Figure 3

Disruption of EphB6 activity prevents induction of apoptosis in response to doxorubicin treatment. (A,B) ΔB6-Jurkat and pc3-Jurkat cells (1 × 10⁶ cells per well in 6 well plates) were treated in triplicates with 5 µM Dox or a matching solvent control in serum-free medium, for 14 hours. Caspase-3 activity was quantified using the EnzChek® CASPASE-3 Assay kit #1 (Invitrogen) according to the manufacturer’s instructions. (A) The graph shows caspase-3 activity in arbitrary units (AU), as measured at A₄₄₂ using a SpectraMax M5 plate reader, and represents analysis of one of three independent experiments. (B) The graph shows fold-change in caspase-3 activity following Dox treatment, compared to a matching solvent control and represents analysis of three independent experiments. (C) ΔB6-Jurkat and pc3-Jurkat cells were treated with 5 µM Dox for 14 hours at 37 °C and 5% CO₂. Cells were lysed and cell lysates were resolved by SDS PAGE. PARP cleavage was analysed by Western blotting with anti-PARP. Western blotting with anti-tubulin was used as a loading control. To optimize presentation, anti-PARP and anti-tubulin Western blot images are shown at different brightness and contrast settings. (D) The indicated cells were treated with 5 µM Dox, or a matching solvent control, for 24 hours. Cells were stained with PE-conjugated Annexin V for 15 minutes in the dark at room temperature and analyzed by flow cytometry. The graphs on the right show median intensities of Annexin V staining of Dox-treated cells after the subtraction of values corresponding to median staining intensities of matching solvent-treated controls. The graphs represent analyses of three independent experiments performed in duplicates. The analyses were done using the FlowJo software. All experiments were done at least three times. Full-length unadjusted Western blot images for this figure are shown in Supplementary Fig. 2A. *, P < 0.05, Student’s t-test. n.s., statistically not significant.