Figure 5.

Induction of HO-1 expression via elevation of intracellular Ca²⁺ levels. (a) RAW 264.7 cells were incubated with BAPTA/AM (B/A, 5 μM), calmidazolium chloride (CC, 5 μM), and nifedipine (Nif, 5 μM) for 1 h and then treated with crocin (500 μM) for 6 h. Equal amounts of cytosolic proteins were subsequently analyzed by Western blotting with HO-1 antibody. Tubulin was used as a loading control. (b) Cells were transfected with HO-1 promoter-luciferase construct and then treated as described above, after which equal amounts of cell extracts were assayed for dual luciferase activity. *P < 0.05 versus the group treated with crocin alone. (c) Intracellular free Ca²⁺ levels were assessed by flow cytometry. Cells were treated with vehicle or crocin (500 μM) for 10 min in either Ca²⁺-containing medium (left) or Ca²⁺-free medium (right). (d) The changes of intracellular Ca²⁺ levels with time were represented as percentages of responding cells. (e) Cells were treated with crocin (500 μM) in the absence or presence of BAPTA/AM (B/A, 5 μM), or nifedipine (Nif, 5 μM) for 10 min, after which intracellular free Ca²⁺ levels (represented as percentage of responding cells) were evaluated by flow cytometry. *P < 0.05 versus the group treated with PBS (vehicle); ^# P < 0.05 versus the group treated with crocin alone in both Ca²⁺-containing and Ca²⁺-free medium.