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), and 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 the 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 nonparametric multiple comparisons Steel test with a control group; *P < .05, **P < .01, ***P < .001.