Fig. 2.

AQP1 localized in the Golgi apparatus and induced cell secretion of ICAM1 and CTSS, leading to the cell local invasion. a Immunofluorescent staining of AQP1 and Golgi marker GM130 or Golgi tracker in Flag-AQP1/MDA-MB-231 cells. Scale bar = 20 μm. b Immunofluorescent staining of GM130/TGN46 together with staining of DAPI in Flag-vector/MDA-MB-231 and Flag-AQP1/MDA-MB-231 cells. In the right panel showed the distribution of cells with different ranges of Golgi ribbon angle (Ɵ: 0°–360°) (n = 80–90 cells per group, ^***P < 0.001). Scale bar = 20 μm. c Trafficking of TS045-VSVG-EGFP in Flag-vector/hela and Flag-AQP1/hela cells (two-tailed Student’s t test, ^*P < 0.05). Scale bar = 20 μm. d Migration and invasion assays showed that Flag-vector/MDA-MB-231 cells treated with the supernatant of Flag-AQP1/MDA-MB-231 cells exhibited the promoted phenotype compared with Flag-vector/MDA-MB-231 cells (two-tailed Student’s t test, ^*P < 0.05). Each bar represented the mean ± SEM from three independent experiments. Scale bar = 100 µm. e Western blot analysis to detect the expression of CTSS and ICAM1 in the whole cell lysates and supernatant medium of Flag-vector/MDA-MB-231 cells and Flag-AQP1/MDA-MB-231 cells. β-actin was the loading control. f Western blot of ICAM1 expression in control and ICAM1-downexpressing cells. g Representative migration or invasion images of control and ICAM1-downexpressing groups (two-tailed Student’s t test, ^*P < 0.05, ^**P < 0.01). Scale bar = 100 µm. h Representative migration or invasion images of control and CTSS inhibition groups (two-tailed Student’s t test, ^**P < 0.01). Scale bar = 100 µm. i, j Representative migration or invasion images from Flag-vector/MDA-MB-231 cells treated with the supernatant medium samples plus 5 µg/ml or 10 µg/ml ICAM1 neutralizing antibody (anti-ICAM1) or control IgG (two-tailed Student’s t test, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001). Each bar represented the mean ± SEM from three independent experiments. Scale bar = 100 µm. k, l Representative migration or invasion images from Flag-vector/MDA-MB-231 cells treated with the supernatant medium samples plus 5 µl or 10 µl CTSS neutralizing antibody (anti-CTSS) or control IgG (two-tailed Student’s t test, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001). 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