Figure 8: Blockade of NHE6–RACK1 interaction minimizes Dox resistance in a chorioallantoic membrane xenograft assay.

(a) Timeline of the human tumour cell xenograft assay in the CAM of chick embryos. (b) mRNA expression of hypoxic markers CA9, GLUT1 and MCT4 in HT-1080-derived tumours extracted from the chorioallantoic membrane 7 days after implantation. RPLPO was used as loading control (n=4 independent experiments with 5 tumours per experiment). (c) Representative staining of HT-1080 tumours extracted from CAM showing hypoxic regions (Pimo⁺) and hypoxic cells (CAIX⁺). Nuclei were stained with DAPI. Scale bar, 100 μm and original magnification is × 10. (d and e) Tumour volumes from (d) HT-1080 and (e) MDA-MB-231 cells grown onto CAM and treated with various concentrations of Dox (n=5–7 embryos per group). (f) Representative images of tumours grown on CAM from HT-1080 cells transfected with scrambled peptide (HT-1080 scr) or NHE6^527–588 peptide (HT-1080 NHE6^527–588) and treated with 0.5 μM Dox. (g and h) Tumour volumes of (g) HT-1080 and (h) MDA-MB-231 cells grown on CAM in the presence of scrambled sequence (scr) or NHE6^527–588 sequence and treated with (g) 0.5 μM Dox or (h) 1 μM Dox (n=5–9 embryos per group). Bars represent the mean±s.e.m. (*P<0.05, **P<0.01, ***P<0.001, unpaired Student’s t-test).