Figure 4

Expression of EphB6 dominant-negative mutant preserves Akt activation in doxorubicin-treated T-ALL cells. (A) ΔB6-Jurkat and pc3-Jurkat cells (4 × 10⁶ per well, in 6 well plates) were treated with 5 µM Dox in serum-free medium for 14 hours. Cell lysates were resolved by SDS PAGE and activating phosphorylation of the Akt kinase was examined by Western blotting with anti-phospho-Akt, recognising Akt phosphorylation at Ser473 (anti-p-Akt). Western blotting with anti-GAPDH was used as a loading control. Akt phosphorylation was quantified by densitometry using Carestream software and measurements were normalized to matching GAPDH controls. The graph represents a reduction in p-Akt signal intensity in Dox-treated cells as a percentage relative to the matching solvent controls. To optimize presentation, anti-p-Akt and anti-GAPDH images are shown at different brightness and contrast settings. (B) Cells were treated as in (A) and phosphorylation of the p70 S6 kinase was analysed by Western blotting with anti-phospho-S6K (anti-p-S6K). Phosphorylation of p70-S6K was quantified and presented as in (A). To optimize presentation, anti-p-S6K and anti-GAPDH images are shown at different brightness and contrast settings. (C) Cells were treated as in (A) and activating phosphorylation Erk kinases was analysed by Western blotting with anti-phospho-Erk (anti-p-Erk1/2). Western blotting with anti-tubulin used as a loading control. To optimize presentation, anti-p-Erk1/2 and anti-tubulin images are shown at different brightness and contrast settings. (D) ΔB6-Jurkat (4 × 10⁴ per well, in 96 well plates) were treated in triplicates with the indicated concentrations of Dox and with a suboptimal concentration of Perifosine individually, or in combination for 24 hours at 37 °C and 5% CO₂. Cells were stained with resazurin and cell survival was monitored using a SpectraMax M5 plate reader. Data are shown as a percentage of cell survival in drug-treated populations relative to matching solvent controls. The synergistic effect of Dox/Perifosine combinations was calculated using CompuSyn software and is presented as combination indices for each drug combination. Indices below 0.9 indicate synergism. All experiments were done at least three times. Full-length unadjusted Western blot images for this figure are shown in Supplementary Fig. 3A–C. *, P < 0.05, Student’s t-test. n.s., statistically not significant.