Journal: The Febs Journal

Article Title: Mesoglycan connects Syndecan‐4 and VEGFR2 through Annexin A1 and formyl peptide receptors to promote angiogenesis in vitro

doi: 10.1111/febs.16043

Figure Lengend Snippet: EVs interact via ANXA1/FPRs in autocrine manner (A) Bright‐field images (scale bar = 150 μm) and (B) histogram of wound healing assay of HUVEC cells treated or not with EVs ctrl (1 × 10 6 ), EVs mesoglycan (1 × 10 6 ), BOC1 (100 μ m ) and/or their association. (C) Representative images of analysed fields of tube formation assay by HUVEC seeded for 12 h on matrigel and EBM2 medium 1 : 1 and in presence or not of EVs ctrl (1 × 10 6 ), EVs mesoglycan (1 × 10 6 ) and/or BOC1 (100 μ m ). Magnification 20×. Scale bar = 150 μm. Analysis of (D) tube length and (E) number of branches calculated by imagej (Angiogenesis Analyzer tool) software. The data represent a mean of three independent experiments SEM. * P < 0.05 for all treatments vs. untreated cells. § P < 0.05 for all treatment vs. EVs ctrl. ˜ P < 0.05 for all treatments vs. EVs mesoglycan. ° P < 0.05 for all treatment vs. BOC1.

Article Snippet: After removing incubation medium and washing with PBS, cell cultures were incubated in the presence of mesoglycan (0.3 mg·mL −1 , LDO, Laboratori Derivati Organici spa, Trino, Italy), VEGF (10 ng·mL −1 , VEGF‐165 recombinant human for HUVEC cells and VEGF‐164 recombinant mouse for MLEC, R&D Systems, Minneapolis, MN, USA), BOC1 (100 μM, Bachem AG, Bubendorf, Switzerland) and/or EVs ctrl (1 × 10 6 ) and EVs mesoglycan (1 × 10 6 ), both isolated from HUVEC.

Techniques: Wound Healing Assay, Tube Formation Assay, Software

Journal: The Febs Journal

Article Title: Mesoglycan connects Syndecan‐4 and VEGFR2 through Annexin A1 and formyl peptide receptors to promote angiogenesis in vitro

doi: 10.1111/febs.16043

Figure Lengend Snippet: Analysis of EndMT markers on HUVEC cells treated or not with EVs and BOC1. Immunofluorescence analysis of endothelial cells in presence or not of EVs ctrl (1 × 10 6 ), EVs mesoglycan (1 × 10 6 ), BOC1 (100 μ m ) and vesicles and BOC1 (100 μ m ) together. The cells were fixed and labelled with antibody against VE‐cadherin (panels a–f), fibronectin (panels m–r), FAK (panels s–x, and relative ×4 zoom) and with phalloidin (panels g–l). Nuclei were stained with Hoechst 33342 1 : 1000 for 30 min at room temperature (RT) in the dark. Magnification 63 × 1.4 NA. Scale bar = 50 μm. (B) Fluorescence intensity for p‐VEGFR2, VE‐cadherin, F‐actin, FAK and fibronectin signals on HUVEC cells using ImageJ software. The measurements are determined on ten field images from a single coverslip and randomly selected for three coverslips. * P < 0.05, vs. untreated control.

Article Snippet: After removing incubation medium and washing with PBS, cell cultures were incubated in the presence of mesoglycan (0.3 mg·mL −1 , LDO, Laboratori Derivati Organici spa, Trino, Italy), VEGF (10 ng·mL −1 , VEGF‐165 recombinant human for HUVEC cells and VEGF‐164 recombinant mouse for MLEC, R&D Systems, Minneapolis, MN, USA), BOC1 (100 μM, Bachem AG, Bubendorf, Switzerland) and/or EVs ctrl (1 × 10 6 ) and EVs mesoglycan (1 × 10 6 ), both isolated from HUVEC.

Techniques: Immunofluorescence, Staining, Fluorescence, Software

Journal: Nature Communications

Article Title: A common wild rice-derived BOC1 allele reduces callus browning in indica rice transformation

doi: 10.1038/s41467-019-14265-0

Figure Lengend Snippet: a qCBT3 was initially localized to a region between markers RM3131 and RM3766 on the short arm of chromosome 3 ( n = 198 plants of F 2 populations derived from a cross between YIL25 and Teqing). Scale bar, 500 kb. b Positional cloning narrowed the qCBT3 locus to an 18.6 kb region between markers SN11 and SN13 ( n = 6377 individuals used for high-resolution mapping). R1 through R7 are recombinants. The black, white, and gray regions indicate homozygous for the YIL25 genome, homozygous for the Teqing genome, and the interval in the chromosome where crossover took place, respectively. Scale bar, 10 kb. LOC_Os03g12820 is the candidate gene BROWNING OF CALLUS 1 ( BOC1 ). Data are means ( n = 3 biologically independent samples), with error bars showing standard error. c Structure of the candidate gene BOC1 . White box, white box with arrow, black boxes, and lines between boxes indicate the 5′-UTR, 3′-UTR, exons, and introns, respectively. d Mutation sites of the BOC1 promoter in YIL25 and Teqing. The ellipsis indicates deletion of bases. Scale bar, 50 bp. Source data underlying Figs. a , b are provided as a Source Data file.

