Journal: Advanced Science

Article Title: Annexin A3 Represses Endothelial Permeability and Inflammation During Sepsis via Actin Cytoskeleton Modulation

doi: 10.1002/advs.202416904

Figure Lengend Snippet: ANXA3 deficiency aggravates the response to sepsis. A) Survival analysis of male WT and ANXA3 −/− mice after LPS treatment (10 mg kg −1 ), n = 14/group. p = 0.031, log‐rank (Mantel–Cox) test. B) At 24 h after LPS treatment, the mice were scored for the presence or absence of six different macroscopic signs of sepsis. n = 9–12, ** p < 0.01, two‐tailed unpaired Student's t test. C) Survival analysis after establishment of the CLP model in male WT and ANXA3 −/− mice, n = 12, p = 0.045, log‐rank (Mantel–Cox) test. D,E) At 18 h after LPS treatment, representative lung histological changes were assessed by H&E staining in different groups of mice. Scale bar = 500 µm in D) and scale bar = 25 µm in E). G) Lung injury scores of pulmonary damages in different groups. n = 6, *** p < 0.001; two‐way ANOVA with Tukey's post hoc test. F,H) Mouse lung tissue samples from different groups were subjected to immunostaining for MPO and positive cell count analysis. Scale bar = 25 µm, n = 6; *** p < 0.001; two‐way ANOVA with Tukey's post hoc test. I) Evans blue dye was injected into the mice at 18 h after LPS treatment, after which the lung tissue was harvested for imaging. J) The data were quantified as the Evans blue dye quantity (µg) per g of dry tissue weight. K) A representative photograph of FITC‐dextran leakage into the lungs (indicated by the arrows) of a mouse, as determined by the appearance of intravenously injected FITC‐dextran in vivo, is shown. The scale bar represents 20 µm. L) The relative FITC‐dextran leakage of each tissue sample was calculated from the fluorescence intensity (A.U.). *** p < 0.001, two‐way ANOVA with Tukey's post hoc test.

Article Snippet: ANXA3 knockout mice were purchased from Cyagen Biosciences (Suzhou, China).

Techniques: Two Tailed Test, Staining, Immunostaining, Cell Counting, Injection, Imaging, In Vivo, Fluorescence

Journal: Advanced Science

Article Title: Annexin A3 Represses Endothelial Permeability and Inflammation During Sepsis via Actin Cytoskeleton Modulation

doi: 10.1002/advs.202416904

Figure Lengend Snippet: Knockout of ANXA3 exacerbates pulmonary vascular permeability and exacerbates inflammatory gene expression in LPS‐induced lung injury. IF staining analysis of the endothelial cell AJs protein VE‐cadherin A,B), the endothelial TJs marker claudin 5 C,D), and Zo‐1 E,F) in lung tissue sections from the indicated mice. G,J) mRNA levels of vascular activation markers (CD62E, ICAM‐1, VCAM‐1, and CCL2) in the lung tissues of the indicated mice. n = 6, * p < 0.05, ** p < 0.01, *** p < 0.001; two‐way ANOVA with Tukey's post hoc test.

Article Snippet: ANXA3 knockout mice were purchased from Cyagen Biosciences (Suzhou, China).

Techniques: Knock-Out, Permeability, Gene Expression, Staining, Marker, Activation Assay

Journal: Advanced Science

Article Title: Annexin A3 Represses Endothelial Permeability and Inflammation During Sepsis via Actin Cytoskeleton Modulation

doi: 10.1002/advs.202416904

Figure Lengend Snippet: Role of ANXA3 in the regulation of junctional proteins. A,B) Gel images and quantification of WB showing ANXA3, Zo‐1, VE‐cadherin, and claudin 5 expression in HUVECs transiently infected with lentivirus containing a negative control sequence and a shRNA target‐2 sequence. n = 3, ** p < 0.01, *** p < 0.001, two‐tailed unpaired Student's t test. C,D) Immunocytochemical staining for ZO‐1, VE‐cadherin, and claudin 5 in HUVECs infected with lentivirus containing a negative control sequence C) or shRNA target‐2 sequence D). Scale bar = 50 µm, n = 4 independent experiments. E) After ANXA3 was knocked down or overexpressed in HUVECs, the fluorescence intensity of FITC‐DEX leakage from the upper chamber to the lower chamber in the transwell. n = 3, * p < 0.05, *** p < 0.001. One‐way analysis of variance followed by Tukey's test was used for multiple group comparisons. F–I) Rhodamine‐phalloidin staining of the actin cytoskeleton in sh‐CON, sh‐ANXA3, LV–NC, and LV–ANXA3 cells (scale bar = 20 µm). Histograms represent intensity profiles of actin staining along the dotted lines in photomicrographs, emphasizing the differences in cross‐cellular actin expression. J) Molecular docking mimics the ANXA3 (yellow) and β‐actin (blue) complex. (K) Co‐IP experiments confirmed the interaction of ANXA3 with β‐actin.

