Fig. 4: The Effect of FTY720 on KMT2A-R cells is dependent on PP2A activation.
From: SET-PP2A complex as a new therapeutic target in KMT2A (MLL) rearranged AML
A Protein complex -immunoprecipitation showing SET—PP2A interaction in KMT2A-R- AML cell lines upon FTY720 treatment for 24 h. Immunoblotting against SET (39 kDa) was performed after Immunoprecipitation of PP2A (FT: Flow through control; IgG HC: IgG high chains; IgG LC: IgG low chains). B Immunoblot for phospho-AKT1/2 (Ser473) (60 kDa), AKT (pan)(60 kDa), phosphoGSK3β (Ser9) (42 kDa), GSK3β (42 kDa), phosphoERK1/2 (Thr402/Tyr404) (42–44 kDa), ERK1/2 (42–44 kDa) and GAPDH (37 kDa) in K562, Kasumi1, THP1 and MV411 upon 5 µM FTY720 treatment for 24 and 48 h. Densitometry analysis was conducted by Li-cor Image Studio software. GAPDH was used as a loading control. Values are expressed relative to the vehicle at 24 h. C Immunoblot for phospho-AKT1/2 (Ser473) (60 kDa), AKT (pan) (60 kDa), phosphoGSK3β (Ser9) (42 kDa), GSK3β (42 kDa), phosphoERK1/2 (Thr402/Tyr404) (42–44 kDa), ERK1/2 (42–44 kDa) and GAPDH (37 kDa) in K562, Kasumi1,THP1 and MV411 upon 5 µM FTY720 treatment for 24 h. Cells were pre-treated with 2.5 nM Okadaic Acid for 4 h. Densitometry analysis was conducted by LI-COR Image Studio software. GAPDH was used as a loading control. Values are expressed relative to the vehicle. D Analysis of cell death upon FTY720 treatment. Cells were pre-treated with 2.5 nM Okadaic Acid for 4 h and then treated with 5 µM FTY720 for 72 h. GFP signal was used as quantitative reporter of alive, non-apoptotic cells and measured by fluorescent- activated cell sorting (FACS). Data show mean ± SD of triplicate wells and are representative of three independent experiments. 2-Way Anova Dunet’s multiple comparison test **p < 0.01; ***p < 0.001; ****p < 0.0001. E Analysis of cell death of eGFP-MV411 transfected with either shSCRAMBLE or shPP2A upon FTY720 treatment for 72 h. GFP signal was used as quantitative reporter of alive cells and measured by flow cytometry. Data show mean ± SD of triplicate wells and are representative of three independent experiments. 2-Way Anova Tukey’s multiple comparison test **p < 0.01; ***p < 0.001. F qRT-PCR showing the expression of PPP2CA and PPP2CB in eGFP-MV411 shPP2A. Gene expression was normalized by GAPDH control and analyzed by Pfaffl equation. Values are expressed relative to shSCRAMBLE controls. Data represent mean ± SD of three replicas. Two tailed paired t test *** p < 0.001.
