Annexin 5 pi detection kit

The Annexin V‐PI detection kit (Keygen Biotech, Jiangsu, China) was used for apoptosis analysis. Briefly, the cells were treated with different concentrations of LY‐1816 or other agents for 24 hours, then washed with PBS. Annexin V‐FITC and propidium iodide (PI) were then added according to the manufacturer's instructions, and the samples were incubated in the dark for 15 minutes. Pictures were taken by using an Olympus digital camera (Shinjuku, Tokyo, Japan) that was attached to a light microscope.

Recombinant Human IL-6 and Matched IL-6 Antibody Pairs were obtained from eBioscience (San Diego, CA, USA). Camptothecin and cisplatin were purchased from Calbiochem (San Diego, CA, USA). Anti-phospho and total kinase antibodies were acquired from Cell Signaling Technology (Beverly, MA, USA). An Annexin V/PI Detection Kit was obtained from KeyGEN Biotech (Nanjing, China). DAPI was acquired from Vector Laboratories (Burlingame, CA, USA). The siRNA of IL-6, ATM, p65 and controls was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). RPMI-1640, DMEM and FBS were acquired from Hyclone (Logan, UT, USA). Lipofectamine 2000 was purchased from Invitrogen (Grand Island, NY, USA). SYBR Premix Ex Taq, Trizol and Prime-Script Reverse Transcriptase were obtained from TaKaRa Biotechnology (Dalian, China).

Cell cycle was evaluated using the RNase A/Propidium Iodide (PI) Detection kit (KeyGEN, China). The staining buffer of PI/RNase A was prepared at a 9:1 dilution. After precipitation by centrifugation, the cell pellets were suspended using 500 μL of 70% cold ethanol and then fixed for 2 h to overnight at 4°C. After washing with PBS, the cell pellets were incubated with staining solution in the dark for 30–60 min. The percentage of G0/G1, S, and G2/M phases was estimated using flow cytometry (BD LSRFortessa, USA). Apoptosis was determined with the Annexin-V/PI Detection kit (KeyGEN, China). The suspended cells in the culture were collected first, and then the attached cells were collected after being digested with Trypsin. After centrifugation, the cells were resuspended with 500 μL binding buffer. 5 μL of Annexin-V-FITC and 5 μL of PI were added and gently mixed. The reaction was conducted in the dark at room temperature for 5–15 min, and the percentages of early and late apoptosis, as well as viable and dead cells, was detected by flow cytometry (BD LSRFortessa, USA) within 1 h.