Journal: eLife
Article Title: Unveiling the signaling network of FLT3-ITD AML improves drug sensitivity prediction
doi: 10.7554/eLife.90532
Figure Lengend Snippet: ( A ) Cartoon of the in silico simulation conditions. ( B–C ) Bar plot showing the in silico simulation of proliferation activation ( B ) and apoptosis inhibition ( C ) levels in untreated and FLT3i conditions in combination with knock-out of each of 10 crucial kinases in FLT3 ITD-JMD (blue) and -TKD (yellow) cells. ( D–E ) In FLT3 ITD-JMD (blue) and -TKD (yellow) cells treated with 100 nM Midostaurin and/or 10 µM SP600125 (JNK inhibitor) for 24 hr, the percentage of Annexin V positive cells ( D ) and the absorbance values at 595 nm ( E ), normalized on control condition, are reported in bar plots. ( F ) Primary samples from acute myeloid leukemia (AML) patients with the FLT3 ITD-TKD mutation (n=2, yellow bars) or the FLT3 ITD-JMD/TKD mutation (n=3, blue bars) were exposed to Midostaurin (100 nM, PKC412), and JNK inhibitor (10 µM, SP600125) for 48 hr, or combinations thereof. The specific cell death of gated AML blasts was calculated to account for treatment-unrelated spontaneous cell death. The bars on the graph represent the mean values with standard errors. ( G ) In FLT3 ITD-JMD (blue) and FLT3 ITD-TKD (yellow) cells treated with 100 nM Midostaurin and/or 10 µM SP600125, followed by 10’ of TNFα 10 ng/ml, the protein levels of phospho-JNK (T183/Y185), JNK, phospho-CDK1 (Y15), phospho-CDK1 (T161), CDK1, phospho-CDK2 (T160), CDK2, CyclinB1, CycinE2, and Vinculin were evaluated by western blot analysis. ( H ) Cytofluorimetric cell cycle analysis of DAPI-stained FLT3 ITD-JMD (blue) and FLT3 ITD-TKD (yellow) cells treated with 100 nM Midostaurin and/or 10 µM SP600125 (JNK inhibitor) for 24 hr. ( I ) Cartoon of the molecular mechanism proposed for FLT3 ITD-JMD and FLT3 ITD-TKD cells. Figure 4—source data 1. Original files for the western blot analysis in of phospho-JNK(T183/Y185), JNK, phospho-CDK1 (Y15), phospho-CDK1 (T161), CDK1, phospho-CDK2(T160), CDK2, CyclinB1, CycinE2, and Vinculin. Figure 4—source data 2. PDF containing and original scans for the western blot analysis of phospho-JNK(T183/Y185), JNK, phospho-CDK1 (Y15), phospho-CDK1 (T161), CDK1, phospho-CDK2(T160), CDK2, CyclinB1, CycinE2, and Vinculin, with highlighted band.
Article Snippet: Cultures were incubated for 48 hr in the absence or presence of 100 nM PKC412 and 10 μM SP600125 (all Selleck Chemicals LLC, Houston, TX, USA), or combinations of PKC412 with SP600125.
Techniques: In Silico, Activation Assay, Inhibition, Knock-Out, Control, Mutagenesis, Western Blot, Cell Cycle Assay, Staining
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