Journal: Journal of Natural Products

Article Title: Resin Glycosides from Ipomoea funis as Inhibitors of P‑Glycoprotein in Multidrug-Resistant Breast Carcinoma Cells

doi: 10.1021/acs.jnatprod.5c01273

Figure Lengend Snippet: Effect of intrapilosin I ( 2 ) on apoptosis: (A) flow cytometry analysis with double staining using annexin V/PE and 7AAD in MCF-7 and MCF-7/Vin cells, bars represent the percentage of early and late apoptosis in the different cell phenotypes, data are presented as mean ± SD from three independent experiments. *** p < 0.01; (B) Western blot analysis of apoptosis-associated proteins (caspase-3) in MCF-7 and MCF-7/Vin cells after the indicated treatments for 48 h. Abbreviations: N, necrosis; LA, late apoptosis; EA, early apoptosis; V, viable.

Article Snippet: The effect of compound 2 on cell death was evaluated using a double staining assay with the PE Annexin V kit (130–119–353, Miltenyi Biotec) and 7AAD (A9400, Sigma-Aldrich).

Techniques: Flow Cytometry, Double Staining, Western Blot

Journal: BioImpacts

Article Title: Annexin A1, calreticulin and high mobility group box 1 are elevated in secondary progressive multiple sclerosis: Does immunogenic cell death occur in multiple sclerosis?

doi: 10.34172/bi.30264

Figure Lengend Snippet: Fig. 1. ANXA1 level in CSF sample of control group and SPMS patient. Bar graph related to ANXA1 measurement using standard ELISA assay that represents means ± SD measured in each group. A comparison between the results from control group and SPMS patient was done using a t-test. ELISA experiments were repeated at least three times. ** indicates: P < 0.01.

Article Snippet: Human ANXA1 commercial ELISA Kit (Elabscience; E-EL-H5512) was used to determine the level of ANXA1.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Scientific reports

Article Title: Hydrodynamic stress stimulates growth of cell clusters via the ANXA1/PI3K/AKT axis in colorectal cancer.

doi: 10.1038/s41598-019-56739-7

Figure Lengend Snippet: Figure 3. Hydrodynamic stress (HDS) induces upregulation of Annexin 1 (ANXA1) in cancer cell clusters. (A) Gene ontology (GO) analysis of gene-expression microarray data for C45 CTOS treated with single-set pulse HDS. At 6 h after HDS, 123 genes were upregulated by more than 1.5-fold; these genes were analyzed for GO enrichment. Orange bars: the number of genes included in each GO term. Blue bars: the log10(1/P- value) for each GO term. (B) Venn diagram shows overlap of 9 genes that were upregulated, both by mild HDS (single-set pulse HDS), which did not disrupt cell membranes, and by strong HDS, which caused architectural disruptions13. (C) Relative expression of Annexin 1 (ANXA1) mRNA before (pre) and 6 h after single-set pulse HDS, in multiple CTOS lines. Data are the average ± SD; N = 3 for each run. *P < 0.05. (D) Relative expression of ANXA1 mRNA, before and 6 h after SLO treatment in multiple CTOS lines. Data are the average ± SD; N = 3 for each run. *P < 0.05.

Article Snippet: For making short hairpin (sh) RNAs targeting ANXA1, we transferred the target sequences of the ANXA1 human shRNA constructs carried in pRS plasmids (TR314775, OriGene, Rockville, MD) to the pPB vector.

Techniques: Gene Expression, Microarray, Expressing

Journal: Scientific reports

Article Title: Hydrodynamic stress stimulates growth of cell clusters via the ANXA1/PI3K/AKT axis in colorectal cancer.

doi: 10.1038/s41598-019-56739-7

Figure Lengend Snippet: Figure 4. Knock down of Annexin 1 (ANXA1) suppresses growth stimulated by hydrodynamic stress (HDS). (A) Semi-quantitative PCR analysis confirms the knockdown of ANXA1 gene expression. Relative ANXA1 expression levels are shown in C45 CTOS that were either transduced with an ANXA1 shRNA construct (sh#1 or sh#2) or a non-targeting (NT) control construct. ACTB: β-actin, the internal control gene. Data are the average ± SD. *P < 0.05. (B) Immunoblots show proteins from C45 CTOS that were not transduced (NT) or transduced with 2 different ANXA1 shRNAs (sh#1 and sh#2). Proteins were extracted at 1, 3, and 7 days (d1, d3, and d7, respectively) after subculturing. (C) Relative growth (ATP assay) of C45 CTOS transduced with control (NT) or ANXA1 shRNAs (sh#1 and sh#2) and cultured for 7 days after single-set pulse HDS. Data are the average ± SD, normalized to control (ctrl) values; N = 20 for each condition. *P < 0.05. (D,E) Relative growth (ATP assay) of C45 CTOS treated with control (NT) or ANXA1 shRNAs (sh#1 and sh#2), and cultured for 14 days with (D) multiple-set pulse HDS or with (E) continuous HDS. Data are the average ± SD, normalized to ctrl; N = 9 in (D) and N = 6 in (E) for each condition. *P < 0.05.

Article Snippet: For making short hairpin (sh) RNAs targeting ANXA1, we transferred the target sequences of the ANXA1 human shRNA constructs carried in pRS plasmids (TR314775, OriGene, Rockville, MD) to the pPB vector.