Article Snippet: Amino acid sequences homologous to BOC1 were downloaded from the National Center for Biotechnology Information website ( http://blast.ncbi.nlm.nih.gov/ ).

Techniques: Derivative Assay, Cloning, Mutagenesis

Journal: Nature Communications

Article Title: A common wild rice-derived BOC1 allele reduces callus browning in indica rice transformation

doi: 10.1038/s41467-019-14265-0

Figure Lengend Snippet: a – d Phenotypes of calli from pRi- BOC1 -YIL25 transgenic plants ( n = 18). Negative represents segregating plants from the transformation process but without the transgene. Scale bars, 1 cm. e Comparison of the relative expression levels and CBI between a negative transgenic line and 10 RNAi lines ( n = 3 biological replicates). f – i Phenotypes of calli from pCPL- BOC1 -Teqing transgenic plants ( n = 15). Scale bars, 1 cm. j Comparison of the relative expression levels and CBI in a negative transgenic line and 10 complementation lines ( n = 3 biological replicates). k – n Phenotypes of calli from pOE- BOC1 -Teqing transgenic plants ( n = 22). o Comparison of the relative expression levels and CBI in a negative transgenic line and 10 overexpression lines ( n = 3 biological replicates). The black line indicates the trend of the relative expression levels, and the gray line indicates the trend of the CBI. Scale bars, 1 cm. Data are means ± SE, two-tailed Student’s t -tests. Source data underlying Figs. e , j , o are provided as a Source Data file.

Article Snippet: Amino acid sequences homologous to BOC1 were downloaded from the National Center for Biotechnology Information website ( http://blast.ncbi.nlm.nih.gov/ ).

Techniques: Transgenic Assay, Transformation Assay, Comparison, Expressing, Over Expression, Two Tailed Test

Journal: Nature Communications

Article Title: A common wild rice-derived BOC1 allele reduces callus browning in indica rice transformation

doi: 10.1038/s41467-019-14265-0

Figure Lengend Snippet: a Expression analysis of BOC1 in calli at different stages of subculture. Data are means, with bars showing SE, n = 3 biological replicates. Two-tailed Student’s t -tests were performed to determine significant differences. b BOC1 expression patterns in Teqing and YIL25 calli subcultured for 21 days, as revealed by mRNA in situ hybridization. Thirty calli each from Teqing and YIL25 were collected for mRNA in situ hybridization. Scale bars, 100 μm. c Transient expression assays of the effects of InDels and SNPs in the BOC1 promoter. Top, structure of the pGreenII 0800- LUC vector. Left, constructs with site-directed mutagenesis of the BOC1 promoter. Luc , the target promoter controlling the firefly luciferase reporter gene. The Renilla ( Ren ) luciferase reporter gene was used as an internal control. M1 to M5 indicate the constructs with site-directed mutations at the 337 bp InDel, three SNPs, and 1 bp InDel. Right, expression levels of Luc relative to Ren . Data are presented as mean ± SE. Horizontal bars show the SE for all constructs ( n = 10 biological replicates, two-tailed Student’s t -tests). d Subcellular localization of BOC1 protein in rice protoplast cells. OsMADS15 is a nuclear localization marker. Scale bars, 20 μm. P -values of ( a , c ) were calculated with two-tailed Student’s t -tests. The subcellular localization assays were performed with three biologically independent experiments. Source data underlying Figs. a , c are provided as a Source Data file.

Article Snippet: Amino acid sequences homologous to BOC1 were downloaded from the National Center for Biotechnology Information website ( http://blast.ncbi.nlm.nih.gov/ ).

