Fig. 4

Tc52 induces apoptosis in HeLa but not in VH7 fibroblasts. HeLa and VH7 cells were exposed to three different Tc52 concentrations (2 μM, 5 μM, 10 μM) and 10 μM Tc53 for indicated time points, followed by incubation in toxin-free medium. Treatments were compared to controls and significance was calculated by two-way ANOVA with Dunnett's Multiple Comparison Test, p < 0.05 = *, p < 0.01 = **; p < 0.001 = ***. Significance between different time-points was calculated by one-way ANOVA with Tukey-Kramer Multiple Comparisons Test, p < 0.01 = ##; p < 0.001 = ###. a: Cell-cycle distribution of HeLa cells after 6 h Tc52 treatment and 24 h release reveals major increase in the subG1 fraction at 10 μM Tc52 (8.7-fold, accompanied by a loss of cells in G1 (3-fold). Tc53 has no effect. b: Cell-cycle distribution of VH7 after 6 h Tc52 treatment and 24 h release reveals accumulation in G2/M and increased >4 N-fraction accompanied by loss from G1 without increase in subG1 at 5 μM (1.5-fold G2/M, 1.4-fold >4 N, 1.5-fold G1) and 10 μM (1.3-fold G2/M, 1.5-fold >4 N, 1.3-fold G1) Tc52. Tc53 has no effect. c: Representative pictures of HeLa cells displaying dose- and time-dependent appearance of apoptotic figures (open arrows). Cells were treated with 2 μM or 10 μM Tc52 for 2 h, 6 h, or 30 h, washed, fixed with formaldehyde and nuclei were stained with DAPI. d: Representative pictures of normal VH7 fibroblasts displaying a dose- and time-dependent alteration of the nuclear structure, forming DAPI-rich foci (filled arrows). Cells were treated with 2 μM or 10 μM Tc52 for 2 h, 6 h, or 30 h, washed, fixed with formaldehyde and nuclei were stained with DAPI. e: Statistical evaluation of number apoptotic figures in HeLa cells from three independent experiments counting at least 100 cells each. Significant apoptosis induction compared to control (2.3 %) is detected in 10 μM Tc52 samples treated for 2 h (16.4 %) and rate increases with exposure to 6 h (30.6 % at 6 h and 30.5 % at 30 h). Significance of increase compared to control was analyzed by two-way ANOVA and Dunnett’s Multiple Comparison test (*), and differences between 10 μM treatments were calculated by one-way ANOVA and Tukey’s Multiple Comparison Test (#). f: Statistical evaluation of apoptotic figures in VH7 cells from three independent experiments counting at least 100 cells each. Significant increase in apoptotic cells compared to controls (2.2 %) is only detected in cultures exposed to 10 μM Tc52 for the complete 30 h (15.5 %). Significance of increase compared to control was analyzed by two-way ANOVA and Dunnett’s Multiple Comparison test (*), and differences between 10 μM treatment was calculated by one-way ANOVA and Tukey’s Multiple Comparison Test (#). g: Statistical evaluation of VH7 cells nuclei containing DAPI-rich foci. Foci were graded regarding their intensity over the nuclear DAPI background from three independent experiments counting at least 100 cells each. There is a clear dose- and time-dependent increase in the number of cells displaying DAPI-bright foci with a shift from low-grade to high-grade foci. Significance was calculated by two-way ANOVA and Dunnett’s Multiple Comparison Test (*)