Fig. 3.

a-d J82, T24, 5637 and KU-19-19 cells were treated with different concentrations of RRx-001, or DMSO as control, and counted at the indicated times. The values represent the mean of three independent experiments +/− standard deviation. Statistical significance was verified by 2-tailed Student’s t-test (* p < 0.05; ** p < 0.01 and *** p < 0.001). e Protein levels of DNMT1 and CCDC6 in J82 cells treated with different concentrations of RRx-001, as determined by western blot. Vinculin and Tubulin were used as internal controls for sample loading. 5-AZA was used as positive control for inhibition of DNMT1 expression. f Whole cell lysates from J82 shCCDC6 or shCTRL cells, treated with different concentrations of RRx-001, or untreated, were immunoblotted with anti-CCDC6 antibody. γH2AX levels are shown. Tubulin was used as loading control. g J82 cells were treated with RRx-001, P5091 or RRx-001 plus P5091 for 144 h and then assessed for cells viability using a modified MTT assay (MTS), Cell Titer 96 AQueous One Solution assay. Isobologram analysis shows the synergistic antiproliferative activity of RRx-001 plus P5091 at the higher doses. In the left panel the graph derives from the values given in the table (right panel). In the graph the black dots below the dotted line indicate the presence of synergistic interaction between the two drugs. In the tables the Fractional Effect represent a percentage expression of the number of live cells. Values of CI < 1, CI = 1 and CI > 1 indicate respectively synergistic, additive and antagonistic effects