Effect of Bcl-2 down-regulation on levels of ER releasable calcium and CCE in Bcr-Abl–expressing cells. (A) C2 and (B) C4 cells were transfected with either a nonspecific (NC siRNA) or Bcl-2 siRNA and Bcl-2 down-regulation was shown by immunofluorescence. Cells were incubated with anti–Bcl-2 (N-19) antibody followed by an incubation with AlexaFluor-488 anti–rabbit IgG antibody (i). Nuclei were stained with Hoechst 33258 (ii). Analysis was carried out using an Olympus BX51 fluorescence microscope equipped with an FITC filter using an Olympus UPlan Fl 40 ×/0.75 objective lens. Pictures were taken using a DP70 digital microscope camera (Olympus). Olympus DP-Soft 823 version 3.2 image acquisition software was used to acquire images, which were then processed in Adobe Photoshop 7.0 software (Adobe Systems, San Jose, CA). The figure was prepared with Adobe Illustrator 10.0.3 software. ER releasable calcium was measured as described in “Materials and methods” and Figure 1 and data are shown as ΔF/F0 (%). As a negative control, cells were treated with streptolysin O only (SLO) and ER calcium content and CCE were also measured. Data of 3 independent experiments are shown (mean ± SD).
