Combined inhibition of Mcl-1 and A1 results in efficient induction of apoptosis in 1205Lu melanoma cells.

<p>(A) 1205Lu melanoma cells were transfected with A1- or Mcl-1-specific siRNAs or control siRNAs as indicated. Cell death was assessed 72 hours after transfection. Mean +/− SD of 3 independent experiments is shown. Asterisks represent significant increase in cell death compared to single inhibition. (B) 1205Lu melanoma cells were transfected with different siRNA sequences targeting A1 or Mcl-1 (A1b, Mcl-1b). Analysis was carried out as described in A. Asterisks represent significant increase in cell death compared to single inhibition. Also, cell death induction by A1b, Mcl-1b, and A1b/Mcl-1b versus control was significant (not indicated in the figure). (C) Caspase activation of 1205Lu cells treated as described in A was assessed by immunoblotting with antibodies detecting the proform as well as the active subunits. Caspase 9 (upper panel) and caspase 3 (lower panel) were analyzed 48 hours after transfection. (D) 1205Lu cells treated with siRNAs as described in A with or without the pan-caspase inhibitor zVAD-FMK (100 µM). Cell death was measured 72 hours after transfection. Mean +/− SD of 3 independent experiments is shown. Asterisks represent significant decrease in cell death of zVAD-FMK-treated cells compared to cells not treated with zVAD-FMK. AN, Annexin V; PI, propidium iodide.