Fig. 6: The PP2A activator DT-061 reduces N1-ICD levels and CLL cell survival in vitro by promoting GSK3β activity. | Cell Death & Disease

Fig. 6: The PP2A activator DT-061 reduces N1-ICD levels and CLL cell survival in vitro 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. 6

A CLL cells were treated for 24 h with the indicated concentrations of DT-061 or DMSO as control (n = 6). Cell viability and apoptosis were evaluated by flow cytometric analysis of Annexin V/PI (An V/PI) double staining. Left, results are represented as the percentage of viable (An V-/PI-), early apoptotic (An V+/PI-), late apoptotic (An V+/PI+), and necrotic (An V-/PI+) cells. One CLL sample is shown. Middle and right, box and whisker plots with data points of the percentage of viable An V-/PI- (middle) and apoptotic An V+ (An V+/PI- plus An V+/PI+) cells (right) are shown. *P < 0.05 according to Wilcoxon paired test. B Western blot analysis of PARP and Mcl-1 was performed in CLL cells cultured for 24 h with 15 μM DT-061 or DMSO as control (n = 8). GAPDH was analyzed as loading control. Left, the values under the blots indicate the fold change in cleaved PARP (89-kDa) and Mcl-1 levels in DT-061-treated cells compared with control DMSO (set to 1), normalized to levels of full length PARP (116-kDa) and GAPDH, respectively. Three CLL samples are shown. Right, box and whisker plots with data points of densitometry analysis of cleaved PARP (top panel) and Mcl-1 (bottom panel), represented as fold change compared with control DMSO. **P < 0.01 according to Wilcoxon paired test. C CLL cells were cultured for 24 h with 15 µM DT-061 as single agent and in combination with 5 µM SB216763, or with DMSO as control (n = 6). Cell viability and apoptosis data were obtained and represented as in panel A. Left, one CLL sample is shown. Right, box and whisker plots with data points of the percentage of viable An V-/PI- (top panel) and apoptotic An V+ (An V+/PI- plus An V+/PI+) cells (bottom panel) are shown. *P < 0.05 according to Wilcoxon paired test. D Western blot analysis of N1-ICD was performed in CLL cells cultured for 3 h with 15 μM DT-061 or DMSO as control (n = 6). GAPDH was analyzed as loading control. The effect of DT-061 on GSK3β inactivation was assessed by analyzing pS9-GSK3β levels. Left, the values under the blots indicate the fold change in N1-ICD and pS9-GSK3β levels in cells treated with DT-061 compared with control DMSO (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 DMSO. *P < 0.05 according to Wilcoxon paired test. E Western blot analysis of N1-ICD and pS9-GSK3β was performed in CLL cells pretreated for 1.5 h with 5 μM SB216763 or DMSO, and then cultured for further 3 h with 15 μM DT-061 (n = 6). GAPDH was analyzed as loading control. Left, the values under the blots indicate the fold change in N1-ICD and pS9-GSK3β levels in cells treated with DT-061 alone or DT-061 plus SB216763, compared with control DMSO (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 DMSO. *P < 0.05 according to Wilcoxon paired test.

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