Techniques: Knockdown, Real-time Polymerase Chain Reaction, Gene Expression, Expressing, Transduction, shRNA, Construct, Control, Western Blot, Subculturing Assay, ATP Assay, Cell Culture

Journal: Scientific reports

Article Title: Hydrodynamic stress stimulates growth of cell clusters via the ANXA1/PI3K/AKT axis in colorectal cancer.

doi: 10.1038/s41598-019-56739-7

Figure Lengend Snippet: Figure 5. Forced overexpression of Annexin 1 (ANXA1) potentiates hydrodynamic stress (HDS)-induced growth stimulation in colorectal cancer cell clusters. (A) Semi-quantitative PCR analysis confirms ANXA1 expression levels in C132 CTOS, transduced with either the ANXA1 construct (overexpression; OE) or the yellow fluorescent protein (YFP) control construct. Data are the average ± SD. *P < 0.05. (B) Immunoblots show ANXA1 expression in C132 CTOS transduced with ANXA1 (OE) or YFP (ctrl). The transduced ANXA1 had a higher molecular weight, because it was tagged with 3x FLAG. (C) Relative growth (ATP assay) of C132 CTOS with ANXA1 OE and YFP as control. CTOSs were cultured for 14 days with multiple-set pulse HDS (mHDS) or without HDS (ctrl). Data are the average ± SD, normalized to YFP without HDS; N = 8 for each condition. *P < 0.05. (D) Relative growth (ATP assay) of C132 CTOS with ANXA1 OE and YFP as control. CTOSs were cultured for 7 days with continuous HDS (cHDS) or without HDS (ctrl). Data are the average ± SD, normalized to control; N = 6 for each condition *P < 0.05.

Article Snippet: For making short hairpin (sh) RNAs targeting ANXA1, we transferred the target sequences of the ANXA1 human shRNA constructs carried in pRS plasmids (TR314775, OriGene, Rockville, MD) to the pPB vector.

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Expressing, Transduction, Construct, Control, Western Blot, Molecular Weight, ATP Assay, Cell Culture

Journal: Scientific reports

Article Title: Hydrodynamic stress stimulates growth of cell clusters via the ANXA1/PI3K/AKT axis in colorectal cancer.

doi: 10.1038/s41598-019-56739-7

Figure Lengend Snippet: Figure 6. Hydrodynamic stress (HDS) stimulates growth by activating the PI3K/AKT pathway downstream of Annexin 1 (ANXA1). (A) Immunoblot shows changes in the expression of proteins from the C45 line of cancer tissue-originated spheroids (CTOSs), treated with single-set pulse HDS. Proteins were identified by the indicated antibodies over the 6 h following HDS treatment. pAKT: AKT phosphorylated (at the indicated residue); tAKT: total AKT protein; ACTB: β-actin, the internal control gene. (B) Densitometry analysis of immunoblots shows the level of phosphorylated AKT (at S473), relative to the total AKT at each time point. The C45 CTOS line was treated with control (NT) or ANXA1 shRNAs (sh#1 or sh#2), then single-set pulse HDS were applied. Protein samples were extracted at the indicated time points following HDS treatment. Data show the quantification of a single representative result, selected from 3 independent experiments (see Supplementary Fig. S10 for raw immunoblots). (C) Relative growth (ATP assay) of C45 CTOS, treated with 2.5 μM GDC-0941 (GDC), and cultured for 14 days with multiple-set pulse HDS (mHDS). Data are the average ± SD, normalized to the DMSO control (crtl); N = 8 for each condition. *P < 0.05. (D) Relative growth (ATP assay) of C45 CTOS, treated with 1 μM GDC-0941 (GDC), and cultured for 14 days with continuous HDS (cHDS). Data are the average ± SD, normalized to the DMSO control; N = 6 for each condition. *P < 0.05.

Article Snippet: For making short hairpin (sh) RNAs targeting ANXA1, we transferred the target sequences of the ANXA1 human shRNA constructs carried in pRS plasmids (TR314775, OriGene, Rockville, MD) to the pPB vector.

Techniques: Western Blot, Expressing, Residue, Control, ATP Assay, Cell Culture

Journal: PLoS Genetics

Article Title: Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury

doi: 10.1371/journal.pgen.1007070

Figure Lengend Snippet: The sarcolemmal wounding assay was performed on flexor digitorus brevis muscles from WT muscle in the presence or absence of recombinant osteopontin (rOPN; 10μl at 1μg/μl). A) The extent of sarcolemmal damage, monitored as FM4-64 area, was greater in rOPN-treated myofibers compared to vehicle-treated control, both over time and at end-point (arrows). B) Repair cap formation, monitored by GFP-tagged annexin A1 (GFP-ANXA1) was delayed in rOPN-treated myofibers (arrowheads). Repair cap diameter at end-point was smaller in treated compared to control myofibers (arrowheads). Over time image series represents stacked consecutive images of the injured site at 10° orientation to reveal the extent of dye accumulation or repair cap formation . FM4-64 and ANXA1-GFP pictures were acquired simultaneously. Marked line plots, avg±sem; box plots, Tukey distribution; n = 50 myofibers (5 mice)/group; #, P<0.05 vs vehicle, 2way ANOVA + Bonferroni; *, P<0.05 vs vehicle, unpaired t-test with Welch’s correction.

Article Snippet: The plasmid encoding human annexin A1 with a carboxy-terminal GFP was obtained from Origene (Rockville, MD; Cat# RG201569).