Techniques: Expressing, Two Tailed Test, In Situ Hybridization, Plasmid Preparation, Construct, Mutagenesis, Luciferase, Control, Marker

Journal: Nature Communications

Article Title: A common wild rice-derived BOC1 allele reduces callus browning in indica rice transformation

doi: 10.1038/s41467-019-14265-0

Figure Lengend Snippet: a – c Transformed calli after three rounds of selection on Hyg- resistant UINB medium. Scale bars, 1 cm. d Comparison of the selection frequency of Hyg -resistant callus. Each dot represents the selective frequency of Hyg -resistant callus of 10 independent experiments. ( e ) Comparison of the transformation efficiency. Each diamond represents the transformation efficiency of 10 independent experiments. Teqing 1300, Teqing calli were infected with Agrobacterium harboring the binary pCAMBIA1300 empty vector as a control. YIL25 1300, YIL25 calli transformed with the binary vector pCAMBIA1300. Teqing pCPL, Teqing calli infected with Agrobacterium harboring the pCAMBIA1300- BOC1 construct. The hollow box in each column in ( d , e ) represents the means of the selective frequency of Hyg -resistant callus and the transformation efficiency of 10 replicates, respectively. Box edges represent the 0.25 and 0.75 quantiles, with the median values shown by red, blue and yellow lines, respectively. Whiskers extend to data no more than 1.5 times the interquartile range, and the remaining data are indicated by dots. P -values of ( d , e ) were determined by two-tailed Student’s t -tests. Source data are provided as a Source Data file.

Article Snippet: Amino acid sequences homologous to BOC1 were downloaded from the National Center for Biotechnology Information website ( http://blast.ncbi.nlm.nih.gov/ ).

Techniques: Transformation Assay, Selection, Comparison, Infection, Plasmid Preparation, Control, Construct, Two Tailed Test

Journal: Nature Communications

Article Title: A common wild rice-derived BOC1 allele reduces callus browning in indica rice transformation

doi: 10.1038/s41467-019-14265-0

Figure Lengend Snippet: a Association testing of callus browning in 50 O . rufipogon accessions vs. 16 variant sites in the 4.6 kb genomic region. Black dots represent 16 variations. b Comparison of CBI in calli subcultured for 21 d in 31 accessions with the 337 bp deletion vs. 19 accessions with the 337 bp insertion. c Comparison of BOC1 expression in calli subcultured for 21 d in lines harboring the 337 bp deletion vs. the 337 bp insertion ( n = 3 biological replicates). d Association testing of callus browning in 74 Asian rice cultivars with 36 variant sites in the 4.6 kb genomic region. e Comparison of CBI in calli subcultured for 21 d between 59 accessions with the T deletion and 15 accessions with the T insertion. f Comparison of BOC1 expression in callus subcultured for 21 d from rice varieties harboring the T deletion vs. the T insertion ( n = 3 biological replicates). Box edges of ( b , c ) and ( e , f ) represent the 0.25 quantile and 0.75 quantile with the median values shown by red and blue lines. Whiskers extend to data no more than 1.5 times the interquartile range, and remaining data are indicated by dots. P -values of ( b , c ) and ( e , f ) were calculated by two-tailed Student’s t -tests. Source data underlying Figs. b , c , e , f are provided as a Source Data file.

Article Snippet: Amino acid sequences homologous to BOC1 were downloaded from the National Center for Biotechnology Information website ( http://blast.ncbi.nlm.nih.gov/ ).

Techniques: Variant Assay, Comparison, Expressing, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting ANXA1 abrogates Treg-mediated immune suppression in triple-negative breast cancer

doi: 10.1136/jitc-2019-000169

Figure Lengend Snippet: RNA-Seq data of downstream signaling after block ANXA1. (A) Heat map (wild type vs Boc1). (B) Volcano plot (wild type vs Boc1). (C) GO enrichment analysis. (D) KEGG enrichment analysis. (E) qPCR tests. All data represent mean±SD; all data represent mean±SD. *p<0.05, **p<0.01, ***p<0.001 as determined by Student’s t-test.

Article Snippet: Boc1 ((1R,2R)-trans-N-Boc-1,2-cyclohexanediamine, 50 μM, Selleck, S3896) was added to the experiment group, and the same dose of solvent was added to the control group.

Techniques: RNA Sequencing, Blocking Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting ANXA1 abrogates Treg-mediated immune suppression in triple-negative breast cancer

doi: 10.1136/jitc-2019-000169

Figure Lengend Snippet: ANXA1 blocker has no effect on apoptosis rate of 4T1 cells. (A) Apoptosis rate of 4T1 treated with Boc1 (20 µM) in vitro. (B) Apoptosis rate of 4T1 treated with Boc1 (50 µM) in vitro. (C) Apoptosis rate of 4T1 treated with Boc1 (100 µM) in vitro. (D) Statistical analysis of apoptosis rates. Data are presented as mean±SD (n=3). (E) Transwell migration assays investigated the migration capacity of 4T1 cells treated with DMSO or Boc1 (50 μM) for 24 hours. (F) Statistical analysis of migrated cells. Data are presented as mean±SD (n=3). ns (no statistical significance) as determined by Student’s t-test. (G) The dose-dependent cytotoxicity induced by Boc1 on 4T1. Data are presented as mean±SD (n=3).

