71-3400

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Experimental details: Western blot analysis of human fibroblasts using: Lane 1: Pre-immune serum; Lane 2: Rabbit anti-Annexin I Polyclonal Antibody (Product # 71-3400).

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Experimental details: Western blot analysis of Annexin I was performed by loading 20 µg of K562 (lane1), HeLa (lane2), U2OS (lane3) and MCF7 (lane4) cell lysate using NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and iBlot® Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. Annexin I was detected at ~ 35 and 39 kDa using Annexin I Rabbit polyclonal Antibody (Product # 71-3400) at 1:1000 in 5 % skim milk at 4°C overnight on a rocking platform. Goat Anti-Rabbit IgG - HRP Secondary Antibody (G21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

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Experimental details: Knockout of ANXA1 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR799825_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of ANXA1 was performed by loading 30 µg of HeLa wild type (Lane 1), HeLa CAS9 (Lane 2), HeLa ANXA1 KO (Lane 3) whole cell extracts. The samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-Annexin A1 Polyclonal Antibody (Product # 71-3400) using 1:1000 dilution and Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to ANXA1.

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Experimental details: Western blot was performed using Anti-Annexin A1 Polyclonal Antibody(Product # 71-3400) and a 48kDa band corresponding to Annexin A1 was observed across cell lines and tissues tested. Nuclear enriched extracts (30 µg lysate) of K-562 (Lane 1), A-431 (Lane 2), U-2 OS (Lane 3), IMR-32 (Lane 4), SH-SY5Y (Lane 5), HeLa (Lane 6), Mouse Lung (Lane 7), Rat Lung (Lane 8) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 Dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177,1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).Expression of ANXA1 was observed all cell lines and tissues except IMR-32 and SH-5Y5Y which have no expression of ANXA1.

Supportive validation

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Experimental details: Immunohistochemistry analysis of Annexin I showing staining in the cytoplasm and nucleus of paraffin-embedded human breast tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Annexin I polyclonal antibody (Product # 71-3400) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of Annexin I showing staining in the cytoplasm and nucleus of paraffin-embedded human kidney tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Annexin I polyclonal antibody (Product # 71-3400) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of Annexin I showing staining in the cytoplasm and nucleus of paraffin-embedded mouse kidney tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Annexin I polyclonal antibody (Product # 71-3400) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

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Supportive validation

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Supportive validation

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Experimental details: Figure 3 AnxA1 controlled claudin-1 and ZO-1 expressions on uterine epithelial cells via FPR1 and FPR2. Claudin-1 ( A ) and ZO-1 ( B ) expressions on uterine epithelial cells were determined 24 h after incubations. Representative images of claudin-1 and ZO-1 immunofluorescence are shown. (-) means absence and (+) means presence of treatments. The data are expressed as mean +- standard error of mean of three to five independent experiments. a p < 0.05 vs. NT; b p < 0.01 and c p < 0.05 vs. AnxA1.

Supportive validation

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 1 Dexamethasone increases the phosphorylation of Anx-A1 and PKC in U937 cells within 5 min in a concentration-dependent fashion. (Panel A) In U937 cells, 5 min treatment with dexamethasone in concentrations of 0.02-5.0 nM increases phosphorylation of Anx-A1 on Ser 27 (upper panel) by ~4-fold and, in parallel, PKCalpha/beta phosphorylation (lower panel) by ~3-fold compared to untreated controls. Whole U937 cell lysates were prepared as detailed. Anx-A1 phosphorylation was detected using a specific anti-Ser 27 phospho Anx-A1 antibody as described. Total Anx-A1 (sometimes shown as a 33/37 kDa doublet) and PKC is shown for reference purposes. (Panel B) The dexamethasone (2 nM) effect on Anx-A1 phosphorylation is dependent on GC receptor occupation since the co-administration of 1 muM RU 486, blocks this effect. Note that the amount of phospho Anx-A1 is significantly less in samples treated with RU 486 alone than in vehicle treated samples, suggesting that some residual GCs may be present in the culture media. * P < 0.05 relative to control values; SSSS P < 0.01 relative to dexamethasone alone. (Panel C) The action of common kinase inhibitors on Anx-A1 phosphorylation. Of these, only PKC 19-31 (5 muM), an inhibitor of PKC, was able to reduce phosphorylation of Anx-A1 when this was stimulated by 2 nM dexamethasone (D). An inhibitor of PI3 kinase inhibitor LY294002 (LY: 10 nM) or a MAP kinase inhibitor PD98059 (PD: 5 muM) were inactive. Insert: a representative Western blot sho

