Annexin 5 apc 7 aad apoptosis kit

An Annexin V-APC/7-AAD Apoptosis Kit (Abnova, #KA3808) was used for the apoptosis analysis as described previously [18 (link)]. ALDH assays were performed as described previously [22 (link)]. Cell cycle assays were performed as described, and propidium iodide(30 μg/ml) was used as DNA content dye [16 (link), 32 (link)]. For CD117 staining, 1 × 10⁶ primary culture tumor cells were stained based on a protocol recommended by the manufacturer. The anti-mouse CD117-PE(Miltenyi Biotec, #REA791) antibody was used. The REA control-PE (Miltenyi Biotec, # REA293) was used as a background control. A BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA) was used for the analysis. Results were analyzed by the Flow Jo software (Version 10.1, OR, USA). All experiments were performed in triplicates.

To analyze the effect of RNASE2, the cell proliferation rate, level of apoptosis, migration, and invasion capabilities of cells in the shNC, shRNASE2, ovNC, and ovRNASE2 groups were measured. After culturing for 1, 2, 3, and 4 days, the optical density of cells at 450 nm (OD_{450 nm}) was measured according to the method of the Cell Counting Kit-8 (KeyGen, Nanjing, China). The cell proliferation rate was calculated using the OD_{450 nm} in the following equation: Cell proliferation rate = (OD value at other time points/OD value at 0 h − 1) × 100% (same sample). After culturing for 48 h, the level of apoptotic cells was determined using the Annexin V-APC/7-AAD Apoptosis Kit (Abnova Corporation, Taibei, China). Cell migration and invasion were evaluated using Transwell and Transwell-Matrigel assays (22 (link)).
To investigate whether RNASE2 plays a role through the PI3K/AKT pathway, LY294002, a PI3K inhibitor, was used to treat cells in the ovRNASE2 group, termed ovRNASE2+LY294002. The ovNC and ovRNASE2 groups were treated with DMSO for use as controls and were named ovNC+DMSO and ovRNASE2+DMSO, respectively. The cell proliferation rate, level of apoptosis, and migration and invasion capabilities of these groups were measured using the same methods as those described above.