Journal: Vaccines

Article Title: Expression of the Reverse Transcriptase Domain of Telomerase Reverse Transcriptase Induces Lytic Cellular Response in DNA-Immunized Mice and Limits Tumorigenic and Metastatic Potential of Murine Adenocarcinoma 4T1 Cells

doi: 10.3390/vaccines8020318

Figure Lengend Snippet: Immunization of BALB/c mice with TERT DNA, TERT variant with a C-terminal hemagglutinin tag (TERT-HA DNA), and empty vector followed by booster immunization with given plasmids mixed with DNA encoding firefly luciferase (Luc DNA), with follow up of luciferase expression by in vivo imaging. Scheme of the immunization ( A ); results of in vivo bioluminescence imaging of booster sites at days 1–12 post injection, example of 3 mice—one from TERT, one from TERT-HA, and one from empty vector group ( B ); dynamics of bioluminescence signal change in TERT, TERT-HA DNA, and empty vector-immunized mice on days 1–12 post booster injection; each line of different colors corresponds to one site of injection (two per mouse) ( C ); relative average level of bioluminescence signal for each group on days 1 to 12 post booster injection ( D ). Bioluminescence signal is represented by the total flux from site of immunization, mean ± SD. Analyzed by ordinary two-way ANOVA with Dunnett’s multiple comparison test, ** − p < 0.01; **** − p < 0.0001; ns—not significant.

Article Snippet: Total photon flux from the site of coinjection of TERT DNA or TERT-HA DNA and Luc DNA was compared using ordinary two-way ANOVA with Dunnett’s multiple comparison test (GraphPad Prism 6, San Diego, CA, USA).

Techniques: Variant Assay, Plasmid Preparation, Luciferase, Expressing, In Vivo Imaging, In Vivo, Imaging, Injection, Comparison

Journal: Vaccines

Article Title: Expression of the Reverse Transcriptase Domain of Telomerase Reverse Transcriptase Induces Lytic Cellular Response in DNA-Immunized Mice and Limits Tumorigenic and Metastatic Potential of Murine Adenocarcinoma 4T1 Cells

doi: 10.3390/vaccines8020318

Figure Lengend Snippet: Generation of tumors by 4T1luc2 clones expressing rtTERT. Tumor growth rate was assessed using total fluorescence signal from the site of injection of 2500 ( A ), 5000 ( B ), and 10,000 ( C ) cells. Tumor volume was evaluated by total fluorescence signal from the site of cell injection by day 16 ( D ) or by calipering at day 21 ( E ). Histochemical characterization of the solid tumors formed by the parental 4T1luc2 cells ( F ) and their derivatives expressing rtTERT 4T1luc2_rtTERT_C6 ( G ); 4T1luc2_rtTERT_H9 ( H ) after ectopic implantation into BALB/c mice (H&E staining, magnification 200×). Results of tumor growth ( A – C ) were analyzed using RM two-way ANOVA with Dunnett’s multiple comparison test. Data on tumor volumes ( D , E ) were analyzed using Kruskal–Wallis with Dunn’s multiple comparison test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns—not significant.

Article Snippet: Total photon flux from the site of coinjection of TERT DNA or TERT-HA DNA and Luc DNA was compared using ordinary two-way ANOVA with Dunnett’s multiple comparison test (GraphPad Prism 6, San Diego, CA, USA).

Techniques: Clone Assay, Expressing, Fluorescence, Injection, Staining, Comparison

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