FIGURE 4.

Sunitinib synergizes with ABT-199 and S63845 in SEM cells but anti-apoptotic proteins are downregulated. (A) DBP results using sensitizers peptides to study anti-apoptotic dependence of BCL-2 and BCL-xL with BAD 0.1 μM, BCL-xL with HRK 100 μM and MCL-1 with MS1 10 μM after 16 h of incubation with sunitinib 1,000 nM in SEM cell line. (B) Cytotoxicity assessed by Annexin V/DAPI staining after 96 h of sunitinib 100 nM, ABT-199 10 nM, A-1331852 100 nM and S63845 100 nM exposure in SEM cells. BH3 mimetics in combination with sunitinib were added 16 h after treatment. (C) Western blot analysis for anti-apoptotic and BIM proteins in SEM cells after 16 h of treatment with sunitinib 100 nM and sunitinib 1,000 nM. Quantification of optical density for each protein was normalized to actin, and fold-change was calculated comparing to protein expression in the control condition. All results are expressed as the mean ± SEM of at least three biologically independent replicates. Statistical significance was calculated using Student’s t-test compared to control condition and considering *p < 0.05 and **p < 0.01. Significance was also calculated comparing combination conditions with both single agents and considering #p < 0.05 and ##p < 0.01. CI is indicated on top of every combination where CI < 1 indicates a synergistic combination.