Kim et al. BMC Cancer 2012, 12:147 http://www.biomedcentral.com/1471-2407/12/147 Page 4 of 10 Figure 1 Combinational treatment of ovarian cancer cell lines with TM and doxorubicin reduces cell viability via promotion of apoptotic signaling. SKOV-3 (a) or A2780 (b) ovarian cancer cells were treated with TM (0, 30 μM for SKOV-3; 0, 7.5 μM for A2780) for 24 h, after which the cells were treated with doxorubicin (0, 2.5, 5, 10 μM) for another 24 h. Cell viability was evaluated as described in Methods. Data are expressed as the mean of the triplicate determinations (X ± SD) compared to untreated cells [100%]. (c) SKOV-3 cells were treated with TM (0, 30 μM) for 24 h, after which the cells were treated with doxorubicin (0, 5 μM) for another 24 h. The change in the mitochondrial membrane potential (ΔΨm) was measured by flow cytometry. Intact cells = Q4, Loss of ΔΨm = Q3, ruptured cell membrane = Q1 and Q2. (d) SKOV-3 cells (left panel) were treated with TM (0, 30 μM) for 24 h, after which the cells were treated with doxorubicin (0, 5 μM) for 1, 4, 7 or 24 h as indicated. The same procedure was employed to treat A2780 cells (right panel) with different concentrations of TM (0, 7.5 μM) and doxorubicin (0, 2.5 μM). Immunoblotting was carried out with primary antibodies against cleaved PARP, caspase-3, and -7. As an internal standard for equal loading blots were probed with an anti-GAPDH antibody. [7]. By immunoblotting using primary antibodies against native and activated/phosphorylated JNK or p38 MAPK we analyzed the effect of TM/doxorubicin treatment on ovarian cancer cells. SKOV-3 cells were treated with TM (0, 30 μM) for 24 h, after which the cells were trea- ted with doxorubicin (0, 5 μM) for various time periods as indicated (Figure 3a, left panel). The same procedure was employed to treat A2780 cells with different concentrations of TM (0, 7.5 μM) and doxorubicin (0, 2.5 μM) (Figure 3a, right panel). TM alone did not cause a change of JNK or p38 activation in SKOV-3 or A2780 cells. When cells were treated with doxorubicin for 24 h in combination with TM, a drastic increase in the activation of both MAPKs, compared to their activa- tions in the cells treated by either TM or doxorubicin alone, was observed (Figure 3a).