Techniques: Muscles, Recombinant, Control

Journal: PLoS Genetics

Article Title: Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury

doi: 10.1371/journal.pgen.1007070

Figure Lengend Snippet: In addition to Ltbp4 , Anxa6 has also been shown to modify muscular dystrophy in mice [ , ]. The deleterious alleles of Ltbp4 (L4) or Anxa6 (A6) (severe) were compared to those from the protective 129 strain (mild) in the sarcolemma injury assay. A) Doubly homozygous A6 mild /L4 mild myofibers had the least injury while doubly homozygous A6 severe /L4 severe fibers had the greatest injury, marked by FM4-64. Myofibers with mixed homozygous genotypes were intermediate with respect to FM4-64 marked injury. B) A similar pattern was observed for Annexin A1 (ANXA1) repair caps, where doubly homozygous mild alleles of L4 and A6 assembled caps more rapidly and produced larger repair caps than doubly homozygous severe alleles. However, ANXA1 repair caps were smaller with the A6 homozygous severe allele, despite the presence of the mild L4 allele, suggesting that repair cap formation is dominated by the A6 genotype. FM4-64 and ANXA1-GFP images used for the analyses in A-B were acquired simultaneously. Marked line plots, avg±sem; box plots, Tukey distribution; n = 50 myofibers (5 mice)/group; marked line plots: #, P<0.05 vs control (A6 mild /L4 mild ), +, P<0.05 vs A6 severe /L4 mild and A6 mild /L4 severe groups, 2way ANOVA + Bonferroni; boxplots: *, P<0.05 vs control (A6 mild /L4 mild ), +, P<0.05 vs A6 severe /L4 mild and A6 mild /L4 severe groups, 1way ANOVA + Bonferroni.

Article Snippet: The plasmid encoding human annexin A1 with a carboxy-terminal GFP was obtained from Origene (Rockville, MD; Cat# RG201569).

Techniques: Produced, Control

Journal: Scientific reports

Article Title: Hydrodynamic stress stimulates growth of cell clusters via the ANXA1/PI3K/AKT axis in colorectal cancer.

doi: 10.1038/s41598-019-56739-7

Figure Lengend Snippet: Figure 3. Hydrodynamic stress (HDS) induces upregulation of Annexin 1 (ANXA1) in cancer cell clusters. (A) Gene ontology (GO) analysis of gene-expression microarray data for C45 CTOS treated with single-set pulse HDS. At 6 h after HDS, 123 genes were upregulated by more than 1.5-fold; these genes were analyzed for GO enrichment. Orange bars: the number of genes included in each GO term. Blue bars: the log10(1/P- value) for each GO term. (B) Venn diagram shows overlap of 9 genes that were upregulated, both by mild HDS (single-set pulse HDS), which did not disrupt cell membranes, and by strong HDS, which caused architectural disruptions13. (C) Relative expression of Annexin 1 (ANXA1) mRNA before (pre) and 6 h after single-set pulse HDS, in multiple CTOS lines. Data are the average ± SD; N = 3 for each run. *P < 0.05. (D) Relative expression of ANXA1 mRNA, before and 6 h after SLO treatment in multiple CTOS lines. Data are the average ± SD; N = 3 for each run. *P < 0.05.

Article Snippet: For making short hairpin (sh) RNAs targeting ANXA1, we transferred the target sequences of the ANXA1 human shRNA constructs carried in pRS plasmids (TR314775, OriGene, Rockville, MD) to the pPB vector.

Techniques: Gene Expression, Microarray, Expressing

Journal: Scientific reports

Article Title: Hydrodynamic stress stimulates growth of cell clusters via the ANXA1/PI3K/AKT axis in colorectal cancer.

doi: 10.1038/s41598-019-56739-7

Figure Lengend Snippet: Figure 4. Knock down of Annexin 1 (ANXA1) suppresses growth stimulated by hydrodynamic stress (HDS). (A) Semi-quantitative PCR analysis confirms the knockdown of ANXA1 gene expression. Relative ANXA1 expression levels are shown in C45 CTOS that were either transduced with an ANXA1 shRNA construct (sh#1 or sh#2) or a non-targeting (NT) control construct. ACTB: β-actin, the internal control gene. Data are the average ± SD. *P < 0.05. (B) Immunoblots show proteins from C45 CTOS that were not transduced (NT) or transduced with 2 different ANXA1 shRNAs (sh#1 and sh#2). Proteins were extracted at 1, 3, and 7 days (d1, d3, and d7, respectively) after subculturing. (C) Relative growth (ATP assay) of C45 CTOS transduced with control (NT) or ANXA1 shRNAs (sh#1 and sh#2) and cultured for 7 days after single-set pulse HDS. Data are the average ± SD, normalized to control (ctrl) values; N = 20 for each condition. *P < 0.05. (D,E) Relative growth (ATP assay) of C45 CTOS treated with control (NT) or ANXA1 shRNAs (sh#1 and sh#2), and cultured for 14 days with (D) multiple-set pulse HDS or with (E) continuous HDS. Data are the average ± SD, normalized to ctrl; N = 9 in (D) and N = 6 in (E) for each condition. *P < 0.05.

Article Snippet: For making short hairpin (sh) RNAs targeting ANXA1, we transferred the target sequences of the ANXA1 human shRNA constructs carried in pRS plasmids (TR314775, OriGene, Rockville, MD) to the pPB vector.

