Selective Induction of Cell Death in Melanoma Cell Lines through Targeting of Mcl-1 and A1
Figure 2
Inhibition of Mcl-1 or A1 leads to melanoma-specific cell death.
(A) Primary human fibroblasts and 1205Lu melanoma cells were treated with the indicated siRNAs and expression of Bcl-2, Bcl-xL, Bcl-w, and Mcl-1 was analyzed after 48 hours by immunoblotting. (B) A1 mRNA was measured by quantitative RT-PCR in cells treated with A1-specific- or control siRNA for 48 hours (left and middle panel). Non-targeted Bcl-2 proteins were analyzed at the same time point by immunoblotting (right panel). (C) Cell death of fibroblasts was determined by quantification of Annexin V (AN)- and propidium iodide (PI)-positive cells 72 hours after transfection of the indicated siRNAs. (D) Cell death analysis of 1205Lu melanoma cells treated as described in C. Asterisks indicate a significant increase in cell death versus control siRNA-treated cells. (E) Fluorescence-activated cell sorting (FACS) of apoptotic and dead fibroblasts (upper panel) or 1205Lu melanoma cells (lower panel) treated as described in C. Numbers indicate the portion of cells in each quadrant that defines Annexin V- or propidium iodide-positive or negative cells. Mean +/− SD of at least 3 independent experiments is shown in B, C, and D. α-tubulin served as loading control in immunoblots. Blots are representative for 3 independent experiments.
