Pi cell cycle detection kit

The effects of As₄S₄ and As³⁺ on cell cycle distribution were determined using a PI cell cycle detection kit (KeyGEN Biotech, China) [28 (link)]. After treatment with As₄S₄ (2.0 μM), As³⁺ (2.0 μM), or a combination of 1.0 μM As₄S₄ and 1.0 μM As³⁺ for 48 h, NB4 and primary APL cells were collected, washed with Ca²⁺-free PBS and fixed with 70% ethanol at 4°C for 16 h. The fixed cells were then digested in PBS containing 0.5 mg/ml RNase (Sigma-Aldrich, USA) at 37°C for 30 min and stained with 0.05 mg/ml PI in the dark at room temperature for 30 min. Data on the cell cycle distribution were determined using the ModFit LT 3.3 software.

Cell apoptosis was analyzed by Annexin V-FITC and PI apoptosis-detection kits and Annexin V-PE and 7-amino-actinomycin D (7-AAD) apoptosis-detection kits (KeyGEN Biotech, Nanjing, Jiangsu, China). The cell cycle was analyzed by a PI cell cycle detection kit (KeyGEN Biotech, Nanjing, Jiangsu, China). For the silencing groups, MDA-MB-231 cells stably expressing FOXM1 shRNA and NC shRNA were treated with 4.0 μM Olaparib for 7 days. Cells were stained with Annexin V-PE and 7-AAD according to the manufacturer’s instructions to detect apoptosis. For the drug-treated groups, MDA-MB-231 cells and MDA-MB-468 cells were treated with different concentrations of FDI-6, Olaparib, or their combination for 7 days. After treatment, the cells were collected and stained with Annexin V-FITC and PI following the manufacturer’s protocol to analyze cell apoptosis. Cellular DNA was stained with PI following the manufacturer’s protocol. Cell apoptosis and the cell cycle were detected by BD FACSCelesta flow cytometry (New York, USA). Apoptosis data were analyzed by FlowJo 7.6, and the cell cycle data were analyzed by ModFit LT [31 (link)].

For cells transfected with mimics, cell apoptosis were detected using Annexin V-APC/7-AAD Apoptosis Detection Kit (KGA1026, KeyGEN BioTECH) according to the manufacture’s protocol, for cells stably transfected with Lentiviral vectors, cell apoptosis was detected using (KGA1026, KeyGEN BioTECH). Flow cytometry (PI staining) were used for detecting cell cycle using PI cell cycle Detection Kit (KGA107, KeyGEN BioTECH) according to the manufacture’s protocol, experiments were done in triplicates.

1,7-Dihydroxy-3,4-dimethoxyxanthone (XAN) with purity of 99% was isolated and purified from Securidaca inappendiculata Hassk. by our laboratory [5 (link)]. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), BCA protein determination kit, PI cell cycle detection kit, and Annexin V-FITC/PI apoptosis detection kit were purchased from KeyGen Biotech (Jiangsu, China). Rabbit primary antibodies against p-p38, p-JNK, p-ERK, p-AKT, p21, p53, P-gp, bcl-2, cleaved caspase-9 (c-caspase-9), cleaved caspase-3 (c-caspase-3), β-actin, and anti-rabbit HPR linked secondary antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). U0126, SB202190 (SB), and SP600125 (SP) (inhibitors of ERK, p38, and JNK, resp.) were supplied by Hanxiang Biotech (Shanghai, China). Roswell Park Memorial Institute 1640 (RPMI 1640), Phosphate Buffered Saline at pH of 7.4 (PBS), and ECL chemiluminescence detection kit were bought from Thermo Scientific (Rockford, IL, USA). Bovine Calf Serum (CS) was supplied by Wisent Inc. (Canada). All other chemicals and reagents used were of analytical grade.