Journal: Cellular and Molecular Life Sciences

Article Title: Inhibition of the membrane repair protein annexin-A2 prevents tumor invasion and metastasis

doi: 10.1007/s00018-023-05049-3

Figure Lengend Snippet: Highly expressed in MDA-MB-231 cells, ANXA2 promotes membrane repair. A Representative image of western-blot analysis showing the revelation of ANXA2 in MDA-MB-231 and MCF7 cells, as compared to GAPDH (loading control). B The histogram presents mean values (± SEM) of the ratio ANX/GAPDH from five independent experiments, analyzed by the gel analysis plugging of ImageJ. A representative membrane of the detection of ANXA1 is presented in supplementary Fig. . Student t test for independent samples. ** p < 0.01. C ANXA2-deficient MDA-MB-231 cells were generated by shRNA transduction strategy. The cellular content of ANXA2 in MDA-MB-231 cells transduced with lentiviral particles containing shRNA targeting ANXA2 (shA2) or a scrambled shRNA (ctl) was quantified by Western blotting. D Control and shANXA2 MDA-MB-231 cells, which expressed constitutively the tdTomato fluorescent protein were imaged by fluorescence microscopy. Right-hand histograms display mean cell area (in pixels 2 ) and nuclei circumference (in pixels) measured by the imageJ software using Tomato and DAPI images. The mean values (+ / − SEM) were calculated from at least 30 cells from three independent experiments. No statistical difference (student t test) was observed for the two parameters. Scale bar: 20 µm. E , G Sequences of representative images showing the response of a control ( E ) or shANXA2 ( G ) 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 μm. F Kinetic data represent the FM1 − 43 fluorescence intensity for control (black filled circles) or shANXA2 (empty circles) MDA-MB-231 cells, integrated over whole cell sections, averaged for about 30 cells (+ / − SEM). H Recruitment of ANXA2 to the site of membrane injury. MDA-MB-231 cells transfected with the plasmid pA2-GFP were damaged by laser ablation. Red arrow, area before irradiation; white arrow, area after irradiation. I Subcellular localization of endogenous ANXA2 in damaged MDA-MB-231. MDA-MB-231 cells were irradiated with a 110-mW infrared laser (white arrow) in DPBS + Ca 2+ , then fixed and immunostained for ANXA2 and counterstained with DAPI (blue). After laser injury, MDA-MB-231 cells exhibited an accumulation of ANXA2 at the disruption site. The inset displays a magnified image of the disruption site where concentrates ANXA2. Scale bars: 10 µm on the images and 1 µm within the inset

Article Snippet: Semi-dry electrophoretic transfer (Bio-Rad) onto PVDF membrane was performed for 1 h at 100 V. The cellular content in ANXA1 (37 kDa), ANXA2 (36 kDa), ANXA4 (35 kDa), ANXA5 (35 kDa), ANXA6 (68 kDa), and actin (42 kDa) or GAPDH (37 kDa) was detected with rabbit anti-ANXA1 polyclonal antibody (PA1006, BosterBio, Pleasanton, CA, USA), mouse anti-ANXA2 monoclonal antibody (3E8-B6, Sigma-Aldrich), mouse anti-ANXA4 monoclonal antibody (SAB4200121, Sigma-Aldrich), mouse anti-ANXA5 monoclonal antibody (AN5, Sigma-Aldrich), mouse anti-ANXA6 monoclonal antibody (sc-271859, Santa cruz Biotechnology, Dallas, USA), rabbit anti-actin polyclonal antibody (A2066, Sigma-Aldrich), and rabbit anti-GAPDH polyclonal antibody (G9545, Sigma-Aldrich), respectively.

Techniques: Membrane, Western Blot, Control, Generated, shRNA, Transduction, Fluorescence, Microscopy, Software, Irradiation, Transfection, Plasmid Preparation, Disruption