Signaling mechanism of VCR and inhibition by doxorubicin. (A) Western blot of total cellular protein was performed on CEM cells stimulated with doxorubicin and VCR as in Figure 1A. GAPDH served as a loading control. (B) CEM cells were stimulated with doxorubicin and VCR. Loss of mitochondrial membrane potential (left panel) and cytochrome c release (right panel) was measured by FACScan. Caspase cleavage was detected by Western blot (bottom panel). *P < .05 (ANOVA), comparing stimulation with VCR alone with doxorubicin alone or combined stimulation with doxorubicin + VCR. (C) CEM cells were treated with 2,3-DCPE (10μM, left panel) or the phosphatase inhibitor okadaic acid (okadaic, 0.03 ng/mL, right panel) for 8 hours, followed by doxorubicin together with VCR for another 48 hours as indicated. Western blot of total cellular protein was performed after 56 hours. *P < .05 (ANOVA). NS indicates not significant. The concentrations of doxorubicin and VCR, measurement of apoptosis, presentation of data, and statistical analysis were performed as described in Figure 1A. Casp indicates caspase; co, unstimulated control cells; cl., cleaved; d, doxorubicin; p, phosphorylated; h, hour; and V, VCR.