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. Author manuscript; available in PMC: 2014 Feb 7.
Published in final edited form as: Leukemia. 2012 Oct 22;27(2):398–408. doi: 10.1038/leu.2012.308

Figure 2. Genetic disruption of Ptpn11 or mutation of N51-FLT3 tyrosine (Y) 599 and duplicated Y599 to phenylalanine (F) diminishes FLT3-ITD-induced hyperproliferation and STAT5 phosphorylation.

Figure 2

(A) Genomic DNA isolated from polyI:polyC–treated Shp2flox/flox;Mx1Cre− (negative control) and Shp2flox/flox;Mx1Cre+ tail and bone marrow LDMNCs was subjected to PCR for the original flox allele (~1300 bp) and the polyI:polyC-induced recombined null allele (~400 bp) and protein from bone marrow LDMNCs from polyI:polyC-treated Shp2flox/flox;Mx1Cre− and Shp2flox/flox;Mx1Cre+ mice was analyzed by immunoblot to examine Shp2 expression. (B) 3H-thymidine incorporation assay of transduced, sorted bone marrow progenitors in the absence and presence of FL 50 ng/mL; two independent experiments combined with n=4 replicates per experiment, ^p<0.0001 for N51-FLT3 v. WT-FLT at baseline in Cre− cells, ^^p<0.0001 for N51-FLT3 v. WT-FLT in response to FL in Cre− cells, *p<0.0001 for N51-FLT3 in Cre+ cells v. Cre− cells at baseline, and **p<0.0001 for N51-FLT3 in Cre+ cells v. Cre− cells in response to FL, statistical analysis performed using random effects ANOVA. (C) Immunoblot analysis of STAT5 phosphorylation and total STAT5 expression in exponentially growing transduced, sorted bone marrow LDMNCs from polyI:polyC-treated Shp2flox/flox;Mx1Cre− and Shp2flox/flox;Mx1Cre+ mice. (D) Schematic diagram indicating mutation of N51-FLT3 to N51-FLT3Y599F1 or N51-FLT3Y599F1/2. (E) Murine bone marrow LDMNCs were transduced with WT-FLT3, N51-FLT3, N51-FLT3Y599F1, or N51-FLT3Y599F1/2, sorted by FACS, and subjected to 3H-thymidine incorporation assay in the absence and presence of FL 50 ng/mL; representative of two independent experiments with similar results, n=5 – 8, *p<0.05 for N51-FLT3Y599F1/2 v. N51-FLT3 at baseline and in the presence of FL 50 ng/mL, statistical analysis using unpaired, two-tailed, student’s t test. (F) Immunoblot analysis of STAT5 phosphorylation, total STAT5, and FLT3 expression in retrovirally transduced and sorted WT-FLT3-, N51-FLT3-, N51-FLT3Y599F1-, or N51-FLT3Y599F1/2-expressing bone marrow-derived macrophages in the absence and presence of FL.