Figure 5. UCHL1-mediated promotion of distant tumour metastases are HIF-1 dependent.

(a–d) Mice were treated with (+) or without (−) the HIF-1 inhibitor, YC-1 (30 mg kg⁻¹ daily) between 3 and 5 days after the intravenous transplantation of EMT6/EF-Luc/EV (EV) and EMT6/EF-Luc/UCHL1 (UCHL1) cells. (a) The total volume of pulmonary metastases was monitored by in vivo imaging as luciferase bioluminescence 7 days after transplantation. n=4. (b) Representative images. (c) Relative number of metastatic lung colonies to the EV and YC-1 (−) group. n=4. (d) Representative images. (e,f) A Transwell migration assay using EMT6/EF-Luc/shNC/EV (upper left in the right panels in e and f), EMT6/EF-Luc/shHIF-1α/EV (upper right), EMT6/EF-Luc/shNC/UCHL1 (lower left) and EMT6/EF-Luc/shHIF-1α/UCHL1 (lower right) cells under normoxic (20%, e) and hypoxia (1%, f) conditions. Mean±s.d. n=4, *P<0.05. Representative images are shown on the right. (g,h) By using two clones of EMT6/EF-Luc/shNC/EV, EMT6/EF-Luc/shHIF-1α/EV, EMT6/EF-Luc/shNC/UCHL1 and EMT6/EF-Luc/shHIF-1α/UCHL1 cells, the same experiments as those described in Fig. 4d,e were performed. (g) Relative number of metastatic lung colonies to the EV and shNC group. n=12. Mean±s.d. *P<0.05, **P<0.01; NS, not significant, Student’s t-test (a,c,e,f,g).