Annexin 5 pi

Antibodies used for immunoblots were MNK2 (1:300 dilution; Abcam), eIF4E (1:5000 dilution; Abcam), P-eIF4E (1:500 dilution; Abcam), ERK1/2 (1:1000 dilution; Abcam), P-ERK1/2 (1:1000 dilution; Abcam), 4EBP1 (1:1000 dilution; Cell Signaling Technology), P-4EBP1 (1:1000 dilution; Cell Signaling Technology), AKT (1:1000 dilution; Affinity), P-AKT (1:1000 dilution; Affinity), β-actin (1:5000 dilution; Abcam), and Vinculin (1:5000 dilution; Abcam). AnnexinV/PI (KeyGEN). MER Inhibitor PD 0325901 and AKT inhibitor BEZ 235 were purchased from Selleck Chemicals.

Treated AML cells were stained with Annexin V/PI (KeyGEN) and subsequently analyzed by FACS. We defined all Annexin V⁺ cells as apoptotic cells, including the early apoptotic cells and the late apoptotic cells. Additionally, we performed terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) to detect apoptotic K562 cells using the corresponding kit (Beyotime).

Annexin V/PI (KeyGEN, Nanjing, China) was used for the detection of cell apoptosis induced by oxaliplatin. The CM-H2DCF-DA probe (C6827, Thermo Fisher Scientific) and BODIPY™ 581/591 C11 probe (D3861, Thermo Fisher Scientific) were added, and the mixture was incubated in the dark at 37 °C for 30 min to measure the cellular ROS and cellular lipid ROS levels. All these measurements were conducted with a flow cytometer (Beckman Coulter, CA, USA) according to the manufacturer’s instructions. The gating strategy for ROS/lipid ROS analysis is provided in Supplementary Fig. 3f.

Cell apoptosis was determined by AnnexinV/PI (KeyGEN, Nanjing, China), followed by flow cytometer analysis (Beckman Coulter, California, USA) according to manufacturer’s instructions.
Caspase activity was measured by Caspase-Glo 3/7 Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol.

Apoptotic cells were determined by FACS analysis after labeling with Annexin-V/PI (Keygen, China). Briefly, cells might be exposed to 0μg/mL, 1.5μg/mL, 3μg/mL of paclitaxel (Harbin Pharmaceutical Group Co., Ltd), 0μM, 5μM, 10μM of cistplatin (Hansoh Pharm, DDP), 0μM, 1.5μM, 3μM of SAHA (Cayman Chem Com, a HDAC inhibitor), 0μM, 0.5μM, 1μM of MG132 (Enzo, proteosome inhibitor) for 48h. The cells were trypsinized, collected and washed by PBS twice. Annexin-V-FITC (5 μl) and 10 μl 50 mg/l PI were added to 500 μl of cell suspension and incubated with cells in the dark for 15 min at room temperature. The cells were analyzed by flow cytometry.

Cell apoptosis was quantified by annexin V/PI staining according to the manufacturer's instructions (KeyGen Biotech, China). After being treated, the NP-MSCs were collected and washed, then a binding buffer was used to resuspend NP-MSCs. 5 μl annexin V and 5 μl PI were used to label NP-MSCs for 15 min, and the cells were analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).

Cells were grown overnight in 6-well plates to 50% confluency. After 48 h of culture with or without 4MOD, cells were harvested and stained with Annexin V/PI (KeyGEN BioTECH, Jiangsu, China) in accordance with the protocol of manufacture. The cell apoptosis rate was detected by flow cytometry (Beckman Coulter, Miami, FL, USA). The FlowJo 10 software (Tree Star, Inc. Ashland, OR) was used for data analysis.