Annexin 5

Cells were seeded in 6-well plates at a density of 2×10⁶/well and exposed to different drugs. After incubation for the indicated time, the cells were treated with 0.25% trypsin without EDTA, and incubated with Annexin V and propidium iodide (PI) (Dojindo) for 15 min at room temperature in the dark. The rate of apoptotic cells was determined by flow cytometer (BD Biosciences) and analyzed by FlowJo software.

NCI-N87 cells or SGC-7901 cells were seeded in 6-well plates and transiently transfected with plasmid DNA or siRNA. Two days after transfection, the cells were treated with certain concentrations of lapatinib for indicated time points. Both floating and adherent cells were harvested and stained with Annexin V and Propidium iodide (Dojindo, Kumamoto, Japan) and further analyzed with a flow cytometry (FACScan, BD Biosciences, USA) equipped with a Cell Quest software (BD Biosciences, USA). Apoptosis was also determined using the TUNEL apoptotic cell detection kit (Roche, Basel, Switzerland), according to the manufacturer's instructions.

The apoptosis-induced morphological changes in both 3DHTMC and 3D RA-SH-SY5Ycells after treatments were detected using Annexin V, FITC and PI apoptosis detection kit (Dojindo Molecular Technology, Inc., Rockville, MD, USA). While Annexin V, a calcium-dependent phospholipid-binding protein, is an early apoptosis marker due to it binding with phosphatidylserine, propidium iodide (PI) is considered a late-stage apoptosis marker due to it entering the cells once cell membrane integrity is lost.
Briefly, cells were freed from Matrigel^®, according to the manufacturer’s instructions. Then, the cell suspension was centrifuged at 1000 rpm for 3 min, removing the supernatant. The cells were then washed twice with PBS and centrifuged at 1000 rpm for a further 3 min. After removing the supernatant, the cells were re-suspended in 100 μL of 10-fold diluted Annexin V binding solution, to which 5 μL of Annexin V, FITC conjugate and 5 μL of PI solution were added. Finally, the apoptotic cells were identified by confocal microscopy after 15 min of incubation at room temperature.

Trichloromethane, triethylamine, and methanol were purchased from Sinopharm Chemical Reagent Co., Ltd. Cobalt nitrate hexahydrate (Co(NO₃)₂ · 6H₂O), dopamine, and 2-methylimidazole were purchased from Aladdin. Anethole trithione was obtained from TargetMol. A membrane and cytosol protein extraction kit, Dil, LysoTracker Green, and a firefly luciferase reporter gene detection kit were obtained from Shanghai Beyotime Biotechnology Co., Ltd. D-Luciferin and potassium salt were purchased from Shanghai Yisheng Biotechnology Co., Ltd. A cell counting kit-8 (CCK-8) was purchased from Nanjing Novazan Biotechnology Co., Ltd. Annexin V and an FITC apoptosis detection kit were obtained from Dojindo Laboratories. Anti-integrin alpha 4 rabbit pAb, anti-integrin beta 1 rabbit pAb, Cy3-labelled goat anti-rabbit IgG, FITC-labelled goat anti-rabbit IgG, and luc-4T1 cells were obtained from Wuhan Service Biotechnology Co., Ltd. A Pierce BCA protein assay kit was purchased from Thermo Fisher Scientific. Bouin’s fixative was purchased from Jinclone Biotechnology Co., Ltd. (Beijing). Distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG₂₀₀₀) was obtained from Avanti. All reagents were analytically pure and used directly after purchase without further purification.

After transfection for 24 h, cell apoptosis was evaluated by staining with Annexin V and PI (Dojindo Molecular Technologies Inc., Shanghai, China) for 15 min at room temperature in the dark, followed by flow cytometry within 1 h (BD Biosciences, Erembodegem, Belgium). Cell apoptosis was analysed by Flowjo 7.6 software (TreeStar Inc., Ashland, OR, USA). After transfection for 48 h, cells were collected and fixed in 70% cold ethanol for at least 12 h at 4 °C. Cells were stained with 50 μg/ml propidium iodide (BD Biosciences, Erembodegem, Belgium) at room temperature for 30 min in the dark, and the cell cycle was analysed using the FACS Calibur system (BD Biosciences, Erembodegem, Belgium) and ModFit 4.0 software (Verity Software House, Topsham, ME, USA).

To measure T cell apoptosis, 1 × 10⁶ splenocytes (SPCs) and lymph node cells (LNCs) were prepared and stained with CD4⁺-APC and CD8⁺-PE/CY7 (eBioscience, San Diego, CA, USA) for 30 min at 4°C. After washing with PBS, the cells were stained with Annexin V and propidium iodide according to the manufacturer’s instructions (Dojindo Laboratories, Japan) and analyzed by flow cytometry. To measure T cell activation, the SPCs and LNCs were freshly stained with CD4⁺-APC, CD8⁺-PE/CY7, CD44⁺-PE, and CD69⁺-PE (eBioscience) for 30 min at 4°C and analyzed by flow cytometry. Isotype control staining was also performed as described (21 (link)). Data were analyzed using FlowJo software version 10 (Tree Star Inc., Ashland, OR, USA).

flow cytometry assay was carried out to detect cell apoptosis. In brief, HT‐1376 and T24 cells transfected with different vectors were harvested and subjected to apoptosis detection using Annexin V(FITC)/Propidium iodide (PI) Apoptosis Detection Kits (Dojindo). Cell apoptosis rate was detected by using flow cytometry (Beckman Coulter) and analyzed using FlowJo 7.6 software (Tree Star, lnc).