Figure 4.

A. Inhibition of apoptosis by Ac-DEVD-CHO in AML K562 cells using Z-DEVD-R110. Cells were treated with IC₅₀, 2xIC₅₀, 3xIC₅₀ of HXR9, with an equivalent CXR9 to the highest concentration of HXR9 or untreated (control) for 2 hours. The cells were then lysed and the Z-DEVD-R110 substrate was added. The results were normalised to lysis buffer alone. There was no statistical difference in caspase-3 activity between both HXR9- or CXR9-treated cells and untreated control cells. Graphs show the mean of 3 independent experiments and error bars show the SEM. B. Nuclear staining of K562 cells either untreated or incubated for 2 hours with 10 μM HXR9. C. Analysis of transcriptional changes of several pro- and anti-apoptotic genes upon HXR9 treatment. K562 cells were treated with the IC₅₀ of HXR9, CXR9 or untreated (control) for 2 hours and gene expression was analysed using RT-PCR. There was no significant change in any of the analysed genes. Results are presented as a ratio with β-actin (x10 000). Statistical analysis was performed using student's t-test by comparing the relative expression of a gene of interest in HXR9 or CXR9 treated cells to its counterpart in untreated cells. Graphs show the mean of 3 independent experiments and error bars show the SEM. D. The effect of ATP depletion on HXR9 cytotoxicity. Cells were pre-incubated with or without 40 mM fructose and then with a range of HXR9 concentrations (2.5 μM - 50 μM) for 2 hours prepared in media either with or without 40 mM fructose, red and blue curves, respectively. HXR9 cytotoxicity was then measured by assessing LDH enzyme activity in cell-free supernatants. ATP depletion did not inhibit HXR9-induced death. Graphs show the mean of 3 independent experiments and error bars show the SEM. E. General inhibition of caspase activity in HXR9 treated AML cell lines by z-VAD-FMK. Cells were pre-treated either with or without 50μM z-VAD-FMK for 1 hour, and then treated with the IC₅₀, or 2xIC₅₀ of HXR9 for 2 hours or with 17.5 μM DNR for 24 hours, which was used as a positive control. There was no statistical difference in terms of sensitivity to HXR9 between pre-treated cells with or without 50μM z-VAD-FMK. Graphs show the mean of 3 independent experiments and error bars show the SEM. ****p < 0.0001 with respect to z-VAD-FMK untreated cells. F. and G. Effect of CsA on HXR9 cytotoxicity. Cells were pre-treated with or without 5μM CsA for 1 hour, and then with the IC₅₀, 2xIC₅₀, 3xIC₅₀ of HXR9, 20 mM H₂O₂ (positive control), or were left untreated (negative control) for 2 hours with or without 5 μM CsA. The cells were then stained with annexin V and 7-AAD and analysed by flow cytometry. There was no statistical difference in the proportion of viable cells between K562 cells F. incubated with or without 5μM CsA, while incubation with 5μM CsA led to a significant decrease in proportion of viable HL-60 cells G.. Graphs show the mean of 3 independent experiments and error bars show the SEM. *p < 0.05, ***p < 0.001 and ****p < 0.0001 with respect to 5 μM CsA untreated cells.