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

Journal: Cell Death & Disease

Article Title: The MEF2A transcription factor interactome in cardiomyocytes

doi: 10.1038/s41419-023-05665-8

Figure Lengend Snippet: A PCMs were transfected with a Flag-MEF2A construct and lysates were assessed for expression by Western blotting. Flag-MEF2A lysates were used for IP using α-Flag magnetic beads and the eluates were blotted with anti-Flag and STAT3 antibodies. IP with lysates from non-transfected PCMs were used as negative controls (Left panel). Non-transfected PCM lysates were used for IP using MEF2A antibody to confirm the interaction between endogenous MEF2A and RBPMS. IP with IgG was used as controls (Top right). Non-transfected PCM lysates were used for IP using a STAT3 antibody IP to confirm the interaction between endogenous STAT3 and MEF2A. IP with IgG was used as control (Bottom right). Number of biological replicates carried out for the Western blot and IP data is n = 3. B Confocal immunofluorescence analysis of endogenous MEF2A and STAT3 indicates a nuclear localization in PCMs. PCMs were fixed and stained for MEF2A in red and counterstained for STAT3 in green. Slides were analyzed by confocal microscopy. The scale bars are 2 and 10 μm. The intensity blot of MEF2A and STAT3 signals is shown over the region shown by the yellow line in the merged image. C STAT3 and MEF2A expressing vectors alone and in combination were ectopically expressed in PCMs along with a 4xMEF2 luciferase reporter gene. Renilla luciferase was used to normalize for transfection efficiency. Lysates were collected at 48 h. The luciferase activity under each condition was measured and normalized to Renilla values to determine the fold change. D PCMs were transiently transfected with 4xMEF2 luciferase reporter gene for 48 h and then treated with STAT3 inhibitor (C188-9;10 µM) for 1 h in serum free medium. The control cells were treated by the solvent (DMSO) in serum free medium. Representative western blots of three independent biological replicates are presented which were carried out to assess the STAT3 inhibitor effect as compared to control condition. A schematic of the 4xMEF2A-Luc construct is shown below the luciferase data. Each condition in the luciferase reporter assay is compared to the respective control condition for three independently transfected samples (technical replicates) to determine the fold change. Each dot represents one biological replicate and corresponds to the mean of three technical replicates. The error bars represent standard error of the mean (SEM). Tukey’s multiple comparisons test in one-way ANOVA and independent two sample t -test using GraphPad Prism 8.0 were used to test for statistical significance. Adjusted p -value *≤0.05, **≤0.01, ***≤0.001****<0.0001, comparing to control.

Article Snippet: Tukey’s Multiple comparisons test in one-way ANOVA using GraphPad Prism 8.0 was used to test for statistical significance.

Techniques: Transfection, Construct, Expressing, Western Blot, Magnetic Beads, Immunofluorescence, Staining, Confocal Microscopy, Luciferase, Activity Assay, Reporter Assay

Journal: Cell Death & Disease

Article Title: The MEF2A transcription factor interactome in cardiomyocytes

doi: 10.1038/s41419-023-05665-8

Figure Lengend Snippet: A PCMs were transfected with a 4xMEF2 luciferase reporter gene along with two independent siRNAs, siSTAT3#1 and #2, to deplete the endogenous STAT3 levels or a scrambled siRNA was used as control. Renilla luciferase was used to normalize for transfection efficiency. Lysates were collected at 48 h. The luciferase activity under each condition was measured and normalized to Renilla values to determine the fold change (Top panel). Corresponding western blot analysis using equal amounts of total protein of the cell lysates were used to confirm siRNA mediated STAT3 depletion as compared to scrambled controls (Bottom panel). Number of biological replicates for western blotting was n = 3. A schematic of 4xMEF2A-Luc construct is shown below the luciferase results. B PCMs were transfected with an α-MHC-Luc reporter gene with two independent siRNAs, siSTAT3#1 and #2, or scrambled siRNA as control. Luciferase activity was assessed after 48 h and normalized to Renilla activity (Top panel). Corresponding western blot analysis of cell lysate to confirm the depletion of STAT3 protein level as compared to the control is shown (Bottom panel). Number of biological replicates for this analysis was n = 3. Schematic of α-MHC-Luc construct is shown below the luciferase data. C PCMs were transiently transfected with 2xSTAT-Luc reporter gene with two independent siRNAs, siMEF2A#1 and #2, or scrambled siRNA as control. Luciferase activity was assessed after 48 h and normalized to Renilla activity (Top panel). Corresponding western blot analysis of cell lysate to confirm the depletion of MEF2A protein level as compared to the control (Bottom panel). Number of biological replicates carried out for western blotting was n = 3. A schematic of the 2xSTAT-Luc construct used is shown below the luciferase data. D PCMs were transiently transfected with a Flag-HDAC3 construct and lysates were assessed for protein expression by Western blotting. Flag-MEF2A lysate was used for IP using α-Flag magnetic beads and the eluates were blotted with anti-Flag and STAT3 antibodies. IP with lysate from non-transfected PCMs were used as negative controls. Number of biological replicates for western blot and IP carried out was n = 3. Each condition in the luciferase reporter assay is compared to the control condition for three independently transfected samples (technical replicates) to determine the fold change. Each dot represents one biological replicate and corresponds to the mean of three technical replicates. The error bars represent standard error of the mean (SEM). Dunnett’s multiple comparisons test in one-way analysis of variance using GraphPad Prism 8.0 was used to test for statistical significance. Adjusted p -value *≤0.05, **≤0.01, ***≤0.001****<0.0001, comparing to control.

