Supplementary Materialscancers-12-01694-s001. or 4NQO only remained practical for 24 h as assessed by real-time impedance assay (Shape 3a). Another cell viability assay, which quantified intracellular ATP, proven that the result of A-1155463-4NQO was synergistic (ZIP synergy rating, 14 3; Shape 3b). Similar outcomes were acquired with 4NQO in combination with another Bcl-xL-specific inhibitor, A-133852 (ZIP synergy score, 17 0; Figure 3c). ABT-263, a pan-Bcl-2 inhibitor, also showed synergy with 4NQO (ZIP synergy score, 8 1; Figure 3c). In contrast, combinations of 4NQO with the Bcl-2- or Mcl-1-specific inhibitors did not show a synergy (ZIP synergy scores, 3 1 and 2 1, respectively; Figure 3c). These results suggested that the DNA-damaging agent combined with Bcl-xL-, but not Bcl-2- or Mcl1-specific inhibitors facilitated the death of human non-malignant cells. Open in a separate window Figure 3 Combination of DNA-damaging agent 4NQO with Bcl-xL-, but not Bcl-2- or Mcl-1-specific inhibitors, exhibit synergistic toxicities on human nonmalignant RPE, cancer A549, H460, Caco-2 and mononuclear cells (MNCs) isolated from AML patients as well as on monkey Vero-E6 and dog MDCK cells. (a) Real-time impedance traces for RPE cells exposed to 1 M 4NQO, 1 M A-1155463 or their Kanamycin sulfate combination. Control trace represents cells exposed Mouse monoclonal to TrkA to 0.1% DMSO (Mean SD; n = 8). (b) The interaction landscapes of Kanamycin sulfate A-1155463-4NQO combination. It represents the net combinational effects on viability of RPE cells, as measured with CTG assays. (c) Synergy scores of combinations of 4NQO and 5 Bcl-2 inhibitors on RPE cells (Mean SD; n = 3). Cells were treated with increasing concentrations of a Bcl-2 inhibitor and 4NQO. After 24 h cell viability was measured using the CTG assay. Synergy scores were quantified based on the ZIP model. (d) Synergy scores for combinations of 4NQO and 2 Bcl-xL inhibitors on human cancer A549, H460, Caco-2 cells. Cells were treated with increasing concentrations of a Bcl-xL inhibitor and 4NQO. After 24 h cell viability was measured using the CTG assay. Synergy scores were quantified based on the ZIP model. (e) Synergy scores for 4NQO- A-1155463 combination on MNCs. Cells were treated with increasing concentrations of a A-1155463, 4NQO or both agents. After 24 h cell viability was measured using the CTG assay. Synergy scores were quantified based on the ZIP model. (f) Synergy scores for 4NQO-A-1155463 combination for Vero-E6 and MDCK cells. Cells were treated with increasing concentrations of a A-1155463, 4NQO or both agents. After 24 h cell viability was measured using the CTG assay. Synergy scores were quantified based on the ZIP model. We also tested the combinations of 4NQO-A-1155463 and 4NQO-A-1331852 in human cancer cell lines A549, H460 and Caco-2 (Figure 3d). In addition, we tested 4NQO-A-1155463 in a panel of patient-derived primary cell cultures (Figure 3e), monkey Vero-E6 and dog MDCK cells (Figure 3f). The combinations showed synergy in all tested cells, indicating that the DNA-damaging agent combined with Bcl-xL-specific inhibitor facilitated the death of monkey, dog, human non-malignant and malignant cells. 2.3. Concerted Action of 4NQO and A-1155463 Leads to Overexpression of p53, Release of Bad and Bax from Bcl-xL and Activation of MOMP Immunoblot analysis of whole-cell extracts, nuclear and cytoplasm fractions showed that p53, a Kanamycin sulfate key regulator of DNA-damage response and Bcl-xL-dependent apoptosis, was over-expressed and accumulated in the.