Figure 4: CD44 splicing enhances ROS defence and lung colonization potential.

(a) Flow cytometric analysis of xCT expression on 4T1 or 67NR cells. (b) Flow cytometry of xCT expression on CD44v⁺ or CD44v⁻ 4T1 cells (upper left) and on shC CD44v⁺ or shESRP1 CD44v⁺ cells (upper right). CD44v⁻ (Mean RFI; 6.72±0.14), CD44v⁺ (Mean RFI; 10.06±0.93**). shESRP1 CD44v⁺ (Mean RFI; 6.09±0.29), shC CD44v⁺ (Mean RFI; 9.41±0.44**). Mean±s.d. values for RFI from five independent experiments. **P<0.01 (Student's t-test). (c) Intracellular GSH content of 4T1 or 67NR cells (left), CD44v⁺ or CD44v⁻ 4T1 cells (middle), and shC CD44v⁺ or shESRP1 CD44v⁺ cells (right). Data are means±s.d. from five independent experiments. **P<0.01 (Student's t-test). (d) IntegriSense images of lung metastases formed in mice 2 weeks after intravenous injection of CD44v⁺ cells (1×10⁵) and the onset of treatment with sulfasalazine or saline. The fluorescence signal intensity of each lung metastasis was also measured; data are means±s.d. for four animals treated with saline and six animals treated with sulfasalazine. **P<0.01 (Student's t-test). (e) Flow cytometric analysis of human CD44v9 expression on the surface of shESRP1 CD44v⁺ cells stably expressing human CD44v8-10 or stably transfected with the empty vector (mock). (f) Intracellular GSH content of cells transfected as in (e). Data are means±s.d. from five independent experiments. **P<0.01 (Student's t-test). (g) Mock-transfected or CD44v8-10-expressing shESRP1 CD44v⁺ cells were incubated with 500 μM H₂O₂ for 20 min, stained with DCFH-DA, and subjected to flow cytometric analysis. Mock-transfected shESRP1 CD44v⁺ (Mean RFI; 232.73±4.89), CD44v8-10-expressing shC CD44v⁺ (Mean RFI; 160.19±8.82**). Mean±s.d. values of RFI from five independent experiments. **P<0.01 (Student's t-test). (h) IntegriSense images of lung metastases formed 30 days after orthotopic injection of cells (1×10⁵) transfected as in (e) into the mammary gland of recipient mice. The fluorescence signal intensity of each lung metastasis was also measured; data are means±s.d. for five animals per group. **P<0.01 (Student's t-test).