Fig. 5.

The interaction of the NT-6 × helix domain of AQP1 with ANXA2 induced cell secretion of ICAM1 and CTSS, leading to cell local invasion. a Western blot analysis to detect the expression of CTSS and ICAM1 in the whole cell lysates of Flag-vector/MDA-MB-231 cells, Flag-AQP1/MDA-MB-231 cells and Flag-AQP1/shANXA2 #1/MDA-MB-231 cells. β-actin was the loading control. b Western blot analysis of the supernatant medium samples isolated from 3 indicated cells was performed to quantify ICAM1 and CTSS proteins. c-d Representative migration or invasion images from Flag-vector/MDA-MB-231 cells treated with 3 different supernatant medium samples (two-tailed Student’s t test, ^*P < 0.05). Each bar represented the mean ± SEM from three independent experiments. Scale bar = 100 µm. e Co-immunoprecipitation experiments were performed to illustrated the interaction between AQP1-NT-6 × helix and ANXA2. The Flag-AQP1-CT/MDA-MB-231 cells were also tagged with GFP. f The abilities of migration and invasion were detected using Flag-AQP1/MDA-MB-231 and Flag-AQP1-∆NT-6 × helix/MDA-MB-231 cells. Each bar represented the mean ± SEM from four independent experiments (two-tailed Student’s t test, ^***P < 0.001). Scale bar = 100 µm. g Western blot analysis to detect the expression of CTSS and ICAM1 in the whole cell lysates of Flag-AQP1/MDA-MB-231 cells and Flag-AQP1-∆NT-6 × helix/MDA-MB-231 cells. β-actin was the loading control. h Western blot analysis of the supernatant medium samples in 2 indicated cells. i-j Representative migration or invasion images from Flag-vector/MDA-MB-231 cells treated with the supernatant medium of Flag-AQP1/MDA-MB-231 cells and that of Flag-AQP1-∆NT-6 × helix/MDA-MB-231 cells (two-tailed Student’s t test, ^*P < 0.05, ^**P < 0.01). Each bar represented the mean ± SEM from three independent experiments. Scale bar = 100 µm. All in vitro experiments were repeated at least 3 or 4 times