased from Beyotime (Shanghai, China). Other reagents were of analytical grade and purchased from Beijing Chemical Reagents Company (Beijing, China) unless stated otherwise.

Lung tissues were fixed in 4% paraformaldehyde solution (Beyotime, China) for 24 h and then dehydrated and embedded in paraffin following routine methods. The slides were rinsed with Bond Dewax Solution at 72 °C to deparaffinize the tissue slices. Then, the sections were treated with Enhanced Endogenous Peroxidase Blocking Buffer (Beyotime) for 5 min to block endogenous peroxidase activity before they were incubated with Nrf2 antibody (1:500 dilution, Abcam, USA), 4-HNE antibody (1:200 dilution, Abcam) or GPX4 antibody (1:500 dilution, Abcam) overnight, after which DAB was quickly added. The color rendering time was controlled for 5 min. Images viewed under an Olympus microscope were captured. The Nrf2 immunostaining results were scored by multiplying the percentage of positive cells (0, <10%; 1+, 10–25%; 2+, 25–50%; 3+, 50–75% or 4+, >75%) by the staining intensity (0, negative; 1+, weak; 2+, moderate; or 3+, strong), and the 4-HNE and GXP4 immunostaining results were scored as the integrated optical density (IOD)/area as detected by Image-Pro Plus.