Annexin 5

Briefly, transfected NP cells were cultured into 6‐well plates at a density of 2 × 10⁵ cells/well and incubated at 37°C for 24 h under 5% CO₂. Then, 5 μl Annexin V (C1062M, Beyotime) was added for 15 min, followed by 5 μL propidium iodide (PI) (C1062M, Beyotime) and mixing. The reaction was performed at room temperature and protected from light for 20 min. Finally, apoptosis was detected on a Cytomics FC‐500 flow cytometer (Beckman Coulter) using Beckman CXP software within 1 h after the reaction.

CD133+ miapaca-2 cells in logarithmic phase were incubated with different concentrations of Lxn (Abcam, London, UK, ab87145; 0 ng/μL, 5 ng/μL, 10 ng/μL, 20 ng/μL and 40 ng/μL) in SFM for 48 hours; cells of treated and control groups were collected and digested with ethylene diamine tetraacetic acid (EDTA) trypsinase. Cells were collected, washed twice with ice-cold PBS, and then precipitated by centrifugation at 500 g for 10 minutes; the cell pellets were resuspended in 1 × Annexin V binding buffer. To a 100-μL aliquot of the cell suspension, 5 μL of Annexin V (20 μg/mL; Beyotime, Jiangsu, China) and 5 μL of propidium iodide (PI; 50 μg/mL; Beyotime, Jiangsu, China) were added, and the cells were incubated in the dark for 15 minutes at room temperature (25°C). Flow cytometry was performed using FACSCalibur (Becton-Dickinson, San Jose, CA, USA). The data from a total of 10,000 events were analyzed using CellQuest software (Becton-Dickinson Immunocytometry Systems, San Jose, CA, USA). The percentage of Annexin V-positive or PI-positive cells was calculated.

Antimycotics (#15240062), fetal bovine serum (FBS, #10091148), RPMI 1640 medium (#A1049101), and Dulbecco's modified Eagle's high glucose medium (#11965118) were purchased from HyClone (HyClone, Logan, UT, USA). Cell Counting Kit-8 (CCK-8, #CK04) was obtained from Dojindo (Dojindo Laboratories, Kumamoto, Japan). The malondialdehyde (MDA) assay kit (#A003-1-2) and reduced glutathione (GSH) assay kit (#A006-2-1) were purchased from Jiancheng (Jiancheng, Nanjing, China). The DAPI staining solution kit (#MA0127) and zinc protoporphyrin-9 (#MB4231) were purchased from Meilui (Meilui, Dalian, China). Liperfluo (#L248) was purchased from Dojindo (Dojindo Laboratories, Shanghai, China). 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA, #S0033S), Annexin V (#C1062M-1), and Propidium Iodide (PI, #C1062M-3) were purchased from Beyotime (Beyotime, Shanghai, China). For western blot analysis, antibodies against HO-1 (#ab13243), GPX4 (#ab40993), Nrf2 (#ab137550), and β-actin (#ab8226) were purchased from Abcam (Abcam, Cambridge, MA). The Iron Assay Kit (#MAK025), dimethyl sulfoxide (DMSO, #D2650), curcumin (#C1386), z-VAD-fmk (#V116), ferrostatin-1 (#SML0583), and deferoxamine (#D9533) were purchased from Sigma (Sigma, St Louis, USA). These compounds were dissolved in DMSO (at final concentrations less than 0.1% (v/v)).

Methyl thiazolyltetrazolium (MTT) and conventional reagents of cell culture were procured from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Cell Counting Kit-8 was procured from MCE (Shanghai, China). Antibodies for Western blotting were procured from Cell Signal Technology (Danvers, MA, USA). AnnexinV and DHE-DCFDA was purchased from Beyotime Biotechnology (Shanghai, China). Hoechst and MitoSOX were procured from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). Other chemicals and reagents were purchased from local suppliers and were of analytical grade.