CALR mutant (CALR^del52) restores the response of MPL^R464G to ELT. UT-7 and Ba/F3 cells were transduced with retroviruses to overexpress WT MPL (MPL^WT) or mutant MPL (MPL^R464G) together with GFP, and retroviruses overexpressing WT CALR (CALR^WT) or mutant CALR (CALR^del52) with mCherry, and sorted for GFP⁺mCherry⁺ cells. Parental UT-7 cells were used as negative control. (A) Western blot analysis of signaling induced by TPO or ELT. UT-7 cells were cultured in presence of GM-CSF, starved overnight, and stimulated with TPO (10 ng/mL) or ELT (2 μg/mL) for 10 minutes. CALR^del52 enhances signaling in presence of ELT both in MPL^WT- and MPL^R464G- expressing cells. (B) Flow cytometric analysis of MPL expression on Ba/F3 cells using an allophycocyanin-coupled MPL antibody. CALR^del52 does not induce MPL^R464G traffic to the cell membrane. IgG, immunoglobulin G. (C) Proliferation assay in the presence of TPO, ELT, or TPO + ELT or without cytokines (no cytokines). (D) Proliferation assay in presence of ELT and recombinant CALR^del52 (recCALR^del52) (20 μg/mL). In panels C and D, 5 × 10⁴ UT-7 cells were plated in triplicate in 24-well plates and counted every day for 4 days. Shown are averages of 3 independent experiments, each performed in triplicate ± standard error of the mean (error bars). In panel C, the counts are reported for the condition UT-7 MPL^WT/CALR^WT (purple) + TPO and are shown only at day 4 of culture. *P < .05, Mann-Whitney unpaired, nonparametric 1-tailed test. (E) Control (CTRL) and patient CD34⁺ cells (II-2) isolated from peripheral blood were transduced with retrovirus encoding CALR^del52, sorted at day 2 on CD34⁺, and cultured in presence of stem cell factor and ELT for 12 days. At least 20 mCherry⁺ and 20 mCherry⁻ cells were analyzed. All mCherry⁺ cells expressed MK markers, while no mCherry⁻ cells differentiated into MKs (not shown). Representative pictures of mCherry⁺ CTRL and II-2 MK (CD41⁺ and von Willebrand factor [vWF]⁺) are shown. White arrows indicate individual MKs; 2 different plans of the same view are shown (i,ii). Scale bars, 30 μm.