Figure 1

STAT5 phosphorylation at Y694 is associated with decreased NPM1 expression levels. (a) 1 × 10⁶ TF-1 cells were deprived of GM-CSF overnight and subsequently stimulated with IL-3 for 3 h at a concentration of 100 ng/ml. Total protein from each cell lysate (50 μg) was loaded in the immunoblotting assay to determine changes in P-STAT5, T-STAT5 and NPM1 expression. TF-1 cells maintained in the presence of GM-CSF (1000 IU/ml) were analyzed as a control. (b) 1 × 10⁶ TF-1 cells maintained in GM-CSF supplemented medium were incubated with the inhibitor 573108 at the concentration of 170, 255 and 340 μM, respectively, for 3 h. (c) 1 × 10⁶ TF-1 cells deprived of GM-CSF supplement were incubated with the STAT5 inhibitor 573108 at a concentration of 150 μM for 1 h prior to 3 h IL-3 stimulation (100 ng/ml). (d) 1 × 10⁶ HeLa cells were stimulated with hEGF (100 ng/ml) for 3 h, preceded or not by a 1 h incubation with the inhibitor 573108 (200 μM). (e) TF-1 cells (1 × 10⁵ per condition) maintained in GM-CSF supplemented medium were lentivirally transduced with either shSTAT5 or backbone vector for 72 h using a titer of two TU per cell. (f and g) HEK 293T, HeLa cells (6 × 10⁵ per well for HEK 293T or 1 × 10⁶ per T-25 culture flask for HeLa cells) were plated on day 0 and transfected with RFP-STAT5A or RFP-backbone vectors on day 1; the transfected cells were harvested and lysed on day 2 for immunoblotting assays on the expression levels of the indicated proteins. (h) MCF-7 cells (5 × 10⁵ per well) were transfected with RFP-STAT5A or RFP-backbone vectors for 48 h and harvested and lysed for immunoblot as in the preceding panel. (i) A total of 40 μg total protein from each of the four different leukemic cell lysates were loaded on immunoblot assays to measure their STAT5 phosphorylation and NPM1 expression levels. For all experiments shown, densitometry was performed for three independent assays (mean±S.D.) to illustrate the changes of protein expression levels