Fig. 1. Mcl-1 is stabilized in lapatinib-resistant cells.

(A) Photo micrograph of lapatinib-sensitive and resistant BT474 cells (BT474 and rBT474, respectively) treated with lapatinib. Scale bars, 400 μm. n = 3 independent experiments. (B) Cells treated with or without lapatinib in the presence of the caspase inhibitor z-VAD were immunoblotted with antibodies against phospho-HER2 (p-HER2) (Tyr⁸⁷⁷) and total HER2. n = 3 independent experiments. (C) BT474 and rBT474 cells treated with lapatinib and z-VAD for the indicated times were immunoblotted for phospho-Akt (p-Akt)(Thr³⁰⁸)andtotalAkt.n=2independentexperiments.(D)BT474 and rBT474 cellstreated with lapatinib and z-VAD for the indicated times were immunoblotted for phospho-ERK1/2 (p-ERK1/2) (Thr²⁰² and Tyr²⁰⁴) and total ERK1/2. n = 3 independent experiments. (E) BT474 and rBT474 treated with lapatinib for the indicated times were immunoblotted for Mcl-1, Bim, and actin. A representative result is shown (left). Densitometric analysis of the abundance of Mcl-1 and Bim normalized to that of actin is shown (right). The protein abundance at the 0 time point in BT474 cells was set at 100%. n = 5 (Mcl-1) and n = 3 (Bim) independent experiments (means ± SEM). (F) ³⁵S-labeled Mcl-1 protein was incubated in cell-free lysates prepared from BT474 and rBT474. Mcl-1 stability was monitored by detecting ³⁵S-labeled Mcl-1 at the indicated time points. Representative result (top panel) and densitometric analysis of the abundance of Mcl-1 (bottom panel) are shown. The ³⁵S-labeled Mcl-1 abundance at the 0 time point was set at 100%. n = 3 independent experiments (means ± SEM). (G) ³⁵S-labeled Mcl-1 protein was incubated in lysates prepared from rBT474 cells treated with or without lapatinib for 7 days. Mcl-1 stability was monitored by detecting ³⁵S-labeled Mcl-1 at the indicated time points. Representative result (top) and densitometric analysis of the abundance of Mcl-1 (bottom) are shown. The ³⁵S-labeled Mcl-1 abundance at the 0 time point was set at 100%. n = 4 independent experiments (means ± SEM). (H) SKBR3 or rSKBR3 cells expressing FLAG-Mcl-1 and HA-ubiquitin (Ub) were treated with lapatinib in the presence of z-VAD and harvested after treatment with MG132 (Z-Leu-Leu-Leu-CHO). FLAG-Mcl-1 immunoprecipitates were immunoblotted for HA to analyze Mcl-1 ubiquitylation [*nonspecific band, immunoglobulin G (IgG) heavy chain]. IP, immunoprecipitation; IB, immunoblotting. n = 3 independent experiments.