Figure 5.

Tributyltin(IV) butyrate (BT2) activates caspase-dependent apoptotic cell death pathway. (a) Cells were incubated for 48 h in the presence of 0.5 μM BT2 or TBT. At the end of incubation, cells were stained with the vital dye Hoechst 333428 that permits to visualize nuclei. Cells were then visualised under fluorescence microscope Leika equipped with a DAPI filter (magnification of ×400). Micrographs are representative of almost two fields from two independent experiments; (b) Western blot analysis of apoptotic markers, pro-caspase-9, pro-caspase-3 and PARP, in cells treated as in (a). The correct protein loading was ascertained by evaluating γ-tubulin levels. Representative blots of three independent experiments and densitometric analysis are shown; (c) AnnexinV positivity confirmed early apoptosis. Cells were treated with the compounds for 24 h and subjected to Annexin V apoptosis detection kit as reported in Materials and methods. Analysis was performed by flow cytometry using FacsCanto BD. The percentage of annexin V positive cells was evaluated by Flowjo BD software. The results are representative of two independent experiments (*) p-value < 0.05; (**) p-value < 0.01; (***) p-value < 0.001 compared with untreated cells. N.S. not significant.