Figure 2
The effect of CALR mutations on the growth and the response to TPO in human cell line cells expressing MPL. (a) CALR mutation was introduced with the CRISPR-Cas9 system into CMK11-5 cells and F-36P-MPL cells. CMK11-5 clone 751 with CALRdel4/del17, CMK11-5 clone 649 with CALRdel25 and parent CMK11-5 cells (left), as well as F-36P-MPL clone 1 with CALRdel1/ins1, F-36P-MPL clone 2 with CALRdel1/ins1 and parent F-36P-MPL cells (middle, left), were grown in the absence (left and right) or the presence (middle) of GM-CSF. Cell numbers (average of triplicate±s.e.m.) were counted on the indicated days. Both CMK11-5 clones 751 and 649 show increased cell proliferation compared with parent CMK11-5 cells. The growth of F-36P-MPL clones 1 and 2 is less than that of parent F-36P-MPL cells in the presence of 10 ng/ml GM-CSF; however, F-36P-MPL clones 1 and 2 show growth in the absence of GM-CSF. (b) WST-8 assay at various TPO concentrations. CMK11-5 clones 751 and 649, and parent CMK11-5 cells show no response to TPO. In contrast, F-36P-MPL clones 1 and 2, and parent F-36P-MPL demonstrate a response to TPO, and F-36P-MPL clones 1 and 2 show no hypersensitivity to TPO. One representative experiment is shown. Data are presented as means±s.e.m. Tukey’s multiple comparison test with one-way analysis of variance was used. *P<0.05 and **P<0.01vs parent cells. (c) STAT5 phosphorylation. CMK 11-5 clone 751 shows augmented STAT5 phosphorylation compared with parent CMK11-5 cells (left). F-36P-MPL is a cytokine-dependent cell line and STAT5 is phosphorylated in the presence of GM-CSF. Its phosphorylation disappears after withdrawal of GM-CSF (middle). The phosphorylation of STAT5, which transiently decreased after withdrawal of GM-CSF, was re-observed 24 h after GM-CSF depletion in F-36P-MPL clone 1 (right).
