Proapoptotic mechanisms of adavosertib and ruxolitinib. (A) Change in apoptotic dependencies after drug treatment assessed through dynamic BH3 profiling. hBIM is an activator of BAX and BAK, mBAD antagonizes BCL-2, BCL-xL, and BCL-w, HRK specifically antagonizes BCL-xL and MS1 specifically antagonizes MCL-1. DMSO served as a negative control, alamethicin served as a positive control. Significance was assessed with a 2-way ANOVA after Dunnett correction. Bar graphs illustrate mean values of 3 independent experiments. (B) Western blot data of single drug and combination treated cells. Three cell lines were treated for 24 hours with 500 nM adavosertib, 5 μM ruxolitinib, or the combination of both compounds. Immunoblotting shows protein expression changes of antiapoptotic BCL-2 family members and phosphorylation of the proapoptotic sensitizer BAD. Illustrated is a representative example of 3 independent experiments. (C) Drug-induced mitochondrial cytochrome c release in cell lines treated for 24 hours with 500 nM adavosertib, 5 μM ruxolitinib, or the combination of both compounds. Significance was assessed with a 1-way ANOVA and Bonferroni correction. ∗P ≤ .05; ∗∗P ≤ .01, ∗∗∗P ≤ .001, ∗∗∗∗P ≤ .0001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure 6.

Proapoptotic mechanisms of adavosertib and ruxolitinib. (A) Change in apoptotic dependencies after drug treatment assessed through dynamic BH3 profiling. hBIM is an activator of BAX and BAK, mBAD antagonizes BCL-2, BCL-xL, and BCL-w, HRK specifically antagonizes BCL-xL and MS1 specifically antagonizes MCL-1. DMSO served as a negative control, alamethicin served as a positive control. Significance was assessed with a 2-way ANOVA after Dunnett correction. Bar graphs illustrate mean values of 3 independent experiments. (B) Western blot data of single drug and combination treated cells. Three cell lines were treated for 24 hours with 500 nM adavosertib, 5 μM ruxolitinib, or the combination of both compounds. Immunoblotting shows protein expression changes of antiapoptotic BCL-2 family members and phosphorylation of the proapoptotic sensitizer BAD. Illustrated is a representative example of 3 independent experiments. (C) Drug-induced mitochondrial cytochrome c release in cell lines treated for 24 hours with 500 nM adavosertib, 5 μM ruxolitinib, or the combination of both compounds. Significance was assessed with a 1-way ANOVA and Bonferroni correction. ∗P ≤ .05; ∗∗P ≤ .01, ∗∗∗P ≤ .001, ∗∗∗∗P ≤ .0001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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