Article Snippet: Tukey’s Multiple comparisons test in one-way ANOVA using GraphPad Prism 8.0 was used to test for statistical significance.

Techniques: Transfection, Luciferase, Activity Assay, Western Blot, Construct, Expressing, Magnetic Beads, Reporter Assay

Journal: Cell Death & Disease

Article Title: The MEF2A transcription factor interactome in cardiomyocytes

doi: 10.1038/s41419-023-05665-8

Figure Lengend Snippet: A PCMs were transiently transfected with an MMP9 luciferase reporter gene along with siRNA targeting the endogenous MEF2A and STAT3 levels individually and in combination. Scrambled siRNA was used as a control. Renilla luciferase was used to normalize for transfection efficiency. Lysates were collected at 48 h. The luciferase activity under each condition was measured and normalized to Renilla values to determine the fold change. N = 3 biological replicates per condition (Top panel). A representative western blot for three biological replicates for MEF2A, STAT3, MMP9, and β-actin is presented. Corresponding western blot analysis was used to confirm siRNA mediated MEF2A and STAT3 depletion as compared to scrambled controls (Bottom panel). A schematic of the MMP9-Luc construct is shown below the representative western blot. The expression of MEF2A, STAT3, and MMP9 are assessed using western blot analysis. The dot graphs represent the level of MEF2A, STAT3, and MMP9 proteins after normalization to β-actin (right panel). Both prominent bands (hyper and hypophosphrylated) for MEF2A are included together in the quantification. Data are presented as mean ± SEM. n = 3 * P < 0.05 vs control. B PCMs were transfected with the MMP9 luciferase reporter gene and harvested at 48 h. On day1 after transfection, cells were treated with the p38 MAPK inhibitor (SB203580; 10 μM) or its inactive analogue (SB202474; 10 μM) for 24 h in a serum-free medium. Cells were treated with a STAT3 inhibitor (C188-9;10 µM) as indicated, and the control cells were treated with the corresponding solvent (DMSO). Luciferase values were normalized to Renilla. N = 3 biological replicates per condition. Representative western blot for three biological replicates for MEF2A, pSTAT3, STAT3, MMP9, and β-actin. Corresponding western blot analysis of the cell lysates was used to confirm SB203580 and C188-9 treatments as compared to controls as indicated (Bottom Panel). C The plot represents the MMP9 protein levels after normalization to β-actin. Each condition in the luciferase reporter is compared to the respective control condition for three independently transfected samples (technical replicates) to determine the fold change. Each dot represents one biological replicate and corresponds to the mean of three technical replicates. The error bars represent standard error of the mean (SEM). Tukey’s multiple comparisons test in one-way ANOVA using GraphPad Prism 8.0 were used to test for statistical significance. Adjusted p -value *≤0.05, **≤0.01, ***≤0.001, ****<0.0001, comparing to control.

Article Snippet: Tukey’s Multiple comparisons test in one-way ANOVA using GraphPad Prism 8.0 was used to test for statistical significance.

Techniques: Transfection, Luciferase, Activity Assay, Western Blot, Construct, Expressing

Journal: Cell Death & Disease

Article Title: The MEF2A transcription factor interactome in cardiomyocytes

doi: 10.1038/s41419-023-05665-8

Figure Lengend Snippet: A MEF2A and STAT3 are involved in the increase of cardiomyocyte surface area in response to phenylephrine. Immunofluorescence analysis of α-Actinin and DAPI was performed in PCMs transfected with siRNA targeting MEF2A and STAT3 individually and in combination for 48 h then treated with PE (200 μM) or vehicle (H 2 O) for a further 48 h. The figure illustrates 2 representative images for each condition. The scale bar is 20 μm. B The scatter plot represents the quantification of the surface area of α-Actinin-positive cells using Image J software. The blue dots represent PCMs treated with H 2 O and purple dots represent the PCMs treated with PE. The data presented are derived from the surface areas of 100 cells that were randomly selected from four biological replicates (25 cells per biological replicate). Each cell surface area measurement is represented as a dot in each condition and the mean is indicated by a horizontal line. Tukey’s Multiple comparisons test in one-way ANOVA using GraphPad Prism 8.0 was used to test for statistical significance. * represents statistical significance from control vehicle-treated group. # represents statistical significance from PE-treated group. Adjusted p -value *≤0.05, **≤0.01, ***≤0.001, ****<0.0001, comparing to control.

Article Snippet: Tukey’s Multiple comparisons test in one-way ANOVA using GraphPad Prism 8.0 was used to test for statistical significance.

Techniques: Immunofluorescence, Transfection, Software, Derivative Assay