Fig 4. Influence of the therapeutic agents azacitidine, lenalidomide and rigosertib on the separase activity distribution (SAD value) in MDS-L cell culture experiments.

(A) Exponentially growing MDS-L cells were treated with azacitidine, lenalidomide and rigosertib, each at concentrations of 500 nM for 48 hours. Subsequently, separase proteolytic activity was analyzed in the remaining vital cell fraction. Untreated MDS-L cells served as negative control (untr). As in vivo benchmark, the mean over the SAD values of all MDS patients under investigation (n = 37) is presented by the very right column. Method: Unpaired two-tailed t test, p = 0.0067 (CI95%: -3.159 to -0.5398). The data are derived from 11 independent assay experiments, each performed in duplicates. (B) The corresponding cell cycle profiles of treated MDS-L cells were analyzed by flow cytometry after propidium iodide (PI) staining. The cell fractions at G1, S and G2/M phase are represented by white, gray and black fillings, respectively. Data are derived from 2 independent assay experiments performed in duplicates. (C) Assessment of apoptosis in treated MDS-L cells by flow cytometry using annexinV / PI staining. Data from early (annexinV positivity only) and late apoptosis (double-positive fraction for annexinV and PI) were combined and are derived from 2 independent assay experiments performed in duplicates. Abbreviations: SAD, separase activity distribution; untr, untreated; AzaC, azacitidine; Lena, lenalidomide; Rigo, rigosertib.