Apoptosis assay kit

Docetaxel (≥99%) and Cy5.5 were purchased from Nantong Feiyu Biotechnology Co., Ltd. (Nantong, China), while the Cell Counting Kit-8 (CCK-8) was procured from Dojindo (Kumamoto, Japan). The apoptosis assay kit was purchased from KeyGen Biotech (Nanjing, China). The cell cycle assay kit was purchased from Invitrogen (Carlsbad, CA), coumarin-6 (C6) was purchased from Aladdin Reagent Co. (Shanghai, China). Acetonitrile and distilled water (HPLC grade) were obtained from Wenrui Ltd. (Guangzhou, China).
A549 cells were obtained from the ATCC (Manassas, VA). All the reagents were provided by Yike, Life Technologies (Carlsbad, CA). The cells were incubated at 37 °C in a 5% CO₂ incubator.
BALB/c nude mice aged between 3 and 4 weeks were purchased from Guangdong Animal Center (Guangzhou, China). As per the directions released by the China’s Council on Animal Care (room temperature, 20–22 °C; relative humidity (RH), 50–60%), the animals were raised with free access to water and standard laboratory feed. The experiments were carried out in compliance with the guidelines issued by Guangzhou Pharmaceutical University.

After being treated for 48 hours, both adherent and non-adherent cells were harvested, stained with annexin-V-PE and 7-AAD using an apoptosis assay kit (keyGEN, Nanjing, China), and assayed in a Beckman Coulter flow cytometer. Cells were gated for apoptosis assay. The experiment was repeated three times. The cell lysates were also prepared for western blot. Caspase-3 and cleaved caspase-3 antibodies (ab32351,abcam; #9661, Cell Signaling) were tested and β-actin was tested as control. ImageJ2x was used to conduct semi-quantitative analysis. The test was performed as mentioned above.

Cell apoptosis analysis was performed using an Apoptosis Assay Kit (Keygen Biotech) according to the manufacturer's instructions. Briefly, 1×10⁶ cells infected with virus expressing wild-type or mutant DLC1 were trypsinized and resuspended in 500 µL of 1× binding buffer. Then, fluorochrome-conjugated Annexin V was added to the cell suspension and was incubated for 10 min at room temperature, followed by incubation with 5 µL of 7-AAD viability staining solution for 10 min at room temperature. The cells were then subjected to flow cytometry using a FACSAria (BD Biosciences).

Colony formation and CCK-8 assay were conducted to test the cell proliferation ability of Li-7 and Huh-7 cells based on the instructions of the manufacturer. For the CCK-8 analysis, cells were inoculated in the plates (96-well) with 1000 cells/well density. Each well was added with CCK-8 solution (10 μl, Beyotime, Shanghai, China) at a given time (24, 48, 72, and 96 hours). After incubation for 3 h, each well was detected at 450 nm with a spectrophotometer. For colony assay, 150 cells were incubated in every well of 6-well plates, and the medium was refreshed every three weeks. After two weeks, cells in each well were cleaned utilizing PBS, fixed by paraformaldehyde, and next stained through methylrosanilinium chloride solution. Finally, the number of cells in each well was counted. The apoptosis assay kit (KeyGEN BioTECH, Jiangsu) was performed for the apoptosis assay based on the protocols of the manufacturer.