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. Author manuscript; available in PMC: 2011 Aug 1.
Published in final edited form as: Mol Cancer Ther. 2010 Jul 27;9(8):2232–2242. doi: 10.1158/1535-7163.MCT-10-0336

Figure 3. Treatment with 17-DMAG and/or K252a attenuates NGF-mediated autophosphorylation of TrkA and downstream signaling in K562 cells and 32D/wtTrkA cells.

Figure 3

A. K562 and 32D/wtTrkA cells were serum starved for 2 hours, then treated with 100 ng/ml NGF and/or 1.0 μM, 17-DMAG for 12 minutes. Following this, Western blot analysis of p-TrkA, p-AKT and p-ERK1/2 was performed on the total cell lysates. The levels of β-actin served as the loading control. B. 32D cells expressing vector, wt/TrKA or Δ TrkA were exposed to indicated concentrations of 17-DMAG for 48 hours and the percentage of apoptotic cells were assessed by Annexin V and PI staining, followed by flow cytometry. C. TF-1 and K562 cells were exposed to indicated concentrations of K-252a for 48 hours and the percentage of apoptotic cells were determined by Annexin V/PI staining followed by flow cytometry. D. K562 and 32D/wtTrkA cells were serum starved for 2 hours and treated with 100 ng/ml of NGF alone or co-treated with 100 ng/ml of NGF and 17-DMAG or 150 nM of K-252a for K562 cells and 75 nM K-252a for 32D/wtTrkA or NFG, 17-DMAG and K-252a for 12 minutes. Following this, Western blot analysis of p-TrkA was performed on the total cell lysates. The levels of β-actin served as the loading control. E. K562 cells were exposed to 100 nM, 17-DMAG and indicated concentrations of K-252a for 48 hours and percentage of apoptotic cells were measured by Annexin V and PI staining followed by flow cytometry. Isobologram analysis was performed to obtain combination index values for each fraction using Calcusyn software. Combination index values <1.0 correspond to synergistic interactions. The fraction of cells affected (Fa) by the drug combination is presented.