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. 2021 Sep 9;9:695225. doi: 10.3389/fcell.2021.695225

FIGURE 2.

FIGURE 2

Trametinib synergizes with S63845 in NALM-6 cells, increasing BIM and MCL-1 protein expression. (A) DBP results using sensitizer 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 trametinib 100 nM in NALM-6 cell line. (B) Cytotoxicity was assessed by Annexin V/DAPI staining after 96 h incubation with trametinib 100 nM, ABT-199 100 nM, A-1331852 100 nM and S63845 1,000 nM in NALM-6 cell line. BH3 mimetics in combination with trametinib were added 16 h after treatment. (C) Western blot analysis for anti-apoptotic and BIM proteins in NALM-6 cells after 16 h of treatment with trametinib 100 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 ± standard error of 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. Combination Index (CI) is indicated on top of every combination where CI < 1 indicates synergy.