High ANXA5 or ANXA6 expression in advanced cancer patients is a poor prognosis factor. (a,b) Survival curves from Kaplan?Meier plot profiles for breast (n = 201), gastric (n = 305), lung (n = 70), or ovarian (n = 1220) high-grade and -stage cancer patients, stratified by high (red line) and low expression (black line) of ANXA5 (a) or ANXA6 (b) mRNA (Affymetrix identifier, 200782_at and 200982_s_at, respectively). In each analysis, hazard ratio (HR) and log-rank probability were calculated. A HR significantly different from 1 means that survival was better in one of both groups. For example, if a HR is 1.5, the survival probability in group 1 is 1.5 higher than in group 2. To note, cancer grade (typically numbered from 1 to 3) correlates with the velocity of the disease development while stage (from 1 to 4) characterizes the extent of affected tissues. Advanced cancers were triple-negative subtype, stage 3, stage 3 + grade 4, and stage 3, for breast, lung, ovarian, and gastric cancer, respectively. HR, hazard ratio.

Invasiveness properties in breast cancer cells are correlated with high expression of ANXA5 and ANXA6. 10 ?g of protein extracts from MDA-MB-231 and MCF7 cells were analyzed by western-blotting. (a) Representative image of western-blot analysis showing the revelation of ANXA5 and ANXA6 in MDA-MB-231 and MCF7 cells, compared to GAPDH (loading control). The whole membranes are presented in Figure S2. (b) The histogram presents mean values (± SEM) of the ratio ANX/GAPDH from five independent experiments, analyzed by the gel analysis plugging of ImageJ. Student t-test for independent samples. **p < 0.01.

sgANXA5/A6 MDA-MB-231 cells exhibit morphology and growth properties similar to control cells. (a) ANXA5/A6 deficient MDA-MB-231 cells were established by the CRIPSR-Cas9 strategy. The cellular content of ANXA5 and ANXA6 in sgANXA5/A6 MDA-MB-231 cells was quantified by western-blotting. The expression of ANXA5 and ANXA6 is decreased of more than 95% and 85% in sgANXA5/A6 MDA-MB-231 cells, respectively. (b) Control and sgANXA5/A6 MDA-MB-231 cells, which expressed constitutively the tdTomato fluorescent protein (red), were imaged by fluorescence microscopy. Scale bars = 40 ?m. (c) Proliferation of control or sgANXA5/A6 MDA-MB-231 cells was monitored with a lensfree microscope enabling long-term observation of a large cell population (Allier et al., 2017). The averaged number of cells (+/? SEM) normalized to the number of seeded cells from three independent experiments is presented.

ANXA5 and ANXA6 are involved in membrane repair of MDA-MB-231 cells. (a,b) Sequences of representative images showing the response of a control (a) or sgANXA5/A6 (b) MDA-MB-231 cell to a membrane damage performed by 110-mW infrared laser irradiation, in the presence of FM1-43 (green). In all figures, the area of membrane irradiation is marked with a red arrow before irradiation and a white arrow after irradiation. Scale bars: 10 ?m. (c) Kinetic data represent the FM1?43 fluorescence intensity integrated over whole cell sections, averaged for about 30 cells (+/? SEM). For a majority of control MDA-MB-231 cells, the fluorescence intensity reached a plateau after about 60 s (black filled circles). For sgANXA5/A6 MDA-MB-231 cells, a continuous and large increase of the fluorescence intensity was observed (empty circles), indicating the absence of membrane resealing. (d,e) Recruitment of ANXA5-GFP (d) and ANXA6-GFP (e) to the site of membrane injury. Red arrow, area before irradiation; white arrow, area after irradiation. After laser injury, MDA-MB-231 cells exhibited an accumulation of ANXA5 and ANXA6 at the disruption site. Scale bars: 20 ?m.

Silencing of ANXA5 and ANXA6 prevents metastasis process of MDA-MB-231 cells in zebrafish. (a,b) Control (a, n = 24) or sgANXA5/A6 MDA-MB-231 (b, n = 24) cells were injected in the perivitelline space of Casper zebrafish embryos. Tumor imaging was done by fluorescence microscopy at 24- and 48-hpi through the tdTomato fluorescent protein constitutively expressed in MDA-MB-231 cells and merged with bright-field images. Insets display magnified images of a portion of the tail and head with metastases (white arrows). (c) The percentage of embryos, either injected by control or sgANXA5/A6 MDA-MB-231 cells, which presented caudal or head metastases was quantified at 24 hpi. Mortality rate was calculated at 48 hpi.