Figure 4
Thiopurine nucleotide triphosphates of 6 MP enhance Leydig cell death
(a) Immunoblot of pro- and cleaved caspase 9 amounts in cytosolic extracts treated with varying concentration with 6T-ITP, 6MT-GTP and dATP. (b) Time-dependent caspase 3 activation in cytosolic extracts treated with either dATP alone or dATP and 6T-ITP as indicated. (c) Apoptosome formation in the presence of dATP and/or 6T-ITP as visualized by immunoblot of Apaf1 in size exclusion fractions. (d) Activation of caspase 3 by 6T-ITP (α = 1.84), p < 10−11) and (e) 6MT-GTP (α = 0.44, p < 0.05). The solid curve represents the additive response. The dashed curve is the cross-section of the response surface model fit to the data and the dotted curves are the 95% confidence interval of the fitted curve. The corresponding 3D modeling plot, show the additive surface, is seen to the right of each respective group. (f) Structural model showing two individual binding sites for 6T-ITP (cyan) and 6MT-GTP (yellow) as well as dATP. (g) Apaf1 represented in both its inactive (right) and active (left) conformations. Thiopurine nucleotide binding sites are formed only in active Apaf1. A cross section of the response surface modeling plots of dATP plus varying concentrations (h) Model schematic showing the thiopurine nucleotide metabolites of 6 MP, along with dATP, bind and synergize with Apaf1, activating caspase 3 and promoting Leydig cell death.
