FIGURE 4.

Greensporone C-induced mitochondrial signaling pathways in leukemic cells. GC treatment causes alteration in Bcl-2 expression. K562 (A) and U937 (B) cells were treated with increasing doses of GC for 24 h as indicated. After cell lysis, equal amounts of proteins were separated by SDS–PAGE, transfered to PVDF membrane, and immunoblotted with antibodies against caspase-8, cleaved caspase-8, Bid, and GAPDH as indicated. K562 (C) and U937 (D) cells were treated with GC as indicated and expression level of Bax and Bcl-2 was determined by immunoblotting with antibodies against Bax, Bcl-2, and GAPDH. Data obtained from immunoblot analyses of Bax and Bcl-2 in leukemic cell lines were used to evaluate the effects of GC on Bax/Bcl-2 ratio. Densitometric analysis of Bax and Bcl-2 bands in K562 (E) and U937 (F) cells was performed using AlphaImager Software (San Leandro, CA, United States), and data (relative density normalized to GAPDH) were plotted as Bax/Bcl-2 ratio. GC treatment causes the loss of mitochondrial membrane potential in leukemic cells. K562 (G) and U937 (H) cells were treated with indicated doses of GC for 24 h. After JC1 staining cells were analyzed by flow cytometry as described in the section “Materials and Methods.” The graph displays the mean ± SD of three independent of experiments. ^∗P < 0.05 and ^∗∗P < 0.001. GC-induced release of cytochrome c. K562 (I) and U937 (J) cells were treated with and without GC for 24 h. Cytoplasmic fraction was isolated as described in the section “Materials and Methods.” Cell extracts were separated on SDS–PAGE, transferred to PVDF membrane, and immunoblotted with an antibody against cytochrome c and GAPDH.