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. 2016 Oct 20;6:35660. doi: 10.1038/srep35660

Figure 6. Involvement of GSK3β signaling in AKT regulation of hESCs and hiPSCs cell viability and apoptosis.

Figure 6

(a) H9 and FN2.1 cell viability was analyzed by XTT colorimetric assay at 24 hours post-treatment with AKT inhibitors IV (IV, 1 μM), VIII (VIII, 10 μM) and GSKi (GSK, 1 μM) in the presence or absence of CHIRi (CHIR, 3 μM). Mean + SEM from three independent experiments are shown. Statistical analysis was performed by Student’s t test, *p = <0.05; **p = <0.01 and ***p = <0.001 (b) Histogram shows quantitative percentage of surviving cells assessed by Trypan blue exclusion method 24 hours post-AKT inhibitors treatment [IV (1 μM), VIII (10 μM) and GSK (1 μM)] with or without CHIRi (CHIR, 3 μM). Mean + SEM from three independent experiments are shown. Student’s t test, *p = <0.05. (c) Representative histograms, of three independent experiments, of Propidium iodide (PI) staiStatistical analysis was donened H9 and FN2.1 unfixed cells treated for 24 hours with AKT inhibitors [IV (1 μM), VIII (10 μM) and GSK (1 μM)] in combination or not with CHIRi (CHIR, 3 μM). Percentage of PI positive cells (late apoptotic or necrotic) was determined by flow cytometric analysis. Vehicle: DMSO. (d) A representative biparametric flow cytometry analysis, of three independent experiments, of combined fluorescein isothiocyanate (FITC)-conjugated Annexin V and PI staining identifying viable (bottom left), early apoptotic (bottom right), late apoptotic (top right) and necrotic (top left) cells is shown for H9 cells at 8 hours post-AKT inhibitors treatment [IV (1 μM), VIII (10 μM) and GSK (1 μM)] in combination or not with CHIRi (CHIR, 3 μM). Vehicle: DMSO. Percentage of cells in each quadrant is shown. (e) Representative BrdU-APC/7-AAD flow cytometry cell cycle analysis of H9 and FN2.1 undifferentiated cells treated with CHIRi (CHIR, 3 μM) for 24 hours. Vehicle: DMSO. Means + SEM from three independent experiments are graphed for the proportion of cells (%) in each stage of cell cycle (G1, S and G2/M). Statistical analysis was performed by Student’s t-test, *p = <0.05 vs. Vehicle (DMSO).