Cellular Retention of TKI and Apoptosis Commitment Figure 2. Repetitive washing prevents apoptosis after high-dose TKI pulse-exposure. To analyze for residual TKI activity upon HD-TKI pulse-exposure, we employed repetitive wash-out procedures and exposure of previously untreated cells to cell culture supernatants. Therefore, 56104 cells/ml were seeded in a total volume of 2 ml in RPMI 1640 supplemented with 10% FCS and treated with TKI as indicated for 2 hours. Cells exposed to 0.35% DMSO served as controls (‘‘M’’). Then, cells were washed and replated as described in Figure 1B (‘‘1x’’, ‘‘2x’’, ‘‘3x’’; upper panel). To test for residual TKI-activity, the cell culture supernatants were transferred to previously untreated cells (‘‘S1’’, ‘‘S2’’, ‘‘S3’’; lower panel). For further details of the experimental procedure please refer to Figure 1B. Percentage of cells in subG1 phase was measured by flow cytometry after propidium iodide staining 24 hours after start of TKI treatment and is depicted for Ba/F3-BCR-ABL cells (A) and K562 cells (B), respectively. Experiments were performed in triplicate. Data are presented as mean percentage of cells in subG1 phase + SEM. doi:10.1371/journal.pone.0040853.g002 For Western blotting the following antibodies were used: anti- pSTAT5 (pY694) (Millipore, Billerica, MA, USA), anti-STAT5, anti-CRKL, horseradish peroxidase-linked goat anti-mouse im- munoglobulin (Santa Cruz, Heidelberg, Germany), anti-pABL (pY412, pY177), anti-ABL, anti-pCRKL (pY207), total caspase3, cleaved caspase3, horseradish peroxidase-linked goat anti-rabbit (Cell Signaling Technology, Danvers, MA, USA), anti-GAPDH (Meridian Lifescience, Saco, ME, USA). Experimental Design, TKI Treatment and Drug Wash-out Procedures If not otherwise stated, cells were seeded at a density of 56104 cells/ml in a total volume of 2 ml in cell culture media and treated for 2 h with the respective TKI. Following treatment, cells were washed twice with 2 ml PBS at room temperature and subsequently re-seeded in 2 ml cell culture media. The washing procedure was repeated twice, after 2 and 4 h, respectively. The detailed experimental setup is described in Figure 1. Cell Lysate Preparation and Western Blotting 26106 cells were incubated as described above. Cells were then resuspended in 100 ml lysis buffer (20 mM Tris-HCl; pH 7.0, 150 mM NaCl, 10% (v/v) glycerol, 1% Triton X-100, 1.5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.2 mM sodium orthovanadate, 10 mM NaF, 0.4x Complete [Roche Diagnostics, Mannheim, Germany], 1x PhosStop [Roche Diagnostics, Mann- heim, Germany]) and incubated for 30 min at 4uC. Lysates were then cleared by centrifugation and subsequently subjected to SDS- polyacrylamide gel electrophoresis (PAGE) and blotted onto nitrocellulose membranes (ECL membrane, Amersham, Freiburg, Germany) using an SDS electroblotting system (BioRad, Munich, Germany). Membranes were blocked with PBS/0.5% Tween/5% nonfat dry-milk for 1 h, incubated with primary antibody overnight at 4uC in blocking solution and washed twice in PBS/ 0.5% Tween. The membranes were then incubated for 1 h with an appropriate secondary antibody and specific proteins were visualized using the ECL detection system (Amersham, Freiburg, Germany). Flow Cytometry DNA content analysis was performed as described previously. [18] Briefly, cells were harvested, resuspended in 300 ml HFS- buffer (0,05 mg/ml propidium iodide, 1 mg/ml sodium-citrate, 1 ml/ml Triton X-100), and then subjected to FACS analysis. Annexin V staining was done using the PE Annexin V Apoptosis Detection Kit I (Becton Dickinson, Heidelberg, Germany) following the manufacturer’s protocol. For intracellular staining of phospho-proteins and total-proteins 16106 cells were harvested and processed as described elsewhere. [19]. The following antibodies were used for staining: p- STAT5(Y694)-Alexa647, p-CRKL(Y207)-PE, ABCB1-FITC, ABCG2-PE (Becton Dickinson, Heidelberg, Germany), cleaved PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e40853