Effect of AT-101 and fludarabine on survival proteins in CLL cells in suspension culture or on stromal coculture. (A) Effect of AT-101 on Mcl-1 and PARP cleavage in CLL cells (patient 30) growing in suspension culture. CLL lymphocytes were incubated without or with 10 and 20 μM AT-101 for different time periods and the cleaved and uncleaved Mcl-1 and PARP were measured by immunoblotting. Annexin positivity for each sample is given above the immunoblots. (B) Effect of fludarabine on Mcl-1 and PARP cleavage in CLL cells (patient 30) growing in suspension culture. CLL lymphocytes were incubated without or with 10 μM fludarabine for 8, 24, and 72 hours and cleaved and uncleaved Mcl-1 and PARP were measured by immunoblotting. The annexin positivity for each sample is given above the immunoblots. (C) Antiapoptotic protein expression in CLL cells and influence of stromal microenvironment. CLL lymphocytes from patients were either cultured in suspension (C), in suspension with 20 μM AT-101 (C + AT), or with stromal cells in the absence (C + S) or presence of AT-101 (C + S + AT) for 24 hours. Antiapoptotic protein expression (Mcl-1; Bcl-2; Bcl-xl) was analyzed by immunoblotting. Actin was used as a loading control. S denotes stroma alone. Sample numbers are patient identification (Pt) numbers; see Table S1. (D) Effect of stroma and AT-101 on expression level of ERK and AKT proteins in CLL and stromal cells. CLL lymphocytes from patients were either cultured in suspension (C), in suspension with 20 μM AT-101 (C + AT), or with stromal cells in the absence (C + S) or presence of AT-101 (C + S + AT) for 24 hours. Stroma cells were cultured alone (S) or with AT-101 (S + AT) for 24 hours. pERK and pAKT were measured by immunoblotting, and the T-AKT and T-ERK were used for equal loading. Sample numbers are patient identification (Pt) numbers.