Figure 2: Various stimuli produce a significantly long-term and sustained, but not immediate, increase in cytosolic calcium.

(a) Dynamic monitoring of [Ca²⁺]_c immunofluorescence within 30 min after adding doxorubicin (0.2 μg/mL), deferoxamine (100 μM), ionomycin (2 μg/mL) or HBSS (control group) to Huh7 cells incubated with Fluo4-AM calcium indicator. Ionomycin, known as an ionophore Ca²⁺, was used as a positive control to increase [Ca²⁺]_c markedly. (b) Changes in mean cytosolic calcium fluorescence values of Huh7 cells (n = 11–18) treated by doxorubicin (0.2 μg/mL) (blue), deferoxamine (100 μM) (green) and ionomycin (2 μg/mL) (red) are shown, with the mean lines (solid) and 95% CI lines (dashed). Reagents were added at the 3 min. time point. (c) The maximum calcium fluorescence values (mean ± SD) were calculated after adding doxorubicin (blue), deferoxamine (green) and ionomycin (red) within 30 min, respectively. (*p < 0.05, each treatment group vs. control group). (d) Cells were treated by doxorubicin (0.2 μg/mL), hypoxia (1% O₂) and ionizing radiation (10Gy) and the cytosolic calcium immunofluorescence was measured by flow cytometry. Vertical split lines were set at the same fluorescence intensity level and right proportion of cell numbers was calculated. Statistical significances were assessed using Student’s T-Test.