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Experimental details: Fig. 2 Nedocromil potentiates the effect of dexamethasone on Anx-A1 and PKCalpha/beta phosphorylation and protein translocation to the plasma membrane fraction. (Panel A) Nedocromil itself, over a range of concentrations, has a negligible effect on the concentration of Ser 27 phospho Anx-A1 in U937 cell lysates or PKCalpha/beta phosphorylation when compared to untreated samples. Total Anx-A1 (sometimes shown as a 33/37 kDa doublet) and PKC is shown for reference purposes. (Panel B) In the presence of 2 nM dexamethasone, escalating concentrations (0.02-5.0 nM) of nedocromil potentiate (by a further 3-4-fold) the phosphorylation of both Anx-A1 and PKCalpha/beta. Total Anx-A1 (sometimes shown as a 33/37 kDa doublet) and PKC is shown for reference purposes. (Panel C) In the presence of a fixed concentration of dexamethasone (2 nM), nedocromil (0.02-2.0 nM) potentiates the translocation of Ser 27 phospho Anx-A1 from the cytosol into the 13,000 x g particulate fraction of U937 cells. (Panel D) The membrane accumulation of activated PKCalpha/beta is promoted (>2-fold) by the combination of nedocromil (0.5 nM) and dexamethasone (2 nM; see blot insert) and this is reflected in an increase in enzyme activity as measured in the 100,000 x g membrane fraction. * P < 0.05 relative to nedocromil of dexamethasone alone. All experiments were done at least three times and the blots are representatives from one of these experiments. Densitometry was performed as described in the methods and the

Supportive validation

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Experimental details: Figure 4 Up-regulation of Annexin A1 and increase in apoptotic cells in PAX2 knockdown cell lines. ( a ) Reverse phase protein array (RPPA) analysis showed PAX2 knockdown ovarian cancer cell lines had a higher expression of Annexin A1 than control cells; ( b ) Western blot analysis was used to measure Annexin A1 expression in RMUGL and TOV21G ovarian cancer cell lines with or without PAX2 knockdown; ( c ) Flow cytometric analysis of percentage of apoptotic cells using APC-Annexin V staining.

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Experimental details: Figure 6 Western blot analysis of ANXA1 expression in keratinocytes upon overexpression of miR-196a. Total protein extracts from cells overexpressing miR-196a (miR-196a) and negative controls (scramble) were analyzed by western blot for ANXA1. Results show no differences in protein levels between the experiments.

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Experimental details: Figure 4 P2X7R controls different mechanisms in Anxa1, CD14, MMR and TNF- alpha release. ( A ) Representative high intensity projection deconvolved image of a mouse bone marrow-derived macrophages (BMDMs) primed for 4 h with LPS (1 mug/ml), followed by stimulation or not for 5 min with ATP (3 mM) and stained for phosphatidylserine with annexin V-FITC (green), for anxa1 (red) and for nuclei (DAPI, blue) without cell permeabilization; scale bar = 10 muM. ( B ) Western blot for CD14 and MMR in cell-free supernatants and cell lysates from BMDM primed as in ( A ) but with 20 min of ATP (3 mM) stimulation; 10 min before and during ATP stimulation cells were incubated with the general metalloprotease inhibitor GM6001 (100 nM) or with the specific MMP9 inhibitor I (100 nM). Western blots are representative of n = 2 independent experiments and composite is from lanes of the same membrane. ( C,D ) ELISA analysis for released IL-1beta ( C ) or TNF-alpha ( D ) from BMDMs treated as in ( B ) and additionally 10 min before and during ATP stimulation cells were incubated with the Zn 2+ chelator TPEN (50 muM). Data is presented as mean and s.e.m. of n = 3 independent experiments.

