Fig. 6

Rab1b was shown necessary for the AQP1-induced ICAM1/CTSS secretion. a The Venn diagram showed the intersection of three datasheets using 817 RNA-seq data from TCGA database. b Immunoprecipitation experiments were performed by using an anti-Flag M2 affinity gel and then immunoblotted with anti-Rab1b or anti-Flag. c Immunofluorescent staining of Flag together with Rab1b in Flag-AQP1/MDA-MB-231 cells. Scale bar = 20 µm. d Western blot analysis of Rab1b expression in Flag-vector/MDA-MB-231 and Flag-AQP1/MDA-MB-231 cells. e Immunofluorescent assay showed the cellular location of Rab1b in Flag-vector/MDA-MB-231 and Flag-AQP1/MDA-MB-231 cells (n = 60 cells per group, two-tailed Student’s t test, ^***P < 0.001). Scale bar = 20 μm. f Western blot analysis of indicated cell clones. Scr/MDA-MB-231 and Flag-vector/scr/MDA-MB-231 cells were used as control. g-h Immunofluorescent staining of GM130/TGN46 and DAPI in 4 different cell clones. In the right panel showed the distribution of cells with different ranges of Golgi ribbon angle (Ɵ: 0°–360°), respectively. ^**P < 0.01, ^***P < 0.001. Scale bar = 20 μm. i The abilities of migration and invasion were detected using 4 indicated cell clones. Quantitative results were analyzed in the right panel. Each bar represented the mean ± SEM from three independent experiments (two-tailed Student’s t test, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001). Scale bar = 100 μm. j Western blot analysis of the whole cell lysates or supernatant medium samples isolated from 3 indicated cells was performed to quantify ICAM1 and CTSS proteins. k-l Representative migration (k) or invasion (l) images from Flag-vector/MDA-MB-231 cells treated with the supernatant medium 3 different 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 times