Fig. 5: Pharmacologic AKT inhibition reduces N1-ICD levels and CLL cell viability by promoting GSK3β activity. | Cell Death & Disease

Fig. 5: Pharmacologic AKT inhibition reduces N1-ICD levels and CLL cell viability by promoting GSK3β activity.

From: GSK3β is a critical, druggable component of the network regulating the active NOTCH1 protein and cell viability in CLL

Fig. 5

A CLL cells were cultured for 6 h with 5 μM AKTiX (AiX) or complete medium as control (n = 10). Western blot analysis of NOTCH1 was performed using the anti-NOTCH1 (Val1744) and the anti-NOTCH1 (D1E11) antibodies, able to recognize N1-ICD and N1-TM, respectively. Protein loading was assessed using an anti-GAPDH antibody. Left, the values under each blot indicate the fold change in N1-ICD and N1-TM levels in AiX-treated cells compared with control cells (set to 1), normalized to GAPDH levels. AKT Inhibition by AiX was verified by analyzing AKT phosphorylation at Serine 473 (pS473-AKT). The effect of AiX on GSK3β inactivation was assessed by analyzing pS9-GSK3β levels. The values under each blot indicate the fold change in pS473-AKT and pS9-GSK3β levels in AiX-treated cells compared with control cells (set to 1), normalized to levels of total AKT and total GSK3β, respectively. Three CLL samples are shown. Right, box and whisker plots with data points of densitometry analysis of N1-ICD and N1-TM, represented as fold change compared with controls. **P < 0.01; ns, not significant according to Wilcoxon paired test. B CLL cells were cultured for 1.5 h with 5 µM SB216763 or DMSO and for further 6 h with 5 µM AiX (n = 6). Western blot analysis of N1-ICD, pS9-GSK3β, total GSK3β and GAPDH was performed as in panel A. Left, the values under the blots indicate the fold change in N1-ICD and pS9-GSK3β levels in cells treated with AiX alone or AiX plus SB216763, compared with control cells (set to 1), normalized to levels of GAPDH and total GSK3β, respectively. Three CLL samples are shown. Right, box and whisker plots with data points of densitometry analysis of N1-ICD, represented as fold change compared with control. *P < 0.05 according to Wilcoxon paired test. C CLL cells were pretreated for 2 h with 10 μM MG132 or DMSO, and then cultured for further 6 h with or without 5 μM AiX (n = 8). Western blot analysis of N1-ICD, pS9-GSK3β, total GSK3β and GAPDH was performed as in panel A. Top, the values under the blots indicate the fold change in N1-ICD and pS9-GSK3β levels in cells treated with AiX, MG132, or AiX plus MG132, compared with control cells (set to 1), normalized to levels of GAPDH and total GSK3β, respectively. Three CLL samples are shown. Vertical lines inserted in CLL1 and CLL30 blots indicate repositioned gel lanes. Bottom, box and whisker plots with data points of densitometry analysis of N1-ICD, represented as fold change compared with control. **P < 0.01; ns, not significant according to Wilcoxon paired test. D, E CLL cells were cultured with or without different concentrations of AiX (2.5, 5 and 10 µM) or SB216763 (2.5, 5 and 10 µM) alone or in combinations (n = 6). After 18 h, cell viability was measured by MTS assay. D Bar graphs with data points of cell viability (mean ± SD) in treated cells compared with untreated controls, set to 100%. *P < 0.05 according to Wilcoxon paired test. E The antagonism between SB216763 and AiX was calculated by using the SynergyFinder web application and the results were produced with ZIP Synergy model (green indicates an antagonistic effect, white an additive effect, and red a synergistic effect).

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