Annexin 5

Treated cells were collected at select time points for analyses. Cellular viability was evaluated at 48 h post-initial treatment (MET or PBS) by trypan blue exclusion using the Vi-Cell cell viability analyzer (Beckman Coulter Inc.). MET- or PBS-treated cells were stained with annexin V and propidium iodide (Trevigen) at 48 h post-initial treatment and quantified for percent of apoptotic cells in the early (annexin V–positive/propidium iodide–negative) and late (positive for both annexin V and propidium iodide) stages using the Becton Dickinson LSRFortessa Flow Cytometer, as described previously [21 (link)]. RNA isolation, preparation of cDNAs, and QPCR analyses were performed following published studies [22 (link)]. Primers (Supplemental Table S1) were designed to span introns and synthesized by Integrated DNA Technologies. mRNA expression was calibrated to a standard curve generated using pooled cDNA stocks, and TATA-box binding protein (TBP) mRNA was used as the normalizing control.

To determine apoptosis (Annexin V, BD Biosciences, Franklin Lakes, USA) and cell death (PI, Sigma, USA) induction, cells were treated with indicated concentrations of nintedanib for 24 and 48 hours. Cells were trypsinized and stained with APC-labeled Annexin V and PI and subjected to FACS analysis (FACScalibur, Becton Dickinson, Palo Alto, USA). Extent of apoptosis/cell death induction was analyzed by FlowJo software.

DAOY and ONS-76 cells in logarithmic phase of growth were treated with 4-HPR (range 1 μM– 20 μM) for 6 and 24 h. At the end of the incubation, cells were collected and stained with FITC-conjugated Annexin V (Becton Dickinson (BD) Bioscences, San Jose, CA) and 7-AAD (BD) in Annexin V Buffer 1X (0,1 M HEPES pH 7,4, 1,4M NaCl, 25 mM CaCl₂ and H₂O). This method allows discrimination between viable cells (FITC negative, 7-AAD negative), early apoptotic cells with intact cell membranes (FITC positive), late apoptotic (FITC positive, 7-AAD positive), and dead cells (FITC negative, 7-AAD positive). Analysis was performed on 10,000 gated cells to exclude cell debris using a FACSCanto (BD), with excitation set at 488 nm and emission at 518 nm (FITC detector) and 620 nm (7-AAD fluorescence detector).

HepG-2 cells growing on 6-well dishes were treated with chemicals in the presence or absence of harmine and MET-Har-02 for 24 h and total cells were collected. The percentage of apoptotic cells was evaluated by staining 1 × 10⁶ cells with annexin V-FITC (Beyotime) and propidium iodide (Beyotime) in binding buffer for 10 min at room temperature in the dark. The samples were analyzed by flow cytometry (FACScalibur, Becton–Dickinson) within 1 h to determine the percentage of cells displaying annexin V+/propidium iodide-(early apoptosis) or annexin V-/propidium iodide + staining(late apoptosis). Three independent experiments were performed for each assay condition.

In order to evaluate cell death, 2 × 10⁵ cells per well were plated into a 6-well plate. After treating the cells with sodium chloride (sham), 6.25 µM oxaliplatin, 5 mg/mL PRIVIGEN^® IgG, 5 mg/mL Tonglu^® IgG, 100 ng/mL IgG1 or the combination therapy for 48 h, the cells were stained by FITC-conjugated Annexin V (ImmunoTools, Friesoythe, Germany, code 31490013) and propidium iodide (PI, AppliChem, code A2261). The percentage of dead cells was determined with the help of the FACSCalibur device (Becton Dickinson, New Jersey, USA) and CellQuest™ Pro software (Becton Dickinson) using the following formula: 100% × (Annexin V⁺ PI^– + Annexin V⁺ PI⁺ + Annexin V⁻ PI⁺) cells/total cells. In order to investigate if inhibition of ERK1/2 activity could impair the oxaliplatin-induced cell death, HROC277 and HROC285 cells were pretreated with dimethyl sulfoxide (sham), 50 µM PD98059 or 10 µM U0126 for 1 h.