Additional file 1 of Water channel protein AQP1 in cytoplasm is a critical factor in breast cancer local invasion

Additional file 1: Supplementary Fig. 1. Cytoplasmic expression of AQP1 was positively correlated with breast cancer progression. (a) Patients who had a recurrence or metastasis had a higher AQP1 cytoplasmic expression (62.2% vs 38.9%, P = 0.009). Cyto-AQP1: cytoplasmic AQP1 expression. (b) Patients who had a lymph node metastasis (n > 4) had a higher AQP1 cytoplasmic expression (50.0% vs 38.1%, P = 0.038). Cyto-AQP1: cytoplasmic AQP1 expression. (c-d) The relationship between pT stage and AQP1 cytoplasmic expression. Cyto-AQP1: cytoplasmic AQP1 expression. (e) The tumor volume in Flag-vector/MDA-MB-231 and Flag-AQP1/MDA-MB-231 mice group. Values were expressed as mean ± SD (two-tailed Student’s t test and two-way ANOVA, **P < 0.01, ***P < 0.001). (f) Quantitation of the percentage of Ki67-positive cells in tumor sections of Flag-vector/MDA-MB-231 and Flag-AQP1/MDA-MB-231 mice group. Two-tailed Student’s t test, *P < 0.05. Supplementary Fig. 2. Down-regulated expression of AQP1 decreased breast cancer migration and invasion abilities in AQP1-overexpressing MDA-MB-231 cells. (a) Western blot analysis of the expression of AQP1 in Flag-AQP1/MDA-MB-231 cells transfected with AQP1 shRNA. GAPDH was the loading control. (b-c) The abilities of migration and invasion were detected using Flag-AQP1/MDA-MB-231 and Flag-AQP1/shAQP1/MDA-MB-231 cells. Values were expressed as mean ± SEM from three independent experiments (two-tailed Student’s t test, **P<0.01). Scale bar = 100 μm. (d-e) Migration and invasion assay showed that Flag-vector/MDA-MB-231 cells treated with the supernatant of Flag-AQP1/shAQP1/MDA-MB-231 cells reversed the promoted phenotype compared with Flag-AQP1/MDA-MB-231 cells (two-tailed Student’s t test, *P<0.05, ***P<0.001). Each bar represented the mean ± SEM from three independent experiments. Scale bar=100 μm. Supplementary Fig. 3. Over-expression AQP1 increased breast cancer invasion abilities in T47D breast cancer cells. (a) Western blot analysis of the expression of AQP1 in Flag-vector/T47D cells and Flag-AQP1/T47D cells. β-actin was the loading control. (b) The abilities of invasion were detected using Flag-vector/T47D and Flag-AQP1/T47D cells. Values were expressed as mean ± SEM from seven independent experiments (two-tailed Student’s t test, **P<0.01). Scale bar=100 μm. (c) Invasion assay showed that Flag-vector/T47D cells treated with the supernatant of Flag-AQP1/T47D cells exhibited promoted phenotype compared with Flag-AQP1/T47D cells (two-tailed Student’s t test, *P<0.05). Each bar represented the mean ± SEM from four independent experiments. Scale bar=100 μm. (d) Immunofluorescent assay showed the colocation of AQP1 and ANXA2 in Flag-AQP1/T47D cells. Scale bar=20 μm. All experiments were independently repeated at least for three times. Supplementary Fig. 4. Gene enrichment analysis of AQP1 related genes. (a) 1805 differentially expression genes related to AQP1 were associated with extracellular matrix and secretion in up_keywords category of DAVID tool. (b) The biological process enrichment analysis of 1805 differentially expressed genes. (c) The molecular function enrichment analysis of AQP1 related genes. (d) The cellular component enrichment analysis of AQP1 related genes. (e) The KEGG pathway enrichment analysis of AQP1 related genes. Supplementary Fig. 5. Gene enrichment analysis of 76 proteins in the supernatant of AQP1-overexpressing MDA-MB-231 cells. (a) Representative TEM images of Golgi structure of Flag-vector/MDA-MB-231 and Flag-AQP1/MDA-MB-231 cells. Scale bar = 500 nm. (b) Mass