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. 2023 Feb 8;13(2):470. doi: 10.3390/life13020470

Figure 7.

Figure 7

GSC inhibitory capacity of OLE phenolics. (A) The effect of TMZ-only, OLE-only, and OLE phenolics on the size of tumorspheres formed by T98G cells and (B) A172 cells. Tumorspheres were formed in a 96-well round-bottom ultra-low attachment plate in 3 days. (C) Calcein-AM/PI staining determined hypoxia in tumorspheres formed by T98G and A172 cells. Green fluorescence shows metabolically viable and proliferating cells, while red reflects the hypoxia-induced loss of cell membrane integrity and necrotic cells. The tumorspheres were initially imaged untreated and 96 h after treatments with TMZ-only, OLE-only, and OLE phenolics at 40× magnification. ImageJ software measured the size of the captured tumorspheres and the viable/necrotic area ratio. (D) TMZ-only, OLE-only, and OLE phenolics-dependent changes in the RNA expression of GSC marker genes CD133 and OCT4 in T98G and A172 cells. p-values were calculated using two-way ANOVA for tumorsphere formation and an independent samples t-test for RT-qPCR * p < 0.05, ** p < 0.0001 compared to untreated cells; n = 3. UNT: untreated, TMZ: temozolomide, OLE: Olea europaea leaf extract, OL: oleuropein, HT: hydroxytyrosol, TYR: tyrosol.