Techniques: Knockdown, Real-time Polymerase Chain Reaction, Gene Expression, Expressing, Transduction, shRNA, Construct, Control, Western Blot, Subculturing Assay, ATP Assay, Cell Culture

Journal: Scientific reports

Article Title: Hydrodynamic stress stimulates growth of cell clusters via the ANXA1/PI3K/AKT axis in colorectal cancer.

doi: 10.1038/s41598-019-56739-7

Figure Lengend Snippet: Figure 5. Forced overexpression of Annexin 1 (ANXA1) potentiates hydrodynamic stress (HDS)-induced growth stimulation in colorectal cancer cell clusters. (A) Semi-quantitative PCR analysis confirms ANXA1 expression levels in C132 CTOS, transduced with either the ANXA1 construct (overexpression; OE) or the yellow fluorescent protein (YFP) control construct. Data are the average ± SD. *P < 0.05. (B) Immunoblots show ANXA1 expression in C132 CTOS transduced with ANXA1 (OE) or YFP (ctrl). The transduced ANXA1 had a higher molecular weight, because it was tagged with 3x FLAG. (C) Relative growth (ATP assay) of C132 CTOS with ANXA1 OE and YFP as control. CTOSs were cultured for 14 days with multiple-set pulse HDS (mHDS) or without HDS (ctrl). Data are the average ± SD, normalized to YFP without HDS; N = 8 for each condition. *P < 0.05. (D) Relative growth (ATP assay) of C132 CTOS with ANXA1 OE and YFP as control. CTOSs were cultured for 7 days with continuous HDS (cHDS) or without HDS (ctrl). Data are the average ± SD, normalized to control; N = 6 for each condition *P < 0.05.

Article Snippet: For making short hairpin (sh) RNAs targeting ANXA1, we transferred the target sequences of the ANXA1 human shRNA constructs carried in pRS plasmids (TR314775, OriGene, Rockville, MD) to the pPB vector.

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Expressing, Transduction, Construct, Control, Western Blot, Molecular Weight, ATP Assay, Cell Culture

Journal: Scientific reports

Article Title: Hydrodynamic stress stimulates growth of cell clusters via the ANXA1/PI3K/AKT axis in colorectal cancer.

doi: 10.1038/s41598-019-56739-7

Figure Lengend Snippet: Figure 6. Hydrodynamic stress (HDS) stimulates growth by activating the PI3K/AKT pathway downstream of Annexin 1 (ANXA1). (A) Immunoblot shows changes in the expression of proteins from the C45 line of cancer tissue-originated spheroids (CTOSs), treated with single-set pulse HDS. Proteins were identified by the indicated antibodies over the 6 h following HDS treatment. pAKT: AKT phosphorylated (at the indicated residue); tAKT: total AKT protein; ACTB: β-actin, the internal control gene. (B) Densitometry analysis of immunoblots shows the level of phosphorylated AKT (at S473), relative to the total AKT at each time point. The C45 CTOS line was treated with control (NT) or ANXA1 shRNAs (sh#1 or sh#2), then single-set pulse HDS were applied. Protein samples were extracted at the indicated time points following HDS treatment. Data show the quantification of a single representative result, selected from 3 independent experiments (see Supplementary Fig. S10 for raw immunoblots). (C) Relative growth (ATP assay) of C45 CTOS, treated with 2.5 μM GDC-0941 (GDC), and cultured for 14 days with multiple-set pulse HDS (mHDS). Data are the average ± SD, normalized to the DMSO control (crtl); N = 8 for each condition. *P < 0.05. (D) Relative growth (ATP assay) of C45 CTOS, treated with 1 μM GDC-0941 (GDC), and cultured for 14 days with continuous HDS (cHDS). Data are the average ± SD, normalized to the DMSO control; N = 6 for each condition. *P < 0.05.

Article Snippet: For making short hairpin (sh) RNAs targeting ANXA1, we transferred the target sequences of the ANXA1 human shRNA constructs carried in pRS plasmids (TR314775, OriGene, Rockville, MD) to the pPB vector.

Techniques: Western Blot, Expressing, Residue, Control, ATP Assay, Cell Culture

Journal: Journal of neuroendocrinology

Article Title: Annexin A1 Complex Mediates Oxytocin Vesicle Transport

doi: 10.1111/jne.12112

Figure Lengend Snippet: The antibody 4660 recognizes specifically ANXA1 in AtT20 and N11 cells and mouse hypothalamus. Proteins in the cytosols (5 mg/mL) extracted from AtT20 cells (A), untransfected N11 cells (B), and mouse hypothalamus tissues (C) were detected by immunoblotting. Anti-snapin rabbit antibody (αSNAP) and two chicken antibodies (4659 and 4660) were used to detect 15kD snapin (marked with ‘*’) and a 38kD protein (ANXA1) in AtT20 cells (A). The antibodies 4659 and 4660 and mouse anti-ANXA1 antibody (mAb) were used to detect ANXA1 in N11 cells (B) and in mouse hypothalamus (C). (D) The antibody 4660 and mouse anti-ANXA1 antibody (mAb) were used to detect recombinant ANXA1 protein tagged with GST (GST-ANXA1: AN) and GST tag alone (G) that were purified from bacteria.

Article Snippet: Glutathione- s -transferase (GST) tagged ANXA1 was generated by inserting the BamHI-XhoI digest of the PCR product of annexin-1 cDNA in GFP-ANXA1 that was purchased from Origene Co. (Rockville, MD) into pGEX4T-2.

