Figure 2 | Scientific Reports

Figure 2

From: The EphB6 receptor is overexpressed in pediatric T cell acute lymphoblastic leukemia and increases its sensitivity to doxorubicin treatment

Figure 2

Suppression of EphB6 action supports survival of doxorubicin-treated cells. (A) Western blot analysis of EphB6 expression in Jurkat cells stably transfected with a cytoplasmic domain-deleted dominant-negative mutant (DN-EphB6) of EphB6 (ΔB6-Jurkat) or mock-transfected with the empty pcDNA3 expression vector (pc3-Jurkat). Western blotting with anti-tubulin was used as a loading control. (B) pc3-Jurkat and ΔB6-Jurkat cells (4 × 104 cells per well in 96 well plates) were treated with the indicated doxorubicin (Dox) concentrations or a matching solvent control for 24 hours at 37 °C and 5% CO2. Cells were stained with resazurin and cell survival was quantitated using a SpectraMax M5 plate reader. The graph represents the analysis of five replicates and shows the percentage of survival of Dox-treated cells relative to matching solvent-treated cells. (C) E6.1 cells were transduced with EphB6-targeting shRNA (E6.1-B6-shRNA) or non-silencing shRNA (E6.1-NS). EphB6 expression was analysed by Western blotting with anti-EphB6. Western blotting with anti-tubulin was used as a loading control. (D) E6.1-NS and E6.1-B6-shRNA cells (4 × 104 cells per well in 96 well plates) were treated with Dox or a matching solvent control for 24 hours. Cells were stained with resazurin and cell survival was measured using a SpectraMax M5 plate reader. The graph represents analysis of five replicates and shows the percentage of survival of Dox-treated cells relative to the survival of solvent-treated cells. (E) Human malignant lymphoblastic T cells, H9, were electroporated with the pcDNA3 expression vector encoding a DN-EphB6 mutant (ΔB6-H9) or mock-transfected with empty pcDNA3 as a control (pc3-H9), and cells were subjected to 30 days selection with 1 mg/ml of G418. Expression of DN-EphB6 was confirmed by Western blotting. (F) ΔB6-H9 and pc3-H9 cells were treated with the indicated concentrations of Dox or solvent control and cell survival was analysed by resazurin staining as in (B). All experiments were performed at least three times. To optimize presentation, anti-EphB6 and anti-tubulin Western blot images are shown at different brightness and contrast settings. Full-length unadjusted Western blot images for this figure are shown in Supplementary Fig. 1A–C. *P < 0.05, Student’s t-test. n.s., statistically not significant.

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