Fig 1: High-dose midostaurin pulse exposure leads to prolonged inhibition of oncogenic signaling and induction of apoptosis. (a) Ba/F3 cells expressing a FLT3 internal tandem duplication (Ba/F3-FLT3-ITD)22 were seeded into six-well plates (5 × 104 cells per ml, total volume 2 ml) in RPMI 1640+10% FCS. Cells were treated for 2 h or 24 h with midostaurin as indicated. Cells exposed to 0.35% DMSO (0 h) served as controls. Cells treated for 2 h and control cells were washed 2 × with 2 ml phosphate-buffered saline (PBS) at room temperature and replated in fresh cell culture media (2 ml final volume). Twenty four hours after start of the experiment percentage of cells in subG1 phase was measured by flow cytometry after propidium iodide staining (PI). Experiments were performed in biological triplicates. Depicted is the mean percentage of cells in subG1 phase +s.e.m. of biological triplicates. (b) Ba/F3-FLT3-ITD cells were treated with 35, 100 and 3500 nM midostaurin for different periods of time as indicated. Upon treatment, cells were lysed immediately in lysis buffer. Cellular lysates were subjected to 10% SDS–polyacrylamide gel electrophoresis (PAGE) and subsequently immunoblotted with antibodies against cleaved caspase 3 (#9664; Cell Signaling Technology, Danvers, MA, USA), caspase 3 (#7148; Santa Cruz, Dallas, TX, USA) and beta-actin (#A5441-2, Sigma-Aldrich, Munich, Germany). (c) Schematic overview of experimental setup for high-dose pulse exposure experiments. (d, e) Cells (d: Ba/F3-FLT3-ITD; e: MV4–11) were treated with midostaurin for 2 h. Cells initially pulse-exposed to midostaurin were washed twice with 2 ml PBS at room temperature and were then replated in 2 ml fresh media (density: 5 × 104 cells per ml) as described in Figure 1A (1 ×). To test for residual TKI activity, the cell culture supernatant was transferred to previously untreated cells (S1), which were subsequently incubated for 24 h. Two hours after replating, a second drug wash out was performed (2 × 2 ml PBS). Cells were again replated in 2 ml fresh media (2 ×). Again, supernatants were transferred to previously untreated cells (S2). This procedure was repeated for a third time (3 ×, S3). Cells exposed to 0.35% DMSO (M) or to midostaurin for 24 h served as controls. Twenty four hours after start of TKI exposure percentage of cells in subG1 phase was measured by flow cytometry after PI. Depicted is the mean percentage of cells in subG1 phase +s.e.m. of biological triplicates. (f) Ba/F3-FLT3-ITD cells were treated with 3500 nM midostaurin as described in c. Untreated cells served as positive controls for phosphorylation signals (M). Cells treated continuously with TKI for 2 h or 10 h (2 and 10 h) served as positive controls for midostaurin activity. Lysates were prepared 2 h post drug wash out, allowing recovery of phosphorylation signals in case of absent midostaurin activity. S1, S2 and S3: lysates were prepared upon incubation of previously untreated cells for 2 h with the respective cell culture supernatants obtained upon each washing procedure. Lysates were subjected to 10% SDS–PAGE and subsequently immunoblotted with antibodies against phospho-ERK (#9106; pThr202/pTyr204; Cell Signaling) and phospho-STAT5 (#05-495; pY694/pY699; Merck Millipore, Darmstadt, Germany). To control for equal loading, blots were stripped and reprobed with anti-ERK (#9102; Cell Signaling), anti-STAT5 (#1081; Santa Cruz) and anti-GAPDH (#H86504M; Meridian Lifescience, Memphis, TN, USA) antibodies.