Cytokine-independent cell growth induced by mutant CALR. (A) Retroviral vector constructs for the introduction and expression of CALR genes. These constructs produce C terminus FLAG-tagged WT CALR, mutant CALR type 1 (Del52), or mutant CALR type 2 (Ins5).1 Mutant proteins share distinctive amino acid sequences generated by frameshift mutations at the C terminus end of the protein. IRES, internal ribosomal entry site; LTR, long terminal repeat. (B) Immunoblot analysis of extracts (20 µg) prepared from UT-7/TPO cells (-), UT-7/TPO cells infected with mock vector (vect), or UT-7/TPO cells infected with viruses expressing the indicated CALR proteins. β-Actin was used as a loading control. *Indicates nonspecific bands detected in uninfected cell extracts. Cell proliferation assay in the absence or presence of TPO (C) or EPO (D) for UT-7/TPO (C) or UT-7/EPO (D) cells expressing vect (diamond), CALR WT (circle), CALR Del52 (triangle), and CALR Ins5 (square). Absorbance was measured at 450 nm to detect formazan dye produced by viable cells, and the mean value ± standard deviation (SD) from 3 replicates is depicted.