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Experimental details: Fig 6 Shotgun proteomic analyses (MS/MS) of AJ WT, AJ -/- NT and 0.11 muM rHsGal-1 treated myotubes. A. Heatmap of top 20 and bottom 20 expressed proteins for WT myotubes based on Log 2 fold change (FC) from the mean value all conditions. Box denotes cluster of proteins that had similar FC values between WT and rHsGal-1 treatment. B. GO-Term Slim analysis of rescued proteins. C. Relative amount of proteins found in the selected GO-Terms(B). D. Log 2 FC of Annexin family proteins (calculated based on WT and Gal-1 FC from NT). E. Quantification of ANXA6 after 48h differentiation in WT, NT, and 0.11 muM rHsGal-1 treated myotubes. F. Quantification of ANXA1 after 48h in WT, NT, and 0.11 muM rHsGal-1 treated myotubes. G. Western blot images of ANXA1 and ANXA6. p values were measured by 2-way ANOVA multiple comparison test and indicated by *p< 0.05, **p< 0.01, and ***p< 0.001. Error bars represent SEM. n = 3. All proteins were preselected based on protein ID significance values of >=20% (p

Supportive validation

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Experimental details: Figure 3 AnxA1 controlled claudin-1 and ZO-1 expressions on uterine epithelial cells via FPR1 and FPR2. Claudin-1 ( A ) and ZO-1 ( B ) expressions on uterine epithelial cells were determined 24 h after incubations. Representative images of claudin-1 and ZO-1 immunofluorescence are shown. (-) means absence and (+) means presence of treatments. The data are expressed as mean +- standard error of mean of three to five independent experiments. a p < 0.05 vs. NT; b p < 0.01 and c p < 0.05 vs. AnxA1.

Supportive validation

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Experimental details: Figure 4 AnxA1 controlled Muc-1 and Claudin-1 expressions on uterine epithelial cells via ERK1/2 phosphorylation. The effect of AnxA1 on ERK ( A ) and STAT1alpha ( B ) phosphorylation and NF-kappaB expression ( C ) were investigated by flow cytometry. The inhibition on ERK1/2, evoked by PD98059 incubation, was investigated on Muc-1 ( D ), Claudin-1 ( F ) and ZO-1 ( H ) expressions using immunofluorescence. Representative images of Muc-1, Claudin-1 and ZO-1 are shown in ( E ), ( G ) and ( I ), respectively. (-) means absence and (+) means presence of treatments. The data are expressed as mean +- standard error of mean of three to five independent experiments. a p < 0.05 and b p < 0.01 vs. NT; c p < 0.05 and d p < 0.01 vs. AnxA1.

Supportive validation

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Experimental details: Figure 5 AnxA1 induced F-actin polymerization via FPR1. F-actin was quantified on uterine epithelial cells 2 h after incubations, using phalloidin-rhodamine by confocal microscopy. (-) means absence and (+) means presence of treatments ( A ). Representative images of F-actin polymerization are shown ( B ). The data are expressed as mean +- standard error of mean of three to five independent experiments. a p < 0.05 vs. NT; b p < 0.05 vs. AnxA1.

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Experimental details: Figure 7 AnxA1 is expressed at embryo-implantation site in vivo. Implantations sites were obtained from C57bl/6 at gestational day 5.5 ( A ). Representative image of AnxA1 expression at implantation site (arrow) and luminal tissue by confocal microscopy is shown ( B ).

