Ba/F3-EpoR cells expressing JAK2 wild-type displayed a low activity in the absence of ligand, but responded well upon stimutation with 50 U/ml Epo (Figure 3B).
The mean Kit-specific fluorescence (MFI) level was increased about 2.5 fold in 663 and 812 cells cultured with SCF compared to cells grown with Epo (Figure 1C).
CFU-E from BM or spleen of these recipients showed increased sensitivity to Epo, with about 25 30% of CFU-E growing in the absence of exogenous Epo (Figure 1H), demonstrating the presence of EEC in the polycythemic mice.
Immunoblotting of cell extracts with the anti-lyn antibody revealed that Lyn expression was induced in a dose dependent manner with levels readily detectable at 0.25 U/mL of Epo (Figure 4B).
Although we were unable to directly demonstrate the phosphorylation of the truncated EPOR in the absence of the cytokine, we observed, under this condition, the activation of EPOR-dependent pathway by detecting STAT5 phosphorylation in CD34+ cells cultured without EPO (Figure 5D).
To target erythropoietin using a different strategy, R3230-GFP cells were engineered to constitutively express and secrete mutant erythropoietin protein R103A-EPO (figure S3).
By staining for HSCs in the marrow (CD150+CD41−CD48−Lin− cells), it was noted that the number of HSCs that express BMP2 was increased in the marrow at 2 weeks of Epo treatment (Figure 6A, and quantified in Figure 6B).
Mutants F595A and F595V exhibit a small inhibition to high Epo doses (Figure 2B), while in the V617F context these mutants do not affect Epo response (Figure 2C).
Accordingly, incubation of blood eosinophils with increasing ratios of Mycobacterium bovis-BCG induced dose-dependent ROS production and EPO release (Figure 8A B), that could be inhibited, in a dose-dependent manner, by an anti-γδTCR antibody [40] (Figure 8C D).
As expected, all mutants displayed strong pSTAT5 and pJAK2 activation upon Epo stimulation (Figure 3C).
