Cromones promote secretion of Anx-A1 from CDMC cells.

<p>Panels A and B. Aliquots of 2×10⁵ CDMC cells were cultured as described and incubated for 5 min with vehicle alone (‘control’), nedocromil (Ned; 10 nM), cromoglycate (Crom; 10 nM), ketotifen (Ket; 10 nM), dexamethasone (Dex; 2 nM) or okadaic acid (Okad; 10 nM). Anx-A1 (panel A) and PKC phosphorylation (panel B) were assessed by western blotting. There is no detectable change in the absolute intracellular amounts of these proteins at 5 min. Panels D and E. Densitometry data from 3 such experiments was analysed graphically in the corresponding bar graphs. Panel E. Anx-A1 released into the supernatant after incubation with the drugs was assessed using an ELISA assay and expressed as ng/ml culture fluid. * Signifies <i>P</i><0.05; **<i>P</i><0.01 and ***<i>P</i><0.001 relative to vehicle treated cells.</p

Cromoglycate, nedocromil, dexamethasone, and human recombinant Anx-A1 inhibit IgE/anti-IgE - induced histamine and PGD₂ release from CDMCs.

<p>CDMCs were cultured, sensitised with IgE and challenged with anti-IgE for 1h as described. The cell culture supernatant was sampled and PGD₂ release (pg/ml) or net % release of histamine was assessed by ELISA. Panels A and B. Cromoglycate 0.001–100 µM produces a concentration-dependent inhibition of histamine (panel A) or PGD₂ (panel B) release. Each data point is expressed as the mean ± SEM (n = 3). Panels C and D. Vehicle (control), nedocromil (Ned; 10nM), dexamethasone (Dex; 2nM) or human recombinant Anx-A1 protein (hr-Anx-A1; 1 nM and 10 nM) was added to the wells to test their inhibitory effect on histamine (panel C) and PGD₂ release (panel D). Each data point is expressed as the mean ± SEM (n = 3).* Signifies <i>P</i><0.05; **<i>P</i><0.01 and ***<i>P</i><0.001 relative to vehicle treated cells.</p

Nedocromil activates PKC_α/β and promotes Anx-A1 phosphorylation.

<p>Panel A. Aliquots of 2×10⁵ CDMC cells were cultured as described and incubated for 5 min with vehicle alone or escalating concentrations of nedocromil (Ned; 2-20 nM). The cell medium was then harvested and the cells lysed. Western blotting was used to assess the intracellular concentration of phospho-PKC and Ser²⁷-phopsho-Anx-A1. A maximum effect was seen at 5 nM nedocromil at this time point. A representative blot from duplicate experiments. Panel B. Using a similar methodology, the time course of activation of PKC was assessed using nedocromil (Ned; 10 nM). A maximum effect was seen at 5 min treatment with the drug at this concentration. A representative blot from duplicate experiments. Panel C. Using samples of lysate prepared from CDMC cells treated with nedocromil (Ned; 5 nM) for 5 min, the relative abundance of three activated isoforms of PKC was assessed using specific antisera. Only PKC α/β was increased by nedocromil treatment. Activated PKC δ oρ θ isoforms were not detected in resting or stimulated cells under these experimental conditions.</p

Nedocromil is inactive in BMDMCs from Anx-A1^-/- mice.

<p>Panel A. BMDMCs were prepared from Anx-A1^-/- and wild-type mice as described. Aliquots were sensitised with anti-DNP-IgE and challenged with DNP-BSA as described (upper graph) or stimulated with 10 µg/ml compound 48/80 (lower graph). Nedocromil was added in increasing concentrations (0.5 – 10 nM) and the release of histamine into the medium was assessed by ELISA. Results are expressed as % net histamine release; mean ± SEM; n = 2 with each sample assayed in triplicate. Panel B: BMDMCs were prepared from Anx-A1^-/- and wild-type mice as described. Aliquots were sensitised with anti-DNP-IgE and challenged with DNP-BSA as described (upper graph) or stimulated with 10 µg/ml compound 48/80 (lower graph). Nedocromil was added in increasing concentrations (1 – 10 nM) and the release of PGD2 into the medium was assessed by ELISA. Results are expressed as PGD2 released in pg/ml. Panel C. BMDMCs from Anx-A1^-/- mice retain their sensitivity to exogenous Anx-A1. BMDMCs were prepared from Anx-A1^-/- and aliquots were sensitised with anti-DNP-IgE and challenged with DNP-BSA as described, in the presence of escalating concentrations of human recombinant Anx-A1 (0.1–20 nM). Histamine and PGD₂ release into the cell culture fluid was assessed by ELISA. The data is plotted as a concentration-inhibition curve. Each point was the mean of triplicates.</p