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Experimental details: Figure 5 Differential ANXA1 expression in porcine CD4 + T cells. (A-C) Representative flow cytometric images of ANXA1 + CD4 + T cells of a wild-type (A) and an INS C94Y tg pig (B) are shown with reduced ANXA1 expression in wild-type CD4 + T cells. (C) Scatter plots summarizing flow cytometry analyses of PBMC of eleven wild-type and nine INS C94Y tg pigs. ANXA1 was significantly elevated in CD4 + T cells of INS C94Y tg pigs (*p < 0.05). (D-F) Similar ANXA1 levels after permeabilization in purified CD4 + T cells of a wild-type (D) and an INS C94Y tg pig (E) shown by immunofluorescence staining for ANXA1 (red), CD4 (green) and nucleus (blue, counterstained with DAPI) and confirmed by flow cytometry using n=4 per group (F) . (G-I) Representative images of immunofluorescence staining for ANXA1 on the outer cell membrane of porcine CD4 + T cells demonstrate higher ANXA1 expression in CD4 + T cells of the INS C94Y tg pig (H) compared to the wild-type (G) (arrows). (I) A higher ANXA1 expression on the cell surface of diabetic CD4 + T cells compared to CD4 + T cells of wild-types was confirmed by flow cytometry (n=6 per group). Horizontal bars indicate group means.

Supportive validation

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Experimental details: Fig. 3 Nedocromil potentiates dexamethasone inhibition of TxB 2 release from U937 cells. (Panel A) Measurement of TxB 2 generation (left hand Y -axis) and Anx-A1 (right hand Y -axis) by ELISA assay in the medium of U937 cells after treatment with nedocromil (5 nM), dexamethasone (2 nM), or a combination of both. The varying inhibition of TxB 2 release corresponds with the amount of Anx-A1 externalised. *** P < 0.001 relative to control values; SSSSSS P < 0.001 relative to dexamethasone alone values. (Panel B) Dexamethasone itself (0.2-20.0 nM) inhibits TxB 2 release at 5 min (as assessed by ELISA assay) but does not produce a maximal effect. However, when nedocromil (0.05-20 nM) is added to a maximally effective (2 nM) concentration of dexamethasone, a concentration-dependent potentiation of the inhibitory effect occurs with near maximal inhibition achieved at 20 nM nedocromil. (Panel C) The inhibition of TxB 2 release (as assessed by ELISA assay) by escalating concentrations of nedocromil (0.2-10 nM) in the presence of a fixed concentration (2 nM) of dexamethasone is paralleled by increasing amounts of Anx-A1 externalised from U937 cells. Insert: representative Western blot in which the total Anx-A1 in the medium was assessed. (Panel D) Reversal of the TxB 2 inhibitory action of a combination of 2 nM dexamethasone with 0.5 nM nedocromil by a mouse anti-Anx-A1 neutralising monoclonal antibody (mab 1A) but not by an irrelevant isotype matched monoclonal antibody. *** P < 0.001

Supportive validation

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Experimental details: Figure 2 Proteomic (2DE-DIGE) profiling identifies novel proteins and pathways modulated by miR-27a in CRC cell lines. ( a ) miR-27a expression was analysed by quantitative reverse transcriptase-PCR in the indicated CRC cell lines. The histogram reports the fold change with respect to HT29 cells taken as control. U6 RNA was used as calibrator. ( b ) HCT116 cells were stably transfected with the miR-Zip-27a vector, carrying the miR-27a antisense and the GFP reporter gene, or with the empty vector as control. Cells were isolated for GFP expression and for the miR-27a low-expressing clones (miR27a_KD); ** P

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Experimental details: Figure 5 miR-27a acts as an oncogenic miRNA in mouse xenografts. ( a ) Representative photographs of the xenograft tumour masses excised from each experimental group. miR-27a inhibitor (anti-miR-27a; n =5) and scrambled control (miR-Ctrl; n =5) in HCT116 cells or miR-27a mimic (miR-27a; n =5) and scrambled control (miR-Ctrl; n =5) in HT29 cells were intratumourally injected every 7 days for four cycles starting from the day in which tumours reached the volume of 200 mm 3 . The growth curve of the tumours is reported for both types of xenografts. ( b ) Representative photomicrographs of haematoxylin and eosin staining, calreticulin and MHC class I IHC analysis and TUNEL assay, respectively, performed on paraffin-embedded sections of the same tissues; scale bars, 200 mu m. ( c ) The histogram shows the apoptotic index as determined by TUNEL assay. ( d ) Western blotting analysis of the selected proteins gene identified by 2DE-DIGE performed on total protein extracts of mouse xenografts and ( e ) their relative quantification. Data are represented as means+-S.D. from at least two independently treated tumours; * P