Article Snippet: ANXA3 knockout mice were purchased from Cyagen Biosciences (Suzhou, China).

Techniques: Expressing, Infection, Negative Control, Sequencing, shRNA, Two Tailed Test, Staining, Fluorescence, Co-Immunoprecipitation Assay

Journal: Advanced Science

Article Title: Annexin A3 Represses Endothelial Permeability and Inflammation During Sepsis via Actin Cytoskeleton Modulation

doi: 10.1002/advs.202416904

Figure Lengend Snippet: Knockdown of ANXA3 aggravates LPS‐induced endothelial activation. A–D) HUVECs were transiently infected with lentivirus containing a negative control sequence or shANXA3; at 48 h postinfection, the HUVECs were stimulated with LPS (1 µg mL −1 ) for 12 h. Immunoblots showing ICAM‐1 and CD62E expression after ANXA3 knockdown in HUVECs. n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001; two‐way ANOVA followed by Tukey's test. E,F) Effects of ANXA3 knockdown on THP‐1 adhesion to HUVECs. Scale bar = 100 µm. G) The BioGRID ( https://thebiogrid.org/ ) database was used to predict potential proteins that interact with ANXA3. H,I) Phosphorylation of ATF2 in control and ANXA3‐knockdown HUVECs was detected by WB. n = 3, ** p < 0.01, *** p < 0.001; two‐way ANOVA followed by Tukey's test.) pATF2 localization and fluorescence intensity were detected by IF in control and ANXA3‐knockdown HUVECs. Scale bar = 10 µm.

Article Snippet: ANXA3 knockout mice were purchased from Cyagen Biosciences (Suzhou, China).

Techniques: Knockdown, Activation Assay, Infection, Negative Control, Sequencing, Western Blot, Expressing, Phospho-proteomics, Control, Fluorescence

Journal: Advanced Science

Article Title: Annexin A3 Represses Endothelial Permeability and Inflammation During Sepsis via Actin Cytoskeleton Modulation

doi: 10.1002/advs.202416904

Figure Lengend Snippet: Overexpressed ANXA3 limits LPS‐induced endothelial activation. A–D) Control and LV–ANXA3 HUVECs were stimulated with LPS and immunoblotting revealed pATF2 and CD62E expression. n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001; two‐way ANOVA followed by Tukey's test. E,F) The localization and fluorescence intensity of pATF2 and CD62E in LV–NC and LV–ANXA3 HUVECs before and after LPS stimulation were detected by IF staining. Scale bar = 10 µm. G,H) Effects of ANXA3 overexpression on THP‐1 adhesion to HUVECs. Scale bar = 20 µm. *** p < 0.001, two‐way ANOVA followed by Tukey's test. [Correction added on a 8 April 2025, after first online publication: Figure image is replaced with the current version.]

Article Snippet: ANXA3 knockout mice were purchased from Cyagen Biosciences (Suzhou, China).

Techniques: Activation Assay, Control, Western Blot, Expressing, Fluorescence, Staining, Over Expression

Journal: Advanced Science

Article Title: Annexin A3 Represses Endothelial Permeability and Inflammation During Sepsis via Actin Cytoskeleton Modulation

doi: 10.1002/advs.202416904

Figure Lengend Snippet: The pATF2 inhibitor SP600125 rescues ANXA3 loss‐mediated in vitro endothelial activation and in vivo lung injury during sepsis. A–D) shCON‐ and shANXA3‐treated HUVECs were pretreated with SP600125 for 1 h and then stimulated with LPS, after which the protein levels of pATF2 and CD62E were detected by WB. n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001; two‐way ANOVA followed by Tukey's test. E,F) shCON‐ and shANXA3‐treated HUVECs were pretreated with SP600125, and THP‐1 adhesion to HUVECs was observed after LPS stimulation. Scale bar = 100 µm, n = 4; *** p < 0.001; two‐way ANOVA followed by Tukey's test. G) The SP600125 inhibitor was used 1 h before and 12 h after LPS modeling, and survival analysis was performed after LPS treatment. Comparisons of survival curves between untreated and SP600125‐treated ANXA3 −/− mice ( * p < 0.05) via the log‐rank (Mantel–Cox) test. H,I) H&E staining and lung injury scores of lung tissue from ANXA3 −/− and WT male mice treated with SP600125 and vector 18 h after LPS modelling. Scale bar = 20 µm. n = 6, *** p < 0.001; two‐way ANOVA followed by Tukey's test. J,K) MPO staining and quantitative analysis of lung tissues from ANXA3 −/− and WT male mice treated with SP600125 and vector 18 h after LPS modelling. Scale bar = 10 µm. n = 6, *** p < 0.001, * p < 0.05; two‐way ANOVA followed by Tukey's test.

