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

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Hypoglycemic Mechanism of the Berberine Organic Acid Salt under the Synergistic Effect of Intestinal Flora and Oxidative Stress

doi: 10.1155/2018/8930374

Figure Lengend Snippet: Sequence of primers used for the RT-PCR assays.

Article Snippet: Membranes were then incubated for 3 h in blocking buffer (1x tris-buffered saline containing 0.1% Tween-20, and 4% nonfat milk) at room temperature and then overnight in the same buffer containing primary antibodies against TLR4 (1: 1000), p-JNK (1: 1000), GLUT2 (1 : 1000), PI3K (1 : 500), or β -actin (1: 1500) (Boster Biological Technology Ltd., Wuhan, China).

Techniques: Sequencing

Journal: Experimental and Therapeutic Medicine

Article Title: Tumor necrosis factor-α promotes the expression of excitatory amino-acid transporter 2 in astrocytes: Optimal concentration and incubation time

doi: 10.3892/etm.2014.2024

Figure Lengend Snippet: TNF-α treatment elevates the expression of EAAT2 in the brain. (A) Expression levels of the EAATs following TNF-α treatment. (B) Quantification of the expression levels of the EAATs. Data are presented as the mean ± standard deviation and were analyzed by one-way analysis of variance (n=3). ** P<0.01 compared with saline controls. TNF-α, tumor necrosis factor-α; EAAT, excitatory amino-acid transporter.

Article Snippet: The membranes were incubated at 4°C overnight with antibodies against EAAT1 (1:500; Wuhan Boster Biological Technology, Ltd., Wuhan, China), EAAT2 (1:500; Wuhan Boster Biological Technology, Ltd.), GFAP (1:2,000) or β-actin (1:1,000; Cell Signaling Technology, Inc.) diluted in blocking solution.

Techniques: Expressing, Standard Deviation, Saline

Journal: Experimental and Therapeutic Medicine

Article Title: Tumor necrosis factor-α promotes the expression of excitatory amino-acid transporter 2 in astrocytes: Optimal concentration and incubation time

doi: 10.3892/etm.2014.2024

Figure Lengend Snippet: Expression of EAATs in astrocytes treated with different doses of TNF-α. (A) Representative western blot analysis of EAAT1, EAAT2, GFAP and β-actin. (B) Densitometric analysis showing the expression of EAATs or GFAP normalized to β-actin. Data are presented as the mean ± standard deviation (n=5). * P<0.05 and ** P<0.01 compared with the control group (0 ng/ml TNF-α). TNF-α, tumor necrosis factor-α; EAAT, excitatory amino-acid transporter; GFAP, glial fibrillary acidic protein.

Article Snippet: The membranes were incubated at 4°C overnight with antibodies against EAAT1 (1:500; Wuhan Boster Biological Technology, Ltd., Wuhan, China), EAAT2 (1:500; Wuhan Boster Biological Technology, Ltd.), GFAP (1:2,000) or β-actin (1:1,000; Cell Signaling Technology, Inc.) diluted in blocking solution.

Techniques: Expressing, Western Blot, Standard Deviation, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Tumor necrosis factor-α promotes the expression of excitatory amino-acid transporter 2 in astrocytes: Optimal concentration and incubation time

doi: 10.3892/etm.2014.2024

Figure Lengend Snippet: Expression levels of EAATs in astrocytes treated with TNF-α for different time-periods. (A) Representative western blot analysis of EAAT1, EAAT2, GFAP and β-actin. (B) Densitometric analysis showing the expression levels of the EAATs or GFAP normalized to β-actin. Data are presented as the mean ± standard deviation (n=6). ** P<0.01 compared with the control group (prior to TNF-α treatment, 0 h). TNF-α, tumor necrosis factor-α; EAAT, excitatory amino-acid transporter; GFAP, glial fibrillary acidic protein.

Article Snippet: The membranes were incubated at 4°C overnight with antibodies against EAAT1 (1:500; Wuhan Boster Biological Technology, Ltd., Wuhan, China), EAAT2 (1:500; Wuhan Boster Biological Technology, Ltd.), GFAP (1:2,000) or β-actin (1:1,000; Cell Signaling Technology, Inc.) diluted in blocking solution.

Techniques: Expressing, Western Blot, Standard Deviation, Control