STAT5 Transformation Activity. CALR mutants specifically activate TpoR leading to cell transformation. γ2A cells were transfected with JAK2, STAT5 and CALR wild type (wt) or the indicated mutants in the presence of TpoR (A), EpoR (B), GCSFR (C). Luciferase assay was performed using the firefly reporter, Spi-Luc/STAT5 (STAT5-luc) and pRL-TK renilla luciferase for normalization. Ba/F3 cells stably transduced with CALR constructs together with TpoR (D), EpoR (E) or GCSFR (F) were cultured for 3 days, in absence of cytokines. Positive controls of Epo and GCSF treatment are shown for Ba/F3 EpoR and GCSFR cells, respectively. Relative viability was measured using CellTiter-Glo technology. Statistical analysis (jmp pro11) was performed by the non-parametric multiple comparisons Steel’s test with a control group; *p<0.001. Used with permission.