either the design of inhibitors that accumulate and are retained in target cells or, alternatively, co-administration of drugs which result in intracellular enrichment of specific TKIs may improve TKI therapy in the future. Supporting Information Figure S1 Onset of apoptosis is unchanged upon exposure to high TKI concentrations. Ba/F3-BCR-ABL cells were treated using different TKI concentrations. Cells were lysed at different time-points using lysis buffer and prepared for Western blot analysis. Immunoblotting was performed using monoclonal antibodies specific for total caspase3 and cleaved caspase3. To control for equal loading, blots were stripped and re- probed with anti-actin antibody. (A) depicts results obtained using imatinib (B) depicts results obtained using dasatinib. At least two independent experiments were performed and one representative experiment is shown. (PDF) Figure S2 Control cells do not reveal significant cyto- toxic effects upon HD-TKI pulse exposure. (A) Ba/F3- BCR-ABL cells (56104 cells/ml, total volume 2 ml) were treated with TKI as indicated for 2 h followed by extensive drug wash-out using 262 ml PBS. Cells were then re-seeded in 2 ml cell culture medium without TKI. Cells exposed to 0.35% DMSO served as controls (‘‘0 h’’). Cells continuously exposed to TKI served as positive controls (‘‘24 h’’). Twenty-four hours after start of TKI exposure the percentage of cells in subG1 phase was measured by flow cytometry after propidium iodide staining. Three indepen- dent experiments were performed. Data are presented as mean percentage of cells in subG1 phase + SEM. (B) Ba/F3 parental cells (56104 cells/ml, total volume 2 ml) were treated for 2 h with TKI as indicated followed by thorough drug wash-out using 262 ml PBS. Cells were then reseeded in 2 ml cell culture medium without TKI. Twenty-four hours after start of TKI exposure the percentage of cells in subG1 phase was measured by flow cytometry after propidium iodide staining. At least three independent experiments were performed and data are presented as mean percentage of cells in subG1 phase + SEM. (PDF) Figure S3 Repetitive washing prevents apoptosis in K562 cells –effect of a different wash-out protocol. K562 cells were treated either with imatinib or dasatinib as indicated. To control for the effects of different washing protocols, in this case the wash-out procedure was performed as previously described by Shah et al. 2008. In brief, cells (56104 cells/ml, total volume 2 ml for PI staining and 20 ml for AnnexinV and cleaved caspase3 staining) were washed three times with a volume of medium (containing 10% FCS) that consisted of 50% of the volume of the drug exposure. Cells were afterwards replated in fresh medium (+10% FCS) without inhibitor. For repetitive washing procedures under the same conditions, we generally followed the scheme as is depicted in Figure 1B. (A) Results of PI measurement of cells at 48 hours. Three independent experiments were performed and data are presented as mean percentage of cells in subG1 phase + SEM. (B) FACS measurement of AnnexinV and cleaved caspase3 at 48 hours. The Y-axis represents forward scatter (linear scale) and the X-axis depicts the signal intensity of AnnexinV (left) and cleaved caspase3 (right) on a log-scale. Three independent experiments were performed. One representative experiment is shown. (PDF) Cellular Retention of TKI and Apoptosis Commitment Figure S4 Intracellular signaling in K562 cells upon HD-TKI exposure –effect of a different wash-out proto- col. K562 cells (56104 cells/ml, total volume 20 ml) were treated with indicated TKI concentrations. Wash-out was performed as previously described by Shah et al. 2008. In brief, cells were washed three times with medium containing 10% FCS with a volume of medium that consisted of 50% of the volume of the drug exposure. Cells were afterwards replated in fresh medium (+10% FCS) without inhibitor. For repetitive washing procedures under the same conditions we generally followed the scheme as is depicted in Figure 1B. (A) Western Blot analysis of important signaling downstream nodes. Samples were lysed 2 h after each washing step. Untreated cells served as positive controls for phosphorylation signals. Cells treated continuously with TKI for 2 hours or 10 hours (‘‘2 h’’ and ‘‘10 h’’) served as positive controls for TKI activity. (B) Cells were treated for 2 h with 100 nM dasatinib, followed by serum wash-out. At various time points after wash-out cells were lysed and prepared for western blot analysis. Phosphotyrosine content was determined using the phosphotyr- osine antibodies Y100 and 4G10 as well as P-BCR-ABL (Y177) and (Y412). Antibodies against ABL and GAPDH served as loading control. (PDF) Figure S5 Determination of washing efficiency. K562 cells (56104/ ml) were pulse exposed for 2 h with 25 mM 14C-labeled imatinib followed by wash-out with PBS (1 ml per 56104 cells per washing step). Immediately after each washing step the PBS supernatant was subjected to beta-counter analysis to measure the concentration of remaining imatinib. After 4 washing steps, cells were replated into TKI free media. Imatinib concentration was then measured 2 h after the last washing step (‘‘+120’’). Supernatant analyzed at the end of the TKI exposure (‘‘EOE’’) represented a positive control for applied TKI. All measurements were performed in triplicate. Depicted are mean values + SEM of 3 independent experiments. Imatinib concen- trations were calculated by fitting the dpm values to a standard curve. (PDF) Figure S6 ABCB1 expression confers imatinib resis- tance in K562 cells. K562 and K562-ABCB1 cells were treated continuously either with 0.5 mM or 25 mM imatinib in the presence or absence of 10 mM PSC833 as indicated. Cells exposed to 0.35% DMSO or 10 mM PSC833 alone served as a control. Samples were analyzed after 24 h by flow cytometry after propidium iodide staining. Three independent experiments were performed and results are shown as means 6 SEM. (PDF) Table S1 TKI concentrations measured by HPLC/LC- MS/MS in cellular lysates. (A) TKI concentrations 6 SEM measured by HPLC/LC-MS/MS in cellular lysates treated with 25 mM imatinib or 100 nM dasatinib for 2 h as indicated followed by drug wash-out. Intracellular TKI concentrations were calcu- lated based on the mean cellular volume. (B) Mean size in mm 6 SD of Ba/F3-BCR-ABL cells and K562 cells. In total, 100 cells of each cell line were measured. Intracellular imatinib concentration was calculated based on the assumption that cells are spherical (V = 4/36p6r3). Thus, the total cellular volume for 0.26106 cells is 0.19 ml for Ba/F3-BCR-ABL cells and 0.5 ml for K562 cells. (PDF) PLoS ONE | www.plosone.org 14 July 2012 | Volume 7 | Issue 7 | e40853