Bcr-Abl–expressing cells exhibit a reduced amount of releasable calcium from ER stores and decreased CCE. (A) ER calcium release and CCE were measured in untreated 32D, C2, and C4 cells. Cells were loaded with 1 μM CaGreen-1 and the level of fluorescence was measured by flow cytometry on the FL-1 channel as a time function. Cells were treated with 2 μM TG to deplete ER calcium stores followed by 5 mM CaCl₂ addition to the medium to assess the capacitative calcium uptake to the cytosol. Three regions were gated during the time-course analysis corresponding to the steady-state cytosolic calcium level (R1), cytosolic calcium level after ER depletion by TG treatment (R2), and after capacitative calcium uptake (R3). A representative result from 3 independent experiments is shown. (B) ER calcium release and CCE in 32D, C2, and C4 cells shown as histograms corresponding to the 3 selected regions. R1 region is the filled histogram, R2 is black-line histogram, and R3 is the gray-line histogram. (C) Ca²⁺ pool released from the ER (▪) and taken up from the extracellular medium (□, CCE) in 32D, C2, and C4 cells and (D) Jurkat, HL60, K562, and BV173 cells. In the case of 32D, C2, and C4 cells, imatinib treatment at 10 μM was performed for 4 hours prior to analysis (32D, C2, C4 + imatinib). The fluorescence values were obtained by statistical analysis using CellQuest software, and the ER releasable calcium content and CCE were calculated as ΔF/F₀ = 100((F–F₀)/F₀), where F₀ is the median fluorescence of R1 (steady state) and F either the median fluorescence of R2 (ER content) or R3 (CCE). %ΔF/F₀ ratios from all experiments presented as mean %ΔF/F₀ ± SD. *P < .01; **P < .001 versus 32D cells (Student t test).