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

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: CRISPR/Cas9 Editing of Glia Maturation Factor Regulates Mitochondrial Dynamics by Attenuation of the NRF2/HO-1 Dependent Ferritin Activation in Glial Cells

doi: 10.1007/s11481-019-09833-6

Figure Lengend Snippet: Effect of GMF on MPP+ induced oxidative stress in BV2 microglial cells. The BV2 and BV2-G microglial cells were seeded (3×106) in a 96-well plate. Then the cells were pre-incubated with DCFH-DA dye for 45 min and the cells were treated with MPP+ (0.1 mM) for 24 h. After the incubation period, cells were used to measure ROS intensity on a fluorescence microscope. Representative images show the toxic effect of MPP+ induced ROS intensity (green fluorescence; A). Bar graphs show the green fluorescence intensity significantly reduced in GMF deficient BV2-G cells as compared with BV2 microglial cells (B). Statistical significance was assessed by independent student t-test using GraphPad InStat prism-7 software. The p value less than <0.05 was considered as statistically significant in all the experiments; Values are given as mean ± SEM of three experiments in each group. p<0.001 and p<0.01 untreated controls vs MPP+-treated cells; p<0.05 MPP+-treated BV2 cells vs MPP+-treated BV2-G cells. Scale bar 100 µm. AU arbitrary unit.

Article Snippet: Statistical significance was assessed by independent student t -test using GraphPad InStat prism-7 software. p<0.001 and p<0.01 untreated controls vs MPP + -treated cells; p<0.01 and p<0.05 MPP + -treated BV2 cells vs MPP + -treated BV2-G cells.

Techniques: Incubation, Fluorescence, Microscopy, Software

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: CRISPR/Cas9 Editing of Glia Maturation Factor Regulates Mitochondrial Dynamics by Attenuation of the NRF2/HO-1 Dependent Ferritin Activation in Glial Cells

doi: 10.1007/s11481-019-09833-6

Figure Lengend Snippet: Effect of GMF on MPP+ induced apoptosis in BV2 microglial cells. The BV2 and BV2-G cells were seeded (3×106) in a 96-well plate. The cells were treated with MPP+ (0.1 mM) for 24 h. After the incubation period, cells were stained with EtBr/AO solution (1:1 v/v) at a final concentration of 100 µg/ml for 5 min and finally imaged under fluorescence microscope. Images represents the toxic effect of MPP+ -induced apoptotic cell death (dark orange red; Fig 2A). Bar graphs show that apoptotic changes were significantly reduced in BV2-G microglial cells as compared with BV2 cells (B). Values are given as mean ± SEM of three experiments in each group. Statistical significance was assessed by independent student t-test using GraphPad InStat prism-7 software. p<0.001 untreated controls vs MPP+-treated cells; p<0.01 MPP+-treated BV2 cells vs MPP+-treated BV2-G cells. The p value less than <0.05 was considered as statistically significant in all the experiments; Scale bar 100 µm.

Article Snippet: Statistical significance was assessed by independent student t -test using GraphPad InStat prism-7 software. p<0.001 and p<0.01 untreated controls vs MPP + -treated cells; p<0.01 and p<0.05 MPP + -treated BV2 cells vs MPP + -treated BV2-G cells.

Techniques: Incubation, Staining, Concentration Assay, Fluorescence, Microscopy, Software

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: CRISPR/Cas9 Editing of Glia Maturation Factor Regulates Mitochondrial Dynamics by Attenuation of the NRF2/HO-1 Dependent Ferritin Activation in Glial Cells

doi: 10.1007/s11481-019-09833-6

Figure Lengend Snippet: Effect of GMF on MPP+ induced NRF2 translocation in BV2 microglial cells. The BV2 and BV2-G cells were seeded in a T25 culture flask and cultured under standard conditions. Cells were incubated with MPP+ (0.1 mM) for 24 h. After the incubation period, cells were washed with PBS lysed and cytosolic and nuclear fractions separated. Aliquots were subjected to western blot analysis (A). Decreased nuclear and increased cytosolic expression level of NRF2 was found in BV2-G cells as compared with BV2 cells. Bar graphs shows the mean densitometry analysis of bands after normalizing with β-actin as a loading control of each group (B and C). Values are given as mean ± SEM of three experiments in each group. Statistical significance was assessed by independent student t-test using GraphPad InStat prism-7 software. p<0.001 untreated controls vs MPP+-treated cells; p<0.05 and p<0.001 MPP+-treated BV2 cells vs MPP+-treated BV2-G cells. The p value less than <0.05 was considered as statistically significant in all the experiments.

Article Snippet: Statistical significance was assessed by independent student t -test using GraphPad InStat prism-7 software. p<0.001 and p<0.01 untreated controls vs MPP + -treated cells; p<0.01 and p<0.05 MPP + -treated BV2 cells vs MPP + -treated BV2-G cells.