Techniques: Western Blot, Recombinant, Purification

Journal: Journal of neuroendocrinology

Article Title: Annexin A1 Complex Mediates Oxytocin Vesicle Transport

doi: 10.1111/jne.12112

Figure Lengend Snippet: ANXA1 colocalizes with oxytocin vesicles (OXT) at the process tips of N11 cells and in the axons of primary hypothalamic neurons. (A) N11 cells grown on poly-L-lysine-coated coverslips were processed for immunocytochemistry with antibodies to oxytocin (green) and ANXA1 (red). Scale bars = 10 μm. (B–E) Primary mouse hypothalamic neurons (E17) were differentiated on poly-L-lysine- coated coverslips in neurobasal/B27 medium for 14 days (DIV14) and processed for immunocytochemistry with antibody to MAP2 (B), CPE (C), or ANXA1 (D–F) along with anti-oxytocin antibody. (B) Primary oxytocin neurons formed a long axon that was not stained by anti-MAP2 antibody. (C) The image of oxytocin vesicles and CPE at the axon terminals of the neurons was taken by confocal microscope. Scale bars = 5 μm. The inset of the panel (D) showed the co-localization of oxytocin vesicles with ANXA1 in the axon. (D–F) ANXA1 showed significant co-localization with vesicles containing oxytocin at the axon terminals. Scale bars = 10 μm. The insets of panel (F) showed the overlapping between the green pixels of oxytocin and the red pixels of ANXA1. The scale bars of the insets = 0.13 μm (= the diameter of one pixel).

Article Snippet: Glutathione- s -transferase (GST) tagged ANXA1 was generated by inserting the BamHI-XhoI digest of the PCR product of annexin-1 cDNA in GFP-ANXA1 that was purchased from Origene Co. (Rockville, MD) into pGEX4T-2.

Techniques: Immunocytochemistry, Staining, Microscopy

Journal: Journal of neuroendocrinology

Article Title: Annexin A1 Complex Mediates Oxytocin Vesicle Transport

doi: 10.1111/jne.12112

Figure Lengend Snippet: ANXA1 is required for the axon-localization of oxytocin vesicles (OXT) in primary hypothalamic neurons. Primary mouse hypothalamic neurons (E17) differentiated on poly-L-lysine-coated coverslips were transduced with control or anti-ANXA1 shRNA lentiviral particles for 5 days. Transduced neurons were then processed for immunocytochemistry using antibodies to ANXA1 (red) and oxytocin (green). The insets showed that neurons transduced with anti-ANXA1 shRNA (B) had low levels of ANXA1 and oxytocin vesicles in the axons compared to those transduced with control shRNA (A). Scale bars = 10 μm. (C) Bar graphs show the average intensities ± SEMs of oxytocin vesicles in the cell bodies and the axons between 0–50, 50–100, 100–150, 150–200, and 200–250 μm from the cell body of neurons transduced with either control or anti-ANXA1 shRNA (ANXA1 KD). (*: p<0.001)

Article Snippet: Glutathione- s -transferase (GST) tagged ANXA1 was generated by inserting the BamHI-XhoI digest of the PCR product of annexin-1 cDNA in GFP-ANXA1 that was purchased from Origene Co. (Rockville, MD) into pGEX4T-2.

Techniques: Transduction, shRNA, Immunocytochemistry

Journal: Journal of neuroendocrinology

Article Title: Annexin A1 Complex Mediates Oxytocin Vesicle Transport

doi: 10.1111/jne.12112

Figure Lengend Snippet: ANXA1, AKAP150, and microtubule motors form a complex in mouse hypothalamus and N11 cells. The cytosols (5 mg/mL) of mouse hypothalamus tissues (A) or N11 cells (B–D) that were treated with DMSO (B), 10 μM forskolin (C, Forsk), or 10 nM PMA (D) were mixed with the antibody 4660 along with protein A beads (60 μL) at 4°C for 24 h. Proteins pulled down by anti-ANXA1 antibody (αAN) were probed by immunoblotting. (E) The cytosols (5 mg/mL, post-nuclear supernatant [SN]) of N11 cells were mixed with anti-p150 (dynactin) antibody for co-immunoprecipitation. Proteins bound to dynactin were detected by immunoblotting.

Article Snippet: Glutathione- s -transferase (GST) tagged ANXA1 was generated by inserting the BamHI-XhoI digest of the PCR product of annexin-1 cDNA in GFP-ANXA1 that was purchased from Origene Co. (Rockville, MD) into pGEX4T-2.

Techniques: Western Blot, Immunoprecipitation

Journal: Journal of neuroendocrinology

Article Title: Annexin A1 Complex Mediates Oxytocin Vesicle Transport

doi: 10.1111/jne.12112

Figure Lengend Snippet: Vesicles containing oxytocin (OXT) and ANXA1 co-migrate toward the axon terminal in response to PKA activation. Mouse hypothalamic neurons (E17, DIV14) were treated with DMSO or 10 μM forskolin for 10 min (A, F10′), 20 min (B, F20′), and 30 min (C, F30′) and stained using antibodies to oxytocin and ANXA1. The axons were indicated by arrows. The magnified images in the insets show the co-localization between oxytocin and ANXA1 at the distal axons of neurons treated with forskolin for 10 min (inset i), 20 min (inset ii), and 30 min (inset iii). Scale bar = 50 μm. (D) To specify the involvement of PKA in forskolin-induced axon-localization of oxytocin vesicles, neurons were pretreated with 10 μM PKI-ester for 1 h prior to 30-min forskolin treatment (inset iv). (E) The average intensities of oxytocin vesicles in cell bodies and along axons (0–50, 50–100, 100–150, and 150–200 μm) were quantified using Metamorph software (n=30 per condition). Bar graphs show the average intensities ± SEMs of oxytocin vesicles along the axons (0–50, 50–100, 100–150, and 150–200 μm) of neurons treated with forskolin (0, 10, 20, and 30 min). (#: p<0.0001, *: p<0.01compared to control)

