Annexin 5

A total of 1×10⁶ cells treated with Hym and Ven (at the indicated concentrations), either alone or in combination, were collected after 24–48 h of incubation, washed twice with cold phosphate-buffered saline (PBS), resuspended in 300 μL binding buffer, stained with 5 μL Annexin-V and 10 μL PI (Multisciences Biotech, Hangzhou, China), incubated in the dark for 15 min, and subjected to flow cytometry (Becton Dickinson Bioscience, Oxford, UK) to analyze apoptosis. The IC50 value, or half maximal inhibitory concentration, of each cell line was calculated using the Xiantao web data analysis tool (https://www.xiantao.love). To determine whether the two drugs exerted a synergistic effect, the combination index (CI) was calculated using Compusyn software (ComboSyn Inc., NJ, USA), with the definitions of additive effect (CI = 1), synergism (CI < 1), and antagonism (CI > 1).

Cells were treated as described above. After 24 h of incubation, apoptosis was measured via flow cytometry with 5 μL annexin V and 10 μL propidium iodide cell stains (Multi Sciences, China). Data are expressed as the percentage of apoptotic cells.

Apoptosis analysis was performed by Annexin V staining. Briefly, cells were resuspended and incubated with Annexin V (MultiSciences Biotech Co., Ltd, 5300117) for 5 min in the dark. The sample was analyzed by flow cytometry (Beckman Coulter, FC500MCL), and the percentage of Annexin V positive cells was counted.

To examine the cell apoptosis, MV4-11 and MOLM-13 sensitive and resistant cells were inoculated into 24 wells at a final density of 1 × 10⁵/ml. Then cells were collected and washed twice with 1× phosphate-buffered saline (1× PBS) and inoculated into 300 μL 1× binding buffer with 10 μL Propidium (PI) and 5 μL Annexin-V for 30 minutes (Cat: AP101, MULTISCIENCES, Hangzhou, China). For cell cycle analysis, cells were seeded in 24 wells at a final concentration of 3 × 10⁵/ml cells per well and collected and fixed with precooled 75% ethanol at 4°C overnight. The next day, cells were washed twice with 1× PBS. Flow cytometry was utilized to detect the DNA content after PI (Cat: CCS012, MULTISCIENCES, Hangzhou, China) staining for about 30 minutes. For p-gp expression analysis, cells were inoculated into 300 μL of 1× PBS with 10 μL of p-gp antibody (RRID: AB_396548, BD, USA) labeled with APC for 30 minutes. Flow cytometry was performed and analyzed on ACEA NovoCyte (ACEA, USA).

To measure ROS, cells were first labeled with surface markers (Lin⁻Sca1⁺Mac1⁺) to identify the relevant HSC subpopulation, and then incubated at 37 °C for 30 min in PBS containing 10 µm MitoSOX Red (M36008, Invitrogen). The cells were then washed twice with PBS, followed by flow cytometric analysis.
p‐Kap1 was reported as a marker for DNA damage.^[31, ^32] Therefore, cells were first labeled with surface markers (Lin⁻Sca1⁺Mac1⁺), fixed, and then permeabilized. The cells were then stained with a p‐Kap1 antibody and Alexa Fluor 488‐conjugated goat anti‐rabbit antibody (A‐10680, Invitrogen) as the primary and secondary antibodies, respectively. The cells were then washed twice with PBS, and fluorescence was measured using flow cytometry.
To measure apoptosis, the cells were first labeled with surface markers (Lin⁻Sca1⁺CD48⁻CD150⁺Mac1⁺), and then immunostained for annexin V (MultiSciences) and DAPI in accordance with the manufacturer's instructions.
For intracellular Ki67 staining, the cells were first labeled with surface markers (Lin⁻Sca1⁺Mac1⁺), fixed, and permeabilized. The cells were then stained with an Alexa Fluor 488‐conjugated Ki67 antibody (652 417, BioLegend) at 37 °C for 30 min. DAPI (422 801, BioLegend) was added to a final concentration of 20 µg mL⁻¹ immediately before flow cytometry.