Supplementary Figure 10: Pharmacological inhibition of PDHA1 arrests mouse and human prostate tumors.
(a,b) Representative structures, selected from MD simulations of the PDHA1/pyruvate (a) and PDHA1/3-FP (b) complexes. The a and b chains are represented as magenta and green ribbons, respectively. In the picture of PDHA1/3-FP complex, the fluorine atom is depicted in violet. Binding Delta G values, estimated by MM-GBSA, are also reported. (c) Upper panel, PDC activity measurements in the lysate from 22Rv1 cells treated with 3-FP or vehicle and supplemented with indicated amount of pyruvate in activity buffer of dipstick assay kit (n = 3, independent cell cultures). Lower panel, quantification of PDC activity by mean optical density of colored bands shown on dipsticks in upper panel (n = 3, independent cell cultures). (d) Body weight of mice of the indicated genotypes treated with 3-FP or vehicle from 8 weeks old for one month (n = 3, independent mice). (e) Upper panel, PDC activity measurements, and quantification in anterior prostate of indicated genotypes from mice treated with 3-FP or vehicle from 8 weeks old for one month (n = 3, independent prostate samples). Lower panel, quantification of PDC activity by mean optical density of colored bands shown on dipsticks in upper panel (n = 3, independent prostate samples). (f) Comparison of prostate lobe volumes from male mice at age of 12 weeks treated with 3-FP or vehicle for one month (mm3, 2 independent lobes per animal are presented. AP, anterior prostate; VP, ventral prostate; DLP, dorsal and lateral prostate, n = 3, independent prostate samples). (g-j) Representative micrographs of histopathological analysis (haematoxylin/eosin and Ki-67 staining) of anterior prostates (AP) in Ptenpc-/- prostate tumours (n = 3, independent prostate samples, 5 fields, and scale bar represents 50 µm) (g,h), quantification of the percentage of Ki-67 positive cells (n = 3 independent prostate samples, 5 fields) (i) and quantification of the percentage of invasive prostate cancer glands in Ptenpc-/- prostate tissue (n = 3, independent prostate samples, 5 fields) (j) from male mice at age of 8 weeks treated with 3-FP or vehicle for one month. (k) Upper panel, PDC activity measurements and quantification in nuclear and cytoplasmic fractions of prostate tumours of indicated genotypes from mice at age of 8 weeks treated with 3-FP or vehicle for one month (n = 3, independent prostate samples). Lower panel, fractionation was validated by western blot analysis for the indicated nuclear and cytosolic proteins. Uncropped images are in Supplementary Figure 15. (l) Western blot analysis of indicated proteins in prostate tumours from Ptenpc-/- mice at age of 8 weeks treated with 3-FP or vehicle for one month (n = 3, independent prostate samples). (m) Quantitative real time-PCR analysis of mRNA expression for Acly and Sqle from mice of indicated genotypes at age of 8 weeks treated with 3-FP or vehicle for one month (n = 3, independent prostate samples). Error bars indicate s.e.m. *P < 0.05; **P < 0.01. n.s, not significant.