Article Snippet: Glutathione- s -transferase (GST) tagged ANXA1 was generated by inserting the BamHI-XhoI digest of the PCR product of annexin-1 cDNA in GFP-ANXA1 that was purchased from Origene Co. (Rockville, MD) into pGEX4T-2.

Techniques: Activation Assay, Staining, Software

Journal: Journal of neuroendocrinology

Article Title: Annexin A1 Complex Mediates Oxytocin Vesicle Transport

doi: 10.1111/jne.12112

Figure Lengend Snippet: Activation of PKA increases the localization of oxytocin-containing vesicles (OXT) to axon terminals while that PKC decreases it. Mouse hypothalamic neurons (E17, DIV14) were treated with DMSO (A), 10 μM forskolin (B, Forsk), or 10 nM PMA (C) for 30 min and stained using anti-oxytocin antibody. The images of oxytocin vesicles at the axon terminals of the neurons were taken by confocal microscope. Scale bars = 10 μm. (D) Bar graphs show the average intensities ± SEMs of oxytocin vesicles at the axon terminals of neurons treated with DMSO, forskolin, or PMA. (*: p<0.01). (E, F) Mouse hypothalamic neurons treated with DMSO (E) or PMA (F) were stained using antibodies to oxytocin and ANXA1. The axons were indicated by arrows. Scale bars = 10 μm. (G) Bar graphs show the average intensities ± SEMs of oxytocin vesicles in the axons of neurons treated with DMSO, PMA, and forskolin (Forsk.). (n=60 neurons per condition, *: p<0.001: DMSO vs. forskolin)

Article Snippet: Glutathione- s -transferase (GST) tagged ANXA1 was generated by inserting the BamHI-XhoI digest of the PCR product of annexin-1 cDNA in GFP-ANXA1 that was purchased from Origene Co. (Rockville, MD) into pGEX4T-2.

Techniques: Activation Assay, Staining, Microscopy

Journal: Journal of neuroendocrinology

Article Title: Annexin A1 Complex Mediates Oxytocin Vesicle Transport

doi: 10.1111/jne.12112

Figure Lengend Snippet: Schematic illustration of how PKA and PKC affect the interaction of ANXA1 with AKAP150 and kinesin-2, thus changing the direction of movement of oxytocin-containing vesicles. (A) Upon activation of PKA that consists of two catalytic [C] and two regulatory [R] subunits, the regulated subunits withdraw their inhibitory action from the catalytic subunits. Then, the catalytic subunits increase the binding of kinesin-2 and AKAP150 to ANXA1 on light-density oxytocin (OXT) vesicle without affecting the interaction of cytoplasmic dynein-dynactin with ANXA1, resulting in enhancement of anterograde transport of the vesicles to the axon terminal. (B) Activated PKC inhibits the interaction of kinesin-2 with AKAP150-ANXA1 complex without affecting the interaction of cytoplasmic dynein-dynactin with ANXA1 complex, resulting in inhibition of anterograde transport of oxytocin vesicles to the axon terminal. (C) Activation of PKA enhances the anterograde transport of light-density oxytocin vesicles to the axon terminals while activation of PKC inhibits the anterograde transport. Activation of PKC increases somatic exocytosis of oxytocin vesicles. Some heavy density oxytocin vesicles are transported to the axon and dendrites.

Article Snippet: Glutathione- s -transferase (GST) tagged ANXA1 was generated by inserting the BamHI-XhoI digest of the PCR product of annexin-1 cDNA in GFP-ANXA1 that was purchased from Origene Co. (Rockville, MD) into pGEX4T-2.

Techniques: Activation Assay, Binding Assay, Inhibition

Journal: Translational Lung Cancer Research

Article Title: Increased plasma levels of damage-associated molecular patterns during systemic anticancer therapy in patients with advanced lung cancer

doi: 10.21037/tlcr-21-92

Figure Lengend Snippet: Dynamics of plasma DAMP levels in advanced lung cancer patients during the first cycle of systemic anticancer therapy. (A) Time course of the fold change in plasma concentrations (relative to baseline) of HMGB1, CRT, HSP70, annexin A1, and histone H3 in individual patients. Plasma samples were collected before and on days 3 and 8 after the onset of the first cycle as well as before the onset of the second cycle of treatment. (B) Fold change in the plasma levels of HMGB1, CRT, HSP70, annexin A1, and histone H3 during the first treatment cycle relative to baseline. Data are means + SEM. The P values for differences in mean values were determined by repeated-measures ANOVA. (C) Comparison of baseline values and maximum values after the onset of the first cycle of treatment for the fold change in plasma levels of the five DAMPs. The numbers of patients showing a >2-fold increase were 34, 34, 24, 5, and 3 for HMGB1, CRT, HSP70, annexin A1, and histone H3, respectively. CRT and histone H3 were undetectable at baseline in 10 and 97 patients, respectively, and these patients were excluded from the analysis. The P values for differences in mean values were determined with the paired t test. DAMP, damage-associated molecular pattern; HMGB1, high-mobility group box 1; CRT, calreticulin; HSP70, heat shock protein 70; SEM, standard error of the mean; ANOVA, analysis of variance.

