Journal: BMC Genomics

Article Title: The role of nitric oxide during embryonic wound healing

doi: 10.1186/s12864-019-6147-6

Figure Lengend Snippet: Production of NO during wound healing and regeneration. ( a ) Control embryos at stage 26 were injured using a needle, or tails of tadpoles at stage 41 were amputated and incubated in media with DAF-2DA solution for 15 minutes, fixed and imaged. ( b ) NO is produced in the first two layers of cells around wound edge (Scale bar = 20 μm). ( c , d ) NO is produced mainly during first 15 minutes after injury in embryos at stage 26 (Scale bar = 100 μm, five replicates, mean with standard deviation, One-way ANOVA Dunnett’s multiple comparisons test) ( e , f ) and after amputation in embryos at stage 41. (Scale bars = 200 μm, three replicates, mean with standard deviation, One-way ANOVA Dunnett’s multiple comparisons test) ( g ) NO is not produced after injury at stage 1 and stage 5, but NO is produced after injury at stage 8 (blastula), stage 11 (gastrula), stage 14 (early neurula) and stage 20 (late neurula) (Scale bar = 500 μm). CTF – corrected total fluorescence, RFU – relative fluorescent unit, pw – post wounding, pa – post amputation **** - p < .0001, * - p < .05, n.s. - p > .05

Article Snippet: The statistical significance was calculated relative to the control using GraphPad Prism 7 One-way ANOVA with Dunnett’s multiple comparisons test.

Techniques: Control, Incubation, Produced, Standard Deviation, Fluorescence

Journal: BMC Genomics

Article Title: The role of nitric oxide during embryonic wound healing

doi: 10.1186/s12864-019-6147-6

Figure Lengend Snippet: Changes in gene expression during wound healing after inhibition of NO production. ( a ) Graphical description of RNA-Seq experiment comparing control and NO inhibited embryonic wound healing. Only the part marked by red rectangle was collected and used for RNA isolation and sequencing. ( b - g ) DEGs, which were identified in RNA-Seq, were grouped based on their expression profile relatively to 0 minutes and GO analysis was performed. ( b , d , f ) Expression profiles of genes are representative of the z-score of the regularized log transformation of the normalized counts. ( c , e , g ) Genes with annotation and human homolog were used for GO analysis. Numbers of analysed genes are in the table together with the representative GO terms for each group. ( h ) RNA-Seq result of lep expression was verified ( i ) using RT-qPCR, separately for nos1 -MO and nos3 -MO. ( j ) Similarly, RNA-Seq result of fos expression was verified using ( k ) RT-qPCR (data are normalized to 0 minutes pw in controls, three replicates, geometric mean with geometric standard deviation, two-sided t-test from log2 values of relative expression between inhibited samples and control in 120 minutes pw), and ( l ) in situ hybridization. Site of injury is marked with a star and the signal where fos is expressed is circled by dot line (Scale bar = 100 μm) (M) Intensity of blue signal around site of injury were measured (one-way Anova, Dunnett’s multiple comparisons test, minimum 8 replicates). **** - p < .0001, ** - p < .01, * - p < .05, n.s. - p > .05 DEGs – differentially expressed genes, pw – post wounding, RIU – relative intensity unit

Article Snippet: The statistical significance was calculated relative to the control using GraphPad Prism 7 One-way ANOVA with Dunnett’s multiple comparisons test.

Techniques: Gene Expression, Inhibition, RNA Sequencing, Control, Isolation, Sequencing, Expressing, Transformation Assay, Quantitative RT-PCR, Standard Deviation, In Situ Hybridization

Journal: BMC Genomics

Article Title: The role of nitric oxide during embryonic wound healing

doi: 10.1186/s12864-019-6147-6

Figure Lengend Snippet: Monitoring of phenotype changes during wound healing in embryos with inhibited NO production. ( a , b , c ) Control embryos, embryos with inhibited production of NO using TRIM 1 hour before injury and embryos injected with mixture of nos1 + nos3 - MO were injured at stage 26 using forceps or needle in the middle and ventral side. ( d ) Laminin layer was visualized at 180 minutes and 360 minutes pw and ends of the laminin layer are marked by a triangle. Formation of “blob” in TRIM embryos is marked by arrow (Scale bar = 100 μm). ( e ) Staining of β- catenin 360 minutes pw (Scale bar = 100 μm). ( f ) Brightfiled image of wound site in 180 minutes pw (Scale bar = 100 μm). ( b , g ) Actin at 30, 60 and 180 minutes pw visualized using green fluorescent phalloidin. Breaks in actin layer are marked by arrow (Scale bar = 100 μm). ( c , h ) Collagen staining at 60 minutes pw. The beginning of the wound is marked by a red triangle. A red arrow marks the end of the collagen layer, while the end of the wound site is marked by a red star. (Scale bar = 100 μm, measurement of coverage of collagen in wound was made from at least six embryos per condition and at least five slices per embryo, one-way anova, Dunnett’s multiple comparisons test). ( i ) Spatial expression of two matrix metalloproteinases mmp7 and mmp9 was visualized by in situ hybridization in time 360 minutes pw (Scale bars = 500 μm). ( j ) RT-qPCR comparison of temporal expression profiles of mmp1 , mmp8 , mmp7 and mmp9 (data are normalized to 0 minutes pw in controls, three replicates, geometric mean with geometric standard deviation, two-sided ttest from log2 values of relative expression between 360 minutes and 0 minutes). **** - p < .0001, *** - p < .001, ** - p < .01, n.s. - > .05 pw – post wounding

Article Snippet: The statistical significance was calculated relative to the control using GraphPad Prism 7 One-way ANOVA with Dunnett’s multiple comparisons test.

Techniques: Control, Injection, Staining, Expressing, In Situ Hybridization, Quantitative RT-PCR, Comparison, Standard Deviation