Cellquest software 1

An Alexa Fluor 488 Annexin V and PI kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used to assess apoptosis. Briefly, 1×10⁵ U373-MG cells were harvested, washed twice with cold PBS and resuspended in 100 µl binding buffer (Thermo Fisher Scientific, Inc.). A total of 5 µl Annexin V-fluorescein isothiocyanate and 1 µl PI were added to the solution. Following 15 min incubation at 4°C, 400 µl binding buffer was added to the solution and cells were analyzed using the Accuri™ C6 Flow Cytometer. The results were analyzed using CellQuest™ software 1.0 (BD Biosciences). A quadrant dot plot was used to identify whether cells were in the early or late phase of apoptosis and whether they were living or necrotic, as previously described (24 (link)).

For the cell cycle analysis, cells were seeded into 6-well plates at a density of 2×10⁵ cells/well, and transfected according to the protocol described above. The experimental groups and time points were set as described for the proliferation assay above. The cells were obtained after being cultured for an appropriate time by trypsinization, and were then pooled with the floating cells, centrifuged at 1,000 × g for 5 min at 4°C and stored at −20°C overnight. DNA staining solution that contained (PI) iodide and RNaseA reagent [MultiSciences (Lianke) Biotech Co., Ltd., Hangzhou, China] were added to the cells. Moreover, cell cycle analysis was performed by fluorescence-activated cell sorting flow cytometry using CellQuest software 1.0 (BD Biosciences, San Jose, CA, USA).