Supportive validation

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Experimental details: Fig. 4 Immunohistochemical analysis of ANXA1 expression in lymph nodes from head and neck carcinomas. a Non-metastatic lymph node (N0) samples : constitutive expression of ANXA1 in the subcapsular sinus. b Metastatic lymph node (N+) samples : endogenous ANXA1 expression increased in the lymph node tissue and in the metastatic cells (arrows). c Negative control of reaction. Sections: 2 mum. Counterstain: Hematoxylin. d Densitometry of ANXA1. Values expressed as mean +- S.E.M. *** p < 0,001

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Experimental details: Figure 1 Cumulative incidence function plots according to ( A ) ANXA1 and ( B ) ARID1A immunoexpression. Dashed red line and full black line represent positive/high expression and negative/low expression for ANXA1 and ARID1A immunoexpression, respectively. p values obtained by Gray's test for trastuzumab resistance relapse.

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Experimental details: Figure 2 Relationship between ARID1A and ANXA1. Proportion of tumours with ARID1A high or low intensity and ANXA1 negativity or positivity ( A ). Association between ARID1A and ANXA1 (Chi-square: p = 0.183) ( B ). Illustrative images of the different protein intensity scores ( C ).

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Experimental details: Figure 3 Distribution of ARID1A and ANXA1 immunoexpression by molecular subtype. Percentage of cases with low and high ARID1A intensity staining score (1+ and 2+ vs. 3+) (Chi-square p = 0.749) ( A ). Percentage of cases with or without ANXA1 expression (Chi-square p < 0.001) ( B ).

Supportive validation

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Experimental details: Effects on the Annexin A1 (AnxA1) expression in MDA-MB-231 and MDA-MB-157 after the stimulation and inhibition of the IL-6 pathway. The cropped Western blotting of AnxA1 expression in the cytoplasm (C), nuclei (N), and supernatants (S) of MDA-MB-231 and MDA-MB-157 cells. ( A ) Cells were treated for 24 h with 1 muM of recombinant human IL-6 (rIL-6). ( B ) Cells were treated for 24 h with 1 muM of neutralizing antibody anti-human IL-6 (Tocilizumab; TCZ), and ( C ) cells were treated for 24 h with 1 muM of an inhibitor of the IL-6 signaling pathway molecule, STAT3 (STATTIC; STC). beta-Actin and Lamin B2 were used to check the proper cell fractionation and as loading controls of cytoplasmic and nuclear extracts, respectively. Three independent experiments were performed.

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Experimental details: Figure 2 Infiltrating myeloid cell-derived ANXA1 controls muscle repair. ( A ) Western blot analysis of ANXA1 protein in total TA muscle. Muscles were analyzed 0, 1, 2, 4, 7, and 14 days after injury. Shown are representative blots (top) and quantification of ANXA1 to beta-actin (bottom) and ratios. ( B ) Quantitative reverse transcriptase PCR analysis of AnxA1 mRNA level in various cell populations FACS-sorted from TA muscle. Muscles were analyzed 0, 1, 2, 4, 7, and 14 days after injury. EC, endothelial cells; FAP, fibro/adipogenic progenitors; SAT, satellite cells; Mac, macrophages; Neut, neutrophils. ( C ) Experimental setup of bone marrow transplantation (BMT). CX3CR1-GFP mice were irradiated and then transplanted with bone marrow cells isolated from WT or AnxA1 -/- mice. Bone marrow engraftment was checked on a blood sample after around 5 weeks. Then animals were injured in their TA by CTX injection and muscles analyzed 0 or 28 days later. Engraftment was confirmed on the bone marrow of each animal on the day of sacrifice. H&E staining ( D ) and myofiber cross-sectional area ( E ) of TA muscles 28 days after CTX injury. Scale bar: 50 mum. Results are mean +- SEM of at least 2 (D14 in A ) or 3 muscles. * P < 0.05, ** P < 0.01 vs. WT or D0.