spectrometry analysis showed the differentially expressed proteins between the supernatant of Flag-vector/MDA-MB-231 cells and Flag-AQP1/MDA-MB-231 cells. (c) The biological process enrichment analysis of 76 differentially expressed genes. (d) The molecular function enrichment analysis of 76 differentially expressed genes. (e) GO cellular component enrichment results of 76 proteins only expressed in the supernatant of Flag-AQP1/MDA-MB-231 cells. (f) Heatmap of the analyzed differentially expressed proteins using 15 AQP1 high expression cases and 15 AQP1 low expression cases in 817 RNA-seq data. Supplementary Fig. 6. Over-expression ANXA2 exhibited extend Golgi apparatus. (a) Western blot analysis of Hela cells transfected with Flag-AQP1. (b) Western blot analysis of GFP-AQP1/MDA-MB-231 cells transfected with Flag-ANXA2. Flag-vector/MDA-MB-231 cells were regarded as negative control. (c) Western blot analysis of MDA-MB-231 cells transfected with indicated ANXA2 shRNAs or control shRNA (scr). (d) Western blot analysis of indicated cell clones. Flag-vector/scr/MDA-MB-231 cells were used as a control. (e) Western blot analysis of MDA-MB-231 cells stably transfected with Flag-ANXA2. Flag-vector/MDA-MB-231 cells were regarded as negative control. (f) Immunofluorescent staining of GM130/TGN46 and DAPI in Flag-vector/MDA-MB-231 and Flag-AQP1/MDA-MB-23 cells. Quantification of the distribution of cells with different ranges of Golgi ribbon angle (Ɵ: 0°–360°) (n = 77-86 cells per group, ***P < 0.001). Scale bar = 20 μm. All experiments were independently repeated for three times. Supplementary Fig. 7. The extend Golgi morphology induced by AQP1 was not through Rab1b-F-actin axis. Immunofluorescence analysis showed that the Golgi morphology was not altered in shRab1b #1/MDA-MB-231 and Flag-AQP1/shRab1b #1/MDA-MB-231 cells when treated with F-actin depolymerization inhibitor latrunculin B, compared with the cells treated with DMSO. Scale bar=20 μm. Supplementary Fig. 8. Both expression and location of ANXA2 and Rab1b independently of each other. (a-b) Western blot assays were performed to detect Rab1b or ANXA2 expression in different cell clones. (c-d) Immunofluorescence experiments were performed to analyze the location of Rab1b or ANXA2 in different cell clones (n>60 cells per group, two-tailed Student’s t test). Scale bar=20 μm. All experiments were independently repeated for three times. Supplementary Fig. 9. The effect of the expression of AQP1/ANXA2/Rab1b/CTSS/ICAM1 on the prognosis of breast cancer patients. (a) Kaplan–Meier analysis showed the association between AQP1 expression and overall survival of breast cancer patients in GEPIA database (log-rank test). (b) Kaplan–Meier analysis showed the association between ANXA2 expression and overall survival of breast cancer patients in Kaplan-Meier plotter database (log-rank test). (c) Kaplan–Meier analysis showed the association between Rab1b expression and overall survival of breast cancer patients in Kaplan-Meier plotter database (log-rank test). (d) Kaplan–Meier analysis showed the association between CTSS expression and overall survival of breast cancer patients in Kaplan-Meier plotter database (log-rank test). (e) Kaplan–Meier analysis showed the association between ICAM1 expression and overall survival of breast cancer patients in Kaplan-Meier plotter database (log-rank test). Supplementary Fig. 10. Relationship of AQP1 with ANXA2, Rab1b, CTSS and ICAM1. (a) In 194 IDC patients, AQP1 had a positive correlation with ANXA2. (b-d) Correlation analysis using GEPIA (http://gepia.cancer-pku.cn) indicated that AQP1 was positively correlated with Rab1b, CTSS and ICAM1 in breast cancer patients.