Article Snippet: ANXA3 knockout mice were purchased from Cyagen Biosciences (Suzhou, China).

Techniques: In Vitro, Activation Assay, In Vivo, Staining, Plasmid Preparation

Journal: Advanced Science

Article Title: Annexin A3 Represses Endothelial Permeability and Inflammation During Sepsis via Actin Cytoskeleton Modulation

doi: 10.1002/advs.202416904

Figure Lengend Snippet: Cyto D inhibits the increased level of pATF2 induced by ANXA3 knockdown. A) HUVECs in the sh‐CON and sh‐ANXA3 groups were pretreated with Cyto D and then treated with LPS or PBS respectively. The actin cytoskeletons were detected by phalloidin staining. Scale bar = 10 µm. B) Gel images showed the effect of Cyto D on pATF2/CD62E levels in ANXA3‐knockdown cells and control cells, and on pATF2/CD62E levels in both groups after LPS stimulation. C) Statistical analysis of the pATF2 in ANXA3‐knockdown cells and control cells treated with Cyto D. n = 3, *** p < 0.001, two‐way ANOVA followed by Tukey's test was used for multigroup comparison. D,E) Statistical analysis of the pATF2/CD62E levels in ANXA3 knockdown cells and control cells pretreated with Cyto D then stimulated with LPS. n = 3, ** p < 0.01, *** p < 0.001, two‐way ANOVA followed by Tukey's test was used for multigroup comparison.

Article Snippet: ANXA3 knockout mice were purchased from Cyagen Biosciences (Suzhou, China).

Techniques: Knockdown, Staining, Control, Comparison

Journal: Advanced Science

Article Title: Annexin A3 Represses Endothelial Permeability and Inflammation During Sepsis via Actin Cytoskeleton Modulation

doi: 10.1002/advs.202416904

Figure Lengend Snippet: Schematic diagram of ANXA3 plays a protective role in endothelial function in sepsis.

Article Snippet: ANXA3 knockout mice were purchased from Cyagen Biosciences (Suzhou, China).

Techniques:

Journal: Journal of cellular and molecular medicine

Article Title: Secretion of annexin A3 from ovarian cancer cells and its association with platinum resistance in ovarian cancer patients.

doi: 10.1111/j.1582-4934.2011.01316.x

Figure Lengend Snippet: Fig. 1 (A) Annexin A3 levels in the conditional culture medium from the ovarian cancer cells were measured by ELISA. (B) Proteins from the ovarian cancer cell lysates and concentrated cul- ture media were analysed by anti-annexin A3 immunoblotting. Enforced expression of annexin A3 in SKOV3 and A2780 cells resulted in the increased secretion of annexin A3 in the culture medium. Down-regulation of annexin A3 in plat- inum-resistant ovarian cancer cells SKOV3/Cis andA2780/Cis, which express high levels of annexin A3, reduced the secretion of annexin A3. (C) Annexin A3 levels in sera from the 30 normal female donors and the 50 ovarian cancer patients were determined by ELISA [particular high levels of annexin A3 (2.0461, 3.4453, 8.8125 and 18.3081 ng/ml, respectively) cannot be seen in the graph because the Y-axis range is 2.0 ng/ml]. The patients are divided into platinum-sensitive (n 20) and resistant (n 30) groups based on clinical data. There are significant differences in the serum levels of annexin A3 among normal donors, the sensitive group and the resistant group. Data were analysed with a Mann–Whitney U-Test. P values were all two-sided. (D) Progress-free times of the 50 ovarian cancer patients. There is a significant difference between the high annexin A3 group (serum A3 1.13 ng/ml) and the low annexin A3 group (serum A3 1.13 ng/ml) (P 0.009 0.05).

