Figure 3. p.Y371H does not confer cytokine sensitivity or cytokine independent growth until silencing of murine Cbl.

Panel (a) Transduction of p.Y371H or the known murine oncogeneic mutant 70Z in wildtype hematopoietic cells from fetal liver, did not confer hypersensitivity to GM-CSF, nor did expression of these mutants in BaF3-EpoR cells result in cytokine independent growth (b). Panel (c) An shRNA to murine Cbl (cbl.2364) demonstrated near complete shutdown of expression in BaF3-EpoR cells by Western blot with re-expression upon introduction of the human WT, 70Z, or p.Y371H in these same cell lines. Panel (d) Both the p.Y371H and 70Z Cbl conferred cytokine independent growth in the presence of cbl.2364. Controls included the Venus vector pMIV and BaF3-EpoR. Error bars for triplicate replicates (s.e.m.) are shown and when not visible, indicate tight clustering. Using a paired t-test: day 7 comparing 2364+ WT-Cbl versus 2364+p.Y371H, p-value= 0.017, and at day 9: p-value <0.001. Panel (e) Serial transduction of the hairpin (2364) and p.Y371H or 70Z constructs also conferred hypersensitive growth after assessing cell proliferation on day 5 in increasing concentrations of Epo. Using a paired t-test at each concentration of Epo when comparing 2364+WT-Cbl versus 2364+p.Y371H: Epo 0 unit/ml: p = 0.036, Epo 0.01 units/ml: p= 0.0015, Epo 0.1 units/ml: p= 0.029, Epo 1 unit/ml (saturating dose) P= 0.697. Panel (f) Both the p.Y371H and 70Z containing cells demonstrated activation of pERK, pAKT, and pS6 in the absence of Epo or in low dose 0.01 unit/mL of Epo in comparison to negative controls. All cell proliferation work was done in triplicate.