Article Snippet: The plasma levels of CRT (#ELH-CALR-1; RayBiotech, Peachtree Corners, GA), HSP70 (#KE00059; Proteintech, Tokyo, Japan), and annexin A1 (#EK1745; Boster Biological Technology, Pleasanton, CA) were similarly quantified with ELISA kits.

Techniques: Clinical Proteomics, Comparison

Journal: Translational Lung Cancer Research

Article Title: Increased plasma levels of damage-associated molecular patterns during systemic anticancer therapy in patients with advanced lung cancer

doi: 10.21037/tlcr-21-92

Figure Lengend Snippet: Association between the maximum fold change in plasma DAMP levels relative to baseline and clinical response. The maximum fold change in HMGB1 (A), CRT (B), HSP70 (C), or annexin A1 (D) levels is shown as the mean + SEM for patients classified according to tumor response. The numbers of patients who showed a CR, PR, SD or PD according to RECIST criteria were 1, 43, 63, and 13, respectively; one patient not evaluable by RECIST criteria was excluded from the analysis. CRT was undetectable at baseline in seven patients with a PR, in two with SD, and in one with PD, and these patients were also excluded from the analysis. The P values for differences in mean values were determined by one-way ANOVA. DAMP, damage-associated molecular pattern; HMGB1, high-mobility group box 1; CRT, calreticulin; HSP70, heat shock protein 70; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease; RECIST, Response Evaluation Criteria in Solid Tumors; ANOVA, analysis of variance.

Article Snippet: The plasma levels of CRT (#ELH-CALR-1; RayBiotech, Peachtree Corners, GA), HSP70 (#KE00059; Proteintech, Tokyo, Japan), and annexin A1 (#EK1745; Boster Biological Technology, Pleasanton, CA) were similarly quantified with ELISA kits.

Techniques: Clinical Proteomics

Journal: Translational Lung Cancer Research

Article Title: Increased plasma levels of damage-associated molecular patterns during systemic anticancer therapy in patients with advanced lung cancer

doi: 10.21037/tlcr-21-92

Figure Lengend Snippet: Maximum fold changes in plasma DAMP concentrations according to anticancer treatment modality. The maximum fold change in HMGB1 (A), CRT (B), HSP70 (C), or annexin A1 (D) levels is shown as the mean + SEM for patients with a CR (n=1) or a PR (n=43). Treatment modalities included platinum doublet chemotherapy (n=22), single-agent chemotherapy (n=3), CCRT (n=11), and EGFR- or ALK-TKIs (n=8). CRT was undetectable at baseline in four patients receiving platinum doublet chemotherapy, one receiving single-agent chemotherapy, and two receiving CCRT, and these patients were excluded from the analysis. The P values for differences in mean values were determined by one-way ANOVA. DAMP, damage-associated molecular pattern; HMGB1, high-mobility group box 1; CRT, calreticulin; HSP70, heat shock protein 70; SEM, standard error of the mean; CR, complete response; PR, partial response; CCRT, concurrent chemoradiotherapy; EGFR, epidermal growth factor receptor; ALK, anaplastic lymphoma kinase; TKI, tyrosine kinase inhibitor; ANOVA, analysis of variance.

Article Snippet: The plasma levels of CRT (#ELH-CALR-1; RayBiotech, Peachtree Corners, GA), HSP70 (#KE00059; Proteintech, Tokyo, Japan), and annexin A1 (#EK1745; Boster Biological Technology, Pleasanton, CA) were similarly quantified with ELISA kits.

Techniques: Clinical Proteomics

Journal: Translational Lung Cancer Research

Article Title: Increased plasma levels of damage-associated molecular patterns during systemic anticancer therapy in patients with advanced lung cancer

doi: 10.21037/tlcr-21-92

Figure Lengend Snippet: Correlation analysis for maximum fold changes in plasma DAMP concentrations. (A,B,C,D,E,F) Correlation analysis for HMGB1 and either CRT (A), HSP70 (B), or annexin A1 (C), for CRT and either HSP70 (D) or annexin A1 (E), or for HSP70 and annexin A1 (F) is shown. The correlation coefficient (r) and corresponding P values are indicated. CRT was undetectable at baseline in 10 patients, and these patients were excluded from the analysis. (G) Venn diagram for the numbers of patients showing a >2-fold increase in the plasma levels of HMGB1, CRT, or HSP70 during the first treatment cycle. DAMP, damage-associated molecular pattern; HMGB1, high-mobility group box 1; CRT, calreticulin; HSP70, heat shock protein 70.

Article Snippet: The plasma levels of CRT (#ELH-CALR-1; RayBiotech, Peachtree Corners, GA), HSP70 (#KE00059; Proteintech, Tokyo, Japan), and annexin A1 (#EK1745; Boster Biological Technology, Pleasanton, CA) were similarly quantified with ELISA kits.