Article Snippet: The level of annexin A3 in sera of ovarian cancer patients was determined using the ELISA kit for human annexin A3 (Cusabio) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, MANN-WHITNEY

Journal: Journal of cellular and molecular medicine

Article Title: Secretion of annexin A3 from ovarian cancer cells and its association with platinum resistance in ovarian cancer patients.

doi: 10.1111/j.1582-4934.2011.01316.x

Figure Lengend Snippet: Fig. 2 High levels of annexin A3 are associated with increased numbers of vesicles in the cytoplasm. (A) Representative TEM (10,000) photographs of paired ovarian cancer cell lines SKOV3 and SKOV3/Cis and A2780 and A2780/Cis. The arrows indicate the increased numbers of vesicles in plat- inum-resistant SKOV3/Cis and A2780/Cis cells. (B) Higher magnification (50,000) photographs of SKOV3/Cis cells. Arrows indicate the MVB-like vesicle (upper) and exocytosis of a vesicle (lower). (C) Representative TEM (10,000) photographs of ovarian cancer cell lines. Arrows in the upper panel indicated increased vesicles in annexin A3-expressing A2780/Ann and SKOV3/Ann cells. The lower panels are pictures of A2780/Cis/R and SKOV3/Cis/R cells. The vesicles observed in A2780/Cis and SKOV3/Cis (A) have largely disappeared.

Article Snippet: The level of annexin A3 in sera of ovarian cancer patients was determined using the ELISA kit for human annexin A3 (Cusabio) according to the manufacturer’s instructions.

Techniques: Expressing

Journal: Journal of cellular and molecular medicine

Article Title: Secretion of annexin A3 from ovarian cancer cells and its association with platinum resistance in ovarian cancer patients.

doi: 10.1111/j.1582-4934.2011.01316.x

Figure Lengend Snippet: Fig. 3 Immunoelectron microscopy studies of annexin A3 in platinum-resistant ovarian cancer cell SKOV3/Cis. Cell preparation and treatment are described in Materials and Methods. Ultrathin sections were examined by transmission elec- tron microscopy (left, 50,000; right, 100,000). The high-density 12-nm colloidal gold particles, which are indicative of annexin A3, could be observed in the cytoplasm of the cells (small arrows). Some gold particles existed in or around vesicles (medium arrows). (A) Control (sections were treated as described without anti- annexin A3 antibody). (B) Annexin A3 expression in cytoplasm. (C, D) Annexin A3 expression in cytoplamic vesicles. The vesicle in C appears ready to undergo exocytosis.

Article Snippet: The level of annexin A3 in sera of ovarian cancer patients was determined using the ELISA kit for human annexin A3 (Cusabio) according to the manufacturer’s instructions.

Techniques: Immuno-Electron Microscopy, Transmission Assay, Microscopy, Control, Expressing

Journal: Journal of cellular and molecular medicine

Article Title: Secretion of annexin A3 from ovarian cancer cells and its association with platinum resistance in ovarian cancer patients.

doi: 10.1111/j.1582-4934.2011.01316.x

Figure Lengend Snippet: Fig. 4 Annexin A3 is associated with exosomes from ovarian cancer cells SKOV3/Cis. (A) Electron microscopic picture of exosomes from SKOV3/Cis cells. The exosomes are 40–100 nm in diameter and exhibit the typical cup-shaped morphology. (B, C) IEM photographs of exo- somes from SKOV3/Cis cells. The high-density dots represent 12-nm gold particles, which are indicatives of annexin A3. (D) Conditional medium (30 ml) from cultured SKOV3/Cis cells was collected, and divided into two parts. After removing the exosomes from one of them (DMEM 2), both were concentrated and analysed by immunobltting with anti-annexin A3 and anti- Hsp70 antibodies. The exosome preparation and the upper sucrose were also examined by the immunoblotting.

Article Snippet: The level of annexin A3 in sera of ovarian cancer patients was determined using the ELISA kit for human annexin A3 (Cusabio) according to the manufacturer’s instructions.

Techniques: Cell Culture, Western Blot

Journal: Journal of cellular and molecular medicine

Article Title: Secretion of annexin A3 from ovarian cancer cells and its association with platinum resistance in ovarian cancer patients.

doi: 10.1111/j.1582-4934.2011.01316.x

Figure Lengend Snippet: Fig. 5 The amount of exosomes released by the ovarian cancer cells depends on the expression of annexin A3. (A) Exosomes were isolated from condi- tioned medium of cultured ovarian cancer cells. The total amount of protein was used to quantify recovered exosomes. (B) Comparison of the amounts of exosomes released from various ovarian cancer cells by immunobltting with anti-annexin A3 and anti-Hsp70 antibodies.

Article Snippet: The level of annexin A3 in sera of ovarian cancer patients was determined using the ELISA kit for human annexin A3 (Cusabio) according to the manufacturer’s instructions.

