Fig. 8

Effect of Osteoprotegerin on HMEC spheres cell cycle and aneuploid kinases. a Cell cycle analysis of HMEC spheres by PI staining. HMEC spheres were grown in HMEC media, SUM149PT conditioned media, OPG depleted SUM149PT conditioned media, HMEC media reconstituted with 500 pg/ml recombinant human OPG, SUM1315MO2 conditioned media, OPG depleted SUM1315MO2 conditioned media and HMEC media reconstituted with 1100 pg/ml recombinant human OPG in 6 well ultra-low attachment plates for 8 days. Cell cycle analysis was performed after PI staining of the HMEC spheres. Each assay was done in triplicate and each experiment was repeated three times. b Diploid and aneuploid populations of HMEC spheres in the presence of the various media combination as indicated. c Western blot analysis of Aurora-A/IAK-1, Bub1, BubR1 and Mps1 in the HMEC, SUM149PT and SUM1315MO2 sphere culture lysates. d and e Western blot analysis for aneuploidy related kinases. HMEC spheres were grown for 8 days in different media combinations as indicated and total cell lysates were immunoblotted for Aurora-A/IAK-1, Bub1, and BubR1. The levels of aneuploidy kinases were normalized with respect to β-actin used as the loading control