Techniques: Clinical Proteomics

Journal: The American Journal of Pathology

Article Title: Intermittent Glucocorticoid Dosing Improves Muscle Repair and Function in Mice with Limb-Girdle Muscular Dystrophy

doi: 10.1016/j.ajpath.2017.07.017

Figure Lengend Snippet: List of Primers for Real-Time Quantitative PCR

Article Snippet: 23 Plasmids A plasmid encoding human annexin A1 with a carboxy-terminal green fluorescent protein (GFP) was obtained from Origene (Rockville, MD; catalog number RG201569).

Techniques:

Journal: The American Journal of Pathology

Article Title: Intermittent Glucocorticoid Dosing Improves Muscle Repair and Function in Mice with Limb-Girdle Muscular Dystrophy

doi: 10.1016/j.ajpath.2017.07.017

Figure Lengend Snippet: Prednisone-accelerated sarcolemmal repair and repair cap formation in Dysf-null myofibers. A: A single dose of glucocorticoid steroids, either prednisone or deflazacort, decreased the extent of sarcolemmal injury in live myofibers, as quantitated by FM4-64 accumulation over time and injury area at analysis end point (arrows). Top left panels: Time-dependent real-time image stacks of injury site (10-μm-wide area). Top right panels: Z-stack rendering of FM4-64 accumulation at injury site at 300 seconds after injury. Bottom panels: Quantitative data are plotted. B: The single steroid dose associated with faster onset of the sarcolemmal repair cap, as monitored in real-time by green fluorescent protein (GFP)–labeled annexin A1 (ANXA1-GFP) accumulation at injury site (arrows). Top panels: Time-dependent image stacks (10-μm-wide area). Bottom panel: Quantitation of time (after injury) to cap onset is shown. Data are expressed as means ± SEM (marked line plots) or Tukey distribution (box plots). n = 5 mice (50 myofibers) per group (3 males and 2 females per group). ∗P < 0.05 versus vehicle (one-way analysis of variance test with Bonferroni multiple comparison); †P < 0.05 versus vehicle (two-way analysis of variance test with Bonferroni multiple comparison). Scale bar = 5 μm (A, right top panels). AU, arbitrary unit.

Article Snippet: 23 Plasmids A plasmid encoding human annexin A1 with a carboxy-terminal green fluorescent protein (GFP) was obtained from Origene (Rockville, MD; catalog number RG201569).

Techniques: Labeling, Quantitation Assay

Journal: The American Journal of Pathology

Article Title: Intermittent Glucocorticoid Dosing Improves Muscle Repair and Function in Mice with Limb-Girdle Muscular Dystrophy

doi: 10.1016/j.ajpath.2017.07.017

Figure Lengend Snippet: Weekly and daily prednisone regimens enhance sarcolemmal repair in Dysf-null myofibers. A:Dysf-null mice were treated for 4 weeks with either weekly or daily prednisone. Both prednisone regimens resulted in improved sarcolemmal repair after laser-induced injury, as shown by diminished accumulation of FM4-64 over time (10-μm-wide area) and decreased sarcolemmal injury area at end point (arrows). B: Both steroid regimens correlated with faster onset of the annexin A1 (ANXA1) repair cap, as monitored through ANXA1–green fluorescent protein (GFP) accumulation at injury site over time (10-μm-wide area). Data are expressed as means ± SEM (marked line plots) or Tukey distribution (box plots). n = 5 mice (50 myofibers) per group (3 males and 2 females in vehicle and daily groups and 2 males and 3 females in weekly group). ∗P < 0.05 versus vehicle (one-way analysis of variance test with Bonferroni multiple comparison); †P < 0.05 versus vehicle (two-way analysis of variance test with Bonferroni multiple comparison). Scale bar = 5 μm (A). AU, arbitrary unit.

Article Snippet: 23 Plasmids A plasmid encoding human annexin A1 with a carboxy-terminal green fluorescent protein (GFP) was obtained from Origene (Rockville, MD; catalog number RG201569).

Techniques:

Journal: The American Journal of Pathology

Article Title: Intermittent Glucocorticoid Dosing Improves Muscle Repair and Function in Mice with Limb-Girdle Muscular Dystrophy

doi: 10.1016/j.ajpath.2017.07.017

Figure Lengend Snippet: Prednisone treatment of Sgcg-null mice enhances sarcolemmal repair and repair cap formation after injury. A: Laser injury was performed on Sgcg-null myofibers after 4 weeks of either weekly or daily prednisone treatment. Both weekly and daily prednisone reduced the extent of sarcolemmal damage after laser-induced injury, as shown by decreased FM4-64 accumulation over time (10-μm-wide area) and at end point (arrows). B: Both weekly and daily prednisone regimens correlated with faster onset (arrows) of accumulation of annexin A1–green fluorescent protein (ANXA1-GFP) in repair caps on the site of sarcolemmal injury (10-μm-wide area). Data are epressed as means ± SEM (marked line plots) or Tukey distribution (box plots). n = 5 mice (50 myofibers) per group (3 males and 2 females per group). ∗P < 0.05 versus vehicle (one-way analysis of variance test with Bonferroni multiple comparison); †P < 0.05 versus vehicle (two-way analysis of variance test with Bonferroni multiple comparison). Scale bar, 5 μm (A). AU, arbitrary unit.

Article Snippet: 23 Plasmids A plasmid encoding human annexin A1 with a carboxy-terminal green fluorescent protein (GFP) was obtained from Origene (Rockville, MD; catalog number RG201569).

Techniques: