Fig. 1

Ectopic expression of JAK2-V617F increases intracellular (cytosolic) Ca²⁺ levels and enhances Ca²⁺ flux upon EPO re-stimulation following cellular starvation. (A) Schematic illustration of the experimental design of the study describing the EPO stimulation regimen. (B) Ca²⁺ influx in Fura-2 AM loaded 32D-JAK2-WT/EpoR and 32D-JAK2-V617F/EpoR cells under steady state condition. Cells maintained under steady state condition were stimulated with EPO (5 IU/ml) in Ca²⁺-free Ringer solution, followed by addition of 2 mM extracellular Ca²⁺ (left panel). Bar graphs show Ca²⁺ flux response normalized to baseline Ca²⁺ levels (F340/380) as maximum peak and area under the curve (AUC) of store depletion (from 109 to 594 s) and SOCE (from 595 to 1080 s) (right panel). (C) Ca²⁺ influx in Fura-2 AM loaded 32D-JAK2-WT/EpoR and 32D-JAK2-V617F/EpoR cells under starved condition. After 16 h of EPO starvation/without starvation, cells were stimulated with EPO (5 IU/ml) in Ca²⁺-free Ringer solution followed by addition of 2-mM extracellular Ca²⁺ (left panel). Bar graphs show Ca²⁺ flux response normalized to baseline Ca.²⁺ levels (F340/380) as quantification of maximal and area under the curve (AUC) of Store depletion (from 109 to 594 s) and SOCE (from 595 to 1080 s) (right panel). Data represents mean ± SEM from 6 independent experiments. Statistical analysis by unpaired Student’s t-test or Mann–Whitney-U-test. * p ≤ 0.05