Techniques: Expressing, Isolation, Cell Culture, Comparison

Journal: Translational cancer research

Article Title: ANXA3 is upregulated by hypoxia-inducible factor 1-alpha and promotes colon cancer growth.

doi: 10.21037/tcr-20-994

Figure Lengend Snippet: Figure 1 Immunohistochemical staining of HIF-1α and ANXA3 in colon cancer and normal colon tissues. Positive expression was visualized

Article Snippet: Next, the membranes were blocked in 5% non-fat dry milk for 1 h. Then the membranes were probed by antibody for HIF1α (1:1,000), ANXA3 (1:800) or β-actin (1:500, Boster, Biotechnology, Wuhan, China) overnight at 4 °C.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Translational cancer research

Article Title: ANXA3 is upregulated by hypoxia-inducible factor 1-alpha and promotes colon cancer growth.

doi: 10.21037/tcr-20-994

Figure Lengend Snippet: Figure 3 Detection of HIF-1α and ANXA3 protein levels in SW480, SW620, HCT116 and HT29 colon cancer cells under normoxic

Article Snippet: Next, the membranes were blocked in 5% non-fat dry milk for 1 h. Then the membranes were probed by antibody for HIF1α (1:1,000), ANXA3 (1:800) or β-actin (1:500, Boster, Biotechnology, Wuhan, China) overnight at 4 °C.

Techniques:

Journal: Translational cancer research

Article Title: ANXA3 is upregulated by hypoxia-inducible factor 1-alpha and promotes colon cancer growth.

doi: 10.21037/tcr-20-994

Figure Lengend Snippet: Figure 2 Detection of HIF-1α and ANXA3 expression in colon cancer and normal colon tissues. (A) qRT-PCR analysis of HIF-1α and

Article Snippet: Next, the membranes were blocked in 5% non-fat dry milk for 1 h. Then the membranes were probed by antibody for HIF1α (1:1,000), ANXA3 (1:800) or β-actin (1:500, Boster, Biotechnology, Wuhan, China) overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR

Journal: Translational cancer research

Article Title: ANXA3 is upregulated by hypoxia-inducible factor 1-alpha and promotes colon cancer growth.

doi: 10.21037/tcr-20-994

Figure Lengend Snippet: Figure 4 HIF-1α regulates ANXA3 expression in SW480 and SW620 cells. The expression of HIF-1α and ANXA3 was measured by qRT-

Article Snippet: Next, the membranes were blocked in 5% non-fat dry milk for 1 h. Then the membranes were probed by antibody for HIF1α (1:1,000), ANXA3 (1:800) or β-actin (1:500, Boster, Biotechnology, Wuhan, China) overnight at 4 °C.

Techniques: Expressing

Journal: Translational cancer research

Article Title: ANXA3 is upregulated by hypoxia-inducible factor 1-alpha and promotes colon cancer growth.

doi: 10.21037/tcr-20-994

Figure Lengend Snippet: Figure 6 HIF-1α knockdown decreases tumor volume and ANXA3 expression in nude xenograft tumor model. (A) Compared with flank

Article Snippet: Next, the membranes were blocked in 5% non-fat dry milk for 1 h. Then the membranes were probed by antibody for HIF1α (1:1,000), ANXA3 (1:800) or β-actin (1:500, Boster, Biotechnology, Wuhan, China) overnight at 4 °C.

Techniques: Knockdown, Expressing

Journal: PLoS ONE

Article Title: Identifying Resistance Mechanisms against Five Tyrosine Kinase Inhibitors Targeting the ERBB/RAS Pathway in 45 Cancer Cell Lines

doi: 10.1371/journal.pone.0059503

Figure Lengend Snippet: Validation of the top genes by TaqMan RT-PCR in the cell lines.

Article Snippet: ANXA3 , Hs00971411_m1 , 209369_at , annexin A3 , gefitinib , 0.040.

Techniques: Biomarker Discovery, Binding Assay

Journal: PLoS ONE

Article Title: Identifying Resistance Mechanisms against Five Tyrosine Kinase Inhibitors Targeting the ERBB/RAS Pathway in 45 Cancer Cell Lines

doi: 10.1371/journal.pone.0059503

Figure Lengend Snippet: Circos plot of RT-PCR validated correlations for genes associated with resistance against multiple agents as identified by microarray analysis. The thickness of the ribbons correlate to the log(p) of the correlation (see .). Note the high number of genes associated with sunitinib resistance and the single gene associated with lapatinib resistance. The two most informative genes are ANXA3 and RAB25, each associated with resistance against four agents.

Article Snippet: ANXA3 , Hs00971411_m1 , 209369_at , annexin A3 , gefitinib , 0.040.

Techniques: Reverse Transcription Polymerase Chain Reaction, Microarray