Techniques: Translocation Assay, Cell Culture, Incubation, Western Blot, Expressing, Software

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: CRISPR/Cas9 Editing of Glia Maturation Factor Regulates Mitochondrial Dynamics by Attenuation of the NRF2/HO-1 Dependent Ferritin Activation in Glial Cells

doi: 10.1007/s11481-019-09833-6

Figure Lengend Snippet: Effect of GMF on MPP+ induced COX2 and NOS2 expression in BV2 microglial cells. The BV2 and BV2-G microglial cells were seeded in a T25 culture flask. The cells were incubated with MPP+ (0.1 mM) for 24 h. After the incubation period, cells were washed with PBS and cell lysates were prepared from these cells for western blot studies. Decreased expression of COX2 and NOS2 were found in BV2-G cells when compared with BV2 microglial cells (A). Bar graphs show the mean densitometry analysis of bands after normalizing with β-actin as a loading control of each group (B and C). Values are given as mean ± SEM of three experiments in each group. Statistical significance was assessed by independent student t-test using GraphPad InStat prism-7 software. p<0.001 and p<0.01 untreated controls vs MPP+-treated cells; p<0.01 and p<0.05 MPP+-treated BV2 cells vs MPP+-treated BV2-G cells. The p value less than <0.05 was considered as statistically significant in all the experiments.

Article Snippet: Statistical significance was assessed by independent student t -test using GraphPad InStat prism-7 software. p<0.001 and p<0.01 untreated controls vs MPP + -treated cells; p<0.01 and p<0.05 MPP + -treated BV2 cells vs MPP + -treated BV2-G cells.

Techniques: Expressing, Incubation, Western Blot, Software

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: CRISPR/Cas9 Editing of Glia Maturation Factor Regulates Mitochondrial Dynamics by Attenuation of the NRF2/HO-1 Dependent Ferritin Activation in Glial Cells

doi: 10.1007/s11481-019-09833-6

Figure Lengend Snippet: Effect of GMF on MPP+ induced HO-1 and ferritin expression in BV2 microglial cells. The BV2 and BV2-G cells were seeded in a T25 culture flask and cultured under standard conditions. The cells were incubated with MPP+ (0.1 mM) for 24 h. After the incubation period, cells were washed with PBS and prepared for western blot analysis. Decreased HO-1 and ferritin expressions were found in BV2-G cells as compared with BV2 cells (A). Bar graphs show the mean densitometry analysis of bands after normalizing with β-actin as a loading control of each group (B and C). Values are given as mean ± SEM of three experiments in each group. Statistical significance was assessed by independent student t-test using GraphPad InStat prism-7 software. p<0.001 untreated controls vs MPP+-treated cells; p<0.05 MPP+-treated BV2 cells vs MPP+-treated BV2-G cells. The p value less than <0.05 was considered as statistically significant in all the experiments.

Article Snippet: Statistical significance was assessed by independent student t -test using GraphPad InStat prism-7 software. p<0.001 and p<0.01 untreated controls vs MPP + -treated cells; p<0.01 and p<0.05 MPP + -treated BV2 cells vs MPP + -treated BV2-G cells.

Techniques: Expressing, Cell Culture, Incubation, Western Blot, Software

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: CRISPR/Cas9 Editing of Glia Maturation Factor Regulates Mitochondrial Dynamics by Attenuation of the NRF2/HO-1 Dependent Ferritin Activation in Glial Cells

doi: 10.1007/s11481-019-09833-6

Figure Lengend Snippet: Effect of GMF on MPP+ induced HO-1 and ferritin expression in BV2 microglial cells. The BV2 and BV2-G microglial cells were seeded on poly-D-lysine coated coverslips. The cells were incubated with MPP+ (0.1 mM) for 24 h. After the incubation period, cells were washed with PBS and processed for immunocytochemistry to detect HO-(A) and ferritin (D) co-localization along with NRF2 expression. Quantitatively decreased total positive area and total average intensity of HO-1 (B and C) and ferritin (E and F) along with NRF2 translocation to nuclear site were found in BV2-G cells as compared with BV2 microglial cells. Statistical significance was assessed by independent student t-test using GraphPad InStat prism-7 software. p<0.05 untreated controls vs MPP+-treated cells; p<0.05 MPP+-treated BV2 cells vs MPP+-treated BV2-G cells. The p value less than <0.05 was considered as statistically significant in all the experiments; Scale bar 100 µm; au arbitrary units

Article Snippet: Statistical significance was assessed by independent student t -test using GraphPad InStat prism-7 software. p<0.001 and p<0.01 untreated controls vs MPP + -treated cells; p<0.01 and p<0.05 MPP + -treated BV2 cells vs MPP + -treated BV2-G cells.

Techniques: Expressing, Incubation, Immunocytochemistry, Translocation Assay, Software