Zinc chelation abrogates CALR^del52-induced JAK-STAT signaling in hematopoietic cell lines. (A) Viability of BaF3-MPL cells expressing CALR^del52 or MPL^W515L following treatment with TPEN for 48 hours. Each point represents the mean of 3 independent cultures. Testing for statistical significance was performed using a Student t test (*P < .05). (B) Immunoblotting demonstrates decreased Stat3 and Stat5 phosphorylation status following TPEN treatment of 4 hours in CALR^del52-expressing Ba/F3-MPL cells but not in Ba/F3-MPL^W515L cells. (C) FLAG-pulldown assays demonstrating zinc chelator TPEN treatment disrupts CALR^del52-MPL binding in CALR^del52-expressing Ba/F3-MPL cells. (D) Viability of BaF3-MPL cells expressing CALR^del52 or MPL^W515L following treatment with clioquinol (CQ) for 48 hours. Cell viability was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide assays. Each point represents the mean of 3 independent cultures. The data are representative of at least 2 independent experiments. Testing for statistical significance was performed using Student t test (*P < .05). (E) Immunoblotting demonstrates decreased Stat3 and Stat5 phosphorylation status following CQ treatment of 4 hours in CALR^del52-expressing Ba/F3-MPL cells but not in Ba/F3-MPL^W515L cells. (F) Immunoblotting of FLAG-immunoprecipitated proteins in 293T cells expressing CALR^del52 and MPL demonstrates disruption of CALR^del52-MPL binding following CQ treatment of 4 hours.