Fig. 3
ZOT5-1-Me and ZOT5-1-Et induced cell death and apoptosis in U-87 MG cells. (A) The decreased viability of U-87 MG cells after treatment with an increasing concentration (0.95–250 µM) of ZOT5-1-Me and ZOT5-1-Et for 4–48 h was evaluated by CCK-8 assay. Cell viability curves and corresponding IC50 values are visible. The dashed line represents a 50% reduction in CCK-8 signal. The data were normalized to control cells (CTRL, 100%, n = 9). (B,C) ZOT5-1-Me and ZOT5-1-Et induced externalisation of phosphatidylserine in U-87 MG cells as evaluated by Annexin V/PI assay followed by flow cytometry (FACS) quantification (n = 6). Camptothecin (CPT, 20 µM) was used as positive control. (B) Representative FACS dot plots after 48 h treatment with ZOTs are presented with the indicated percentages of necrotic (Q1), late-apoptotic (Q2), early-apoptotic (Q3), and viable cells (Q4). Apoptosis was presented as a percentage of Annexin V-positive cells (Q2 + Q3). FACS dot plots after 4 h and 24 h treatments are presented in Supplementary material (D) ZOT5-1-Me and ZOT5-1-Et activated caspase 3/7 in U-87 MG cells as estimated by Caspase-Glo 3/7 assay (n = 6). The data were normalized to control cells (CTRL, 100%, n = 6). CPT (20 µM) was used as positive control. (E–F) Pre-incubation with pan-caspase inhibitor Z-VAD-FMK (10 µM, 48 h) decreased ZOT5-1-Me and ZOT5-1-Et-evoked apoptosis in U-87 MG cells as determined by Caspase-Glo 3/7 assay (E) and Annexin V/PI assay (F) after 48 h treatment with ZOTs (n = 6). (G) Representative FACS dot plots are presented. DMSO was used as a solvent control. Results presented as bar plots with mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001, ns – not statistically significant.
