Fig. 3
Mitochondria play a role in high TIC killing activity of MitoVES. NeuTL adherent and sphere cells were exposed to 2 μM MitoVES for the times shown and ROS evaluated by flow cytometry using DCF (A) or MitoSOX (B), and expressed as relative mean fluorescence intensity (MFI). The histograms on the right are representative of individual readings. (C) Adherent and sphere NeuTL cells were assessed for routine respiration in the absence or presence of MitoVES at the concentrations shown (μM). (D) Adherent and sphere NeuTL cells were probed by WB for the levels of mitochondrial markers with actin as loading control. Adherent and sphere NeuTL (E) and MCF7 cells (F) were evaluated for ΔΨm,i using TMRM and flow cytometry. The histogram in panel E on the right is an example of a reading for NeuTL cells. (G) Adherent and sphere NeuTL cells were labelled with Hoechst to visualise nuclei and TMRM to document ΔΨm,i, and inspected by confocal microscopy. Sphere NeuTL (H) and MCF7 (I) cells were exposed to 2 μM MitoVES for 24 h in the absence or presence of 10 μM FCCP and apoptosis evaluated. The histogram in panel H on the right is an example of reading for NeuTL cells. Data are mean values ± S.D. (n = 3). The symbol ‘*’ in panels A-C, E and F indicates statistically significant differences for adherent and sphere cells with p < 0.05. The symbol ‘*’ in panels H and I indicates statistically significant differences in apoptosis induced in the presence and absence of FCCP with p < 0.05. Images in panels C and D are representative of three independent experiments