Additional file 5: Figure S5. of The anti-inflammatory Annexin A1 induces the clearance and degradation of the amyloid-β peptide

Correlation of ANXA1 expression with FPR, cytokines and Aβ. A. Scatterplot showing relationship between FPRL1/FPR2 and ANXA1 protein expression assessed by Western blotting in the frontal cortex of neurologically healthy controls and sporadic Alzheimer’s patients (n = 5 controls, 3 males, 2 females, range 81–97 years, mean age 86.8 ± 3 years; n = 7 AD cases, 3 males, 4 females, range 83–98 years, mean age 91.3 ± 2 years). B. Scatterplot showing relationship between FPRL1/FPR2 and ANXA1 protein expression assessed by Western blotting in the motor cortex of 5XFAD mice and wild-type littermates and (n = 6/group, males aged 12 and 26 weeks). C. Scatterplot showing relationship between FPR rs1 and ANXA1 mRNA expression assessed in the frontal cortex of 5XFAD mice and wild-type littermates by qPCR (n = 19, males aged 12 and 26 weeks). D. Scatterplot showing relationship between ANXA1 protein expression assessed by Western blotting and TNFα expression measured by ELISA in in the frontal cortex of neurologically healthy controls and sporadic Alzheimer’s patients (n = 8 controls, 5 males, 3 females, range 40–82 years, mean age 67.1 ± 6 years; n = 10 AD cases, 7 males, 3 females, range 42–98 years, mean age 72.5 ± 6 years). E. Scatterplot showing relationship between Tnf and ANXA1 mRNA expression assessed in the frontal cortex of 5XFAD mice and wild-type littermates by qPCR (n = 18, males aged 12 and 26 weeks). ***p < 0.001, r 2 = 0.5779. F. Scatterplot showing relationship between Il6 and ANXA1 mRNA expression assessed in the frontal cortex of 5XFAD mice and wild-type littermates by qPCR (n = 19, males aged 12 weeks and 26 weeks). ****p < 0.0001, r 2 = 0.6352. G. Scatterplot showing relationship between ANXA1 protein expression assessed by Western blotting and Aβ1–40 expression measured by ELISA in the frontal cortex of neurologically healthy controls and sporadic Alzheimer’s patients (n = 10 controls, 6 males, 4 females, range 40–97 years, mean age 71.5 ± 6 years; n = 12 AD cases, 8 males, 4 females, range 42–98 years, mean age 74.4 ± 5 years). H. Scatterplot showing relationship between Aβ and ANXA1 protein expression assessed by Western blotting in the cortex of 5XFAD mice (n = 11, males aged 12 and 26 weeks). (PDF 111 kb

Весь Тагил. 2011. № 006

Efficiency of ANXA1 shRNA infection in BV2 cells. Representative Western blot image showing protein expression of ANXA1 in BV2 cells infected with control shRNA, and ANXA1 shRNA—clones 492A, 492B, 495A and 495B. Band intensities were determined using ImageJ and normalised to tubulin. (PDF 99 kb

Additional file 2: of The anti-inflammatory Annexin A1 induces the clearance and degradation of the amyloid-β peptide

Supplementary methods. (DOC 22 kb

Additional file 3: Figure S3. of The anti-inflammatory Annexin A1 induces the clearance and degradation of the amyloid-β peptide

ImageStream quantification of 5-FAM-labelled Aβ1–42 phagocytosis by BV2 cells. Histogram showing gating of BV2 cells. B. Scatterplot showing gating of focused BV2 cells. C. Histogram and representative images of negative and positive 5-FAM-labelled Aβ1–42 phagocytosis by BV2 cells. D. Representative histograms of 5-FAM-labelled Aβ1–42 phagocytosis by BV2 cells (WT, control shRNA and shRNA ANXA1) incubated for 3 h with 5-FAM-labelled Aβ1–42 (5 μg/ml) added to BV2-conditioned medium. (PDF 865 kb