Figure 4

The navitoclax–irradiation combination triggers apoptosis and halts proliferation in HNSCC cells. The live cell apoptosis assay was performed for two cell lines (UT-SCC-42A and UT-SCC-24B). Cancer cells were labeled with CellTrace Far Red and the IncuCyte Caspase-3/7 Apoptosis Assay Reagent (green) was applied to detect apoptotic cells. Cells (1000 per well) were seeded to two plates and treated with three navitoclax concentrations (100, 1000 and 10,000 nM). The control group was treated with 0.1% DMSO. One of the plates was irradiated (8 Gy) after 24 h. Plates were imaged using the Incucyte S3 imaging system for a total of three days every other hour at 20 × objective (nine images per well). The number of apoptotic cells (green objects), the proliferation rate (red objects) and the percentage of apoptotic cells (green and red cells divided by red cells multiplied by 100) were calculated using the Incucyte analysis software. (a) Navitoclax induced apoptosis in both cell lines. The navitoclax–irradiation combination increased the number of apoptotic cells. (b) Navitoclax decreased proliferation in both cell lines, and after irradiation proliferation was effectively halted, particularly with 10,000-nM navitoclax. (c) Navitoclax elevated the apoptotic index in both cell lines. The apoptotic index increased rapidly while simultaneously halting proliferation 24 h after exposure to irradiation. In 48 h, the navitoclax–irradiation combination led to apoptosis in 40–50% of cells. Average ± SEM (n = 6).