Article Snippet: Boc1 ((1R,2R)-trans-N-Boc-1,2-cyclohexanediamine, 50 μM, Selleck, S3896) was added to the experiment group, and the same dose of solvent was added to the control group.

Techniques: In Vitro, Migration

Journal: International Journal of Molecular Sciences

Article Title: Annexin-1 Mediates Microglial Activation and Migration via the CK2 Pathway during Oxygen–Glucose Deprivation/Reperfusion

doi: 10.3390/ijms17101770

Figure Lengend Snippet: The extent of microglial activation induced by oxygen–glucose deprivation/reperfusion (OGD/R) was altered by Ac2-26 and Boc1 treatment. ( A – I ) Examples of neuron-specific nuclear protein (NeuN)-labeled neurons ( green ) and ionized calcium binding adapter molecule 1 (Iba1)-labeled microglia ( blue ). Scale bar: 50 μm; ( J ) after OGD/R treatment, the number of Iba1-positive particles was increased by Ac2-26 treatment (1 μM) and significantly decreased by Boc1 (4 μM) in CA1. The data are expressed as the mean ± standard error of the mean (SEM) ( n = 6–8). * p < 0.05.

Article Snippet: The non-selective FPR antagonist Boc1 (N-t-Boc-Phe-Leu-Phe-Leu-Phe) was purchased from Calbiochem (San Diego, CA, USA).

Techniques: Activation Assay, Labeling, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Annexin-1 Mediates Microglial Activation and Migration via the CK2 Pathway during Oxygen–Glucose Deprivation/Reperfusion

doi: 10.3390/ijms17101770

Figure Lengend Snippet: Annexin-1 (ANXA1) mediates BV-2 migration via formyl peptide receptors (FPRs). ( A ) Photographs illustrating the effect of Ac2-26 (1.32 μM) and Boc1 (10 μM) on BV-2 cell migration in trans-well migration assays. Photographs were taken from the lower sides of the chamber’s membrane. Violet particles represent cresyl violet-stained BV-2 cells; ( B ) the relative migration ratio as a percentage relative to the control group; ( C ) the relative migration ratio as a percentage relative to the control + dimethyl sulfoxide (DMSO) group; ( D ) photographs illustrating the effect of Ac2-26 (1.32 μM) and Boc1 (10 μM) on BV-2 cell migration in scratch wound assays. Cells between the two white dotted lines were considered migrating cells; ( E ) the relative number of migrating cells (NMC) as a percentage relative to the control group; ( F ) the relative number of migrating cells (NMC) as a percentage relative to the control + DMSO group. BV-2 cells were cultivated under normal conditions for these experiments. All data are expressed as the mean ± standard error of the mean (SEM) ( n = 5–15). * p < 0.05, ** p < 0.01 (compared to the control group), # p < 0.05.. Scale bar: 40 μm.

Article Snippet: The non-selective FPR antagonist Boc1 (N-t-Boc-Phe-Leu-Phe-Leu-Phe) was purchased from Calbiochem (San Diego, CA, USA).

Techniques: Migration, Membrane, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: Annexin-1 Mediates Microglial Activation and Migration via the CK2 Pathway during Oxygen–Glucose Deprivation/Reperfusion

doi: 10.3390/ijms17101770

Figure Lengend Snippet: Annexin-1 (ANXA1) promotes BV-2 migration via formyl peptide receptors (FPRs) during oxygen–glucose deprivation/reperfusion (OGD/R). ( A ) Photographs illustrating the effect of OGD/R, OGD/R with Ac2-26 (1.32 μM), or Boc1 (10 μM) on the migration of BV-2 cells in trans-well migration assays. Photographs were taken from the lower sides of the chamber’s membrane, and the violet particles represent BV-2 cells; ( B ) the relative migration ratio as a percentage relative to the control group; ( C ) the relative migration ratio as a percentage relative to the OGD/R + dimethyl sulfoxide (DMSO) group; ( D ) photographs illustrating the effect of OGD/R, OGD/R with Ac2-26 (1.32 µM), or Boc1 (10 μM) on the migration of BV-2 cells in scratch wound assays. The particles between the two white dotted lines were considered migrating cells; ( E ) the relative number of migrating cells (NMC) as a percentage relative to the control group; ( F ) the relative number of migrating cells (NMC) as a percentage relative to the OGD/R + DMSO group; All data are expressed as the mean ± standard error of the mean (SEM) ( n = 5–10). * p < 0.05 (compared to the first group). ** p < 0.01 (compared to the control group). ## p < 0.01. Scale bar: 40 μm.

Article Snippet: The non-selective FPR antagonist Boc1 (N-t-Boc-Phe-Leu-Phe-Leu-Phe) was purchased from Calbiochem (San Diego, CA, USA).

Techniques: Migration, Membrane, Control