Development and characterization of CALR DEL and KO cell lines. (A) The strategy to obtain CALR KO (targeting exon 1) and DEL (targeting exon 9) UT7 and UT7/mpl cell lines with CRISPR/Cas9 genome editing; the resulting DNA sequence of genome-edited clones used in the study is reported below the sequence of WT CALR, or a typical del52 mutation, at corresponding DNA regions of interest for KO and DEL targeting. The ATG start codon is in bold and underlined; in the UT7 KO cell line, the ATG is removed whereas in UT7/mpl, the KO conditions were due to insertion of a TAG stop codon (in bold and red). The predicted sequence of the resulting DEL protein for UT7 and UT7/mpl is shown within a rectangle, with the novel sequence in red and the common C terminus underlined. (B) The expression of CALR mRNA and CALR protein in CALR WT (parental), DEL and KO UT7, and UT7/mpl cell lines was assessed, respectively, by qRT-PCR (top panel) and western blotting (bottom panel), using antibodies to either the common N-terminal or the C-terminal KDEL domain. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for loading normalization; the asterisk points to mutant protein. (C-D) The GM-CSF–dependent UT7 and TPO-dependent UT7/mpl cells, either parental, CALR DEL, or KO, were seeded at 2 × 10⁵ cells per milliliter in cytokine-free medium. Living cells (×10⁵/mL) were counted daily by trypan blue dye exclusion (top panels). Cell apoptosis was assessed at day 3 using the Annexin V–Fluoassay, and expressed as the percentage of annexin V⁺ cells over total (for UT7 [E] and UT7/mpl [F] cell lines, respectively). Data are the mean plus or minus SD of 3 independent experiments. All P values were determined by Student t test (*P < .05; **P < .01; ***P < .001).