Figure 2

TAE684-resistant cells are dependent on AXL signaling. (a) Growth curve (left) of untransfected (U), scrambled (S shRNA) or AXL shRNA-expressing SY5Y-TR1 cells. Two different AXL shRNAs (3 and 5) were used. The results represent the mean±s.d. of three separate experiments. ***P<0.001. Western blot analysis (right) of AXL knockdown in the same cells. (b) Western blot analysis of AXL and pERK in SY5Y-TR1 cells expressing either a nonspecific (Ctl) or AXL siRNA at the indicated doses for the indicated times. Untransfected SY5Y-TR1 cells (U) were also used as controls. (c) Dose–response curves for SH-SY5Y and SY5Y-TR1 cells treated with the AXL small molecule inhibitor, R428, for 3 days (IC₅₀=SH-SY5Y, 4.79 μM; SY5Y-TR1, 1.93 μM). (d) Drug matrix illustrating percent inhibition (left) and Delta Bliss (right) for R428 in combination with TAE684 in SH-SY5Y and SY5Y-TR1 cells. Percent inhibition values were derived from the cell viability assay after 3 days of treatment with the two compounds. Considering A and B to be the fractional growth inhibition (percent inhibition divided by 100) of single drug treatment at a given dose (for example, TAE684 and R428, respectively), a Bliss expectation was calculated using the formula: (A+B)−A × B for every drug concentration used. The ‘Delta Bliss’ represents the difference between the Bliss expectation and the observed fractional growth inhibition of the combination of drug A and B at the same dose. A Delta Bliss of 1 indicates an additive effect, whereas a value <0 or >1 indicate antagonistic and synergistic effects of the combination, respectively. Color scales for each specific grid (left=percent inhibition; right=Delta Bliss) are shown. Values represent the mean of two separate experiments. (e) qRT–PCR analysis (left) of AXL expression in untransfected (SH-SY5Y) or SH-SY5Y cells stably expressing either AXL (AXL) or vector control (MSCV). ***P<0.001. Western blot analysis (right) of total and pAXL in these cells. AXL was immunoprecipitated using an anti-AXL antibody and western blotting performed with an anti-phosphotyrosine antibody. (f) Dose–response curves for cells in (e) exposed to TAE684 for 3 days (IC₅₀=SH-SY5Y, 44 nM; SH-SY5Y MSCV, 50 nM; SH-SY5Y AXL, 102 nM). (g) Western blot analysis of total and pERK in the same cells as in (e).