Journal: Nature Metabolism

Article Title: An electrogenetic interface to program mammalian gene expression by direct current

doi: 10.1038/s42255-023-00850-7

Figure Lengend Snippet: a – c , One ( a ), two ( b ) and three ( c ) Duracell or Energizer alkaline 1.5 V AA batteries. d , Two Duracell or Energizer lithium button cells (CR2032) provide about 6 V. e , One Duracell or Energizer 9 V block alkaline battery. f , One A220/504 A Exell battery. SEAP levels were measured 24 h after stimulation. All data are mean ± s.d.; n = 4. P values were calculated between stimulated and non-induced control.

Article Snippet: The charger was directly plugged into mains electricity and the charger wires were cut for connection to the electrodes submerged in the culture medium. i , One Duracell or Energizer 12 volt 23 amp alkaline battery. j , Two Duracell or Energizer 9 volt block alkaline batteries. k , Two Duracell or Energizer 12 volt 23 A alkaline batteries. l , Two A220/504 A Exell batteries provide around 30 volts.

Techniques: Blocking Assay, Battery, Control

Journal: Nature Metabolism

Article Title: An electrogenetic interface to program mammalian gene expression by direct current

doi: 10.1038/s42255-023-00850-7

Figure Lengend Snippet: a-c , One ( a ), two ( b ) and three ( c ) Duracell or Energizer alkaline AAA batteries were used, each providing around 1.5 volts. d , One Duracell or Energizer lithium button cell CR2032 provides about 3 volts. e-f , Four Duracell or Energizer alkaline AA ( e ) and AAA ( f ) batteries provide around 6 volts. g , A Belkin power bank whose universal USB-A 2.4 A port delivers up to 5 volts. h , Apple and Huawei mobile phone chargers. The charger was directly plugged into mains electricity and the charger wires were cut for connection to the electrodes submerged in the culture medium. i , One Duracell or Energizer 12 volt 23 amp alkaline battery. j , Two Duracell or Energizer 9 volt block alkaline batteries. k , Two Duracell or Energizer 12 volt 23 A alkaline batteries. l , Two A220/504 A Exell batteries provide around 30 volts. SEAP levels in supernatants of culture medium were measured at 24 h after electrostimulation for the indicated time. Columns each represent the mean ± SD of the four indicated data points. P values were calculated between stimulated and non-stimulated group.

Article Snippet: The charger was directly plugged into mains electricity and the charger wires were cut for connection to the electrodes submerged in the culture medium. i , One Duracell or Energizer 12 volt 23 amp alkaline battery. j , Two Duracell or Energizer 9 volt block alkaline batteries. k , Two Duracell or Energizer 12 volt 23 A alkaline batteries. l , Two A220/504 A Exell batteries provide around 30 volts.

Techniques: Battery, Blocking Assay

Journal: Scientific Reports

Article Title: The role of glycosyltransferase enzyme GCNT3 in colon and ovarian cancer prognosis and chemoresistance

doi: 10.1038/s41598-018-26468-4

Figure Lengend Snippet: GCNT3 overexpression reduces 5FU resistance in CRC cells. (Panel A) Protein expression levels of GCNT3 in non-infected colorectal cancer (CRC) cells, stable cell lines overexpressing GCNT3 and a battery of shGCNT3. Proteins were detected by western blot using specific antibodies against GCNT3, β-Actin and β-Tubulin, as a loading control. Full-length blots/gels are presented in Supplementary Fig. . (Panel B) mRNA expression levels of GCNT3 measured by RT-QPCR, in non-infected CRC cells, stable cell lines overexpressing GCNT3 and shGCNT3 number 7. Data represent mean ± SEM of three independent experiments. (Panel C) Representative immunofluorescence images of GCNT3 (green) and Tubulin (red) of NoORF, GCNT3, Scrambl and shGCNT3 7 cells. Nuclei were stained with DAPI (blue). Scale bars 50 µm. (Panel D) Comparison of 5-fluoracil (5FU) IC50 values (concentration needed for 50% of viability inhibition) between non-infected CRC cells. Cell viability assays were performed after 72 h treatment. Data represent mean ± SEM of at least two independent experiments each performed in triplicate. (Panel E) Induction of GCNT3 expression by 5FU in CRC cells. Tumour cells were treated with 30 µM 5FU, during 72 h, and their mRNA GCNT3 expression was measured by RT-QPCR and represented in comparison to controls (vehicle-treated cells). Results are expressed as the mean ± SEM. of three independent experiments, each performed in triplicate. Student’s t test was applied to assess statistically significant differences (*p < 0.05). (Panel F) Comparison of 5FU IC50 values between NoORF and GCNT3 cells. Cell viability assays were performed after 72 h treatment. Data represent mean ± SEM of at least three independent experiments each performed in triplicate. Asterisk indicates statistically different values in GCNT3 cells respect to the control (NoORF cells), (*p < 0.05).

Article Snippet: In clinical samples, GCNT3 gene expression was analyzed using the specific TaqMan probe (Hs00953355_m1, Life Technologies, Carlsbad CA, USA).

Techniques: Over Expression, Expressing, Infection, Stable Transfection, Battery, Western Blot, Control, Quantitative RT-PCR, Immunofluorescence, Staining, Comparison, Concentration Assay, Inhibition

Journal: Scientific Reports

Article Title: The role of glycosyltransferase enzyme GCNT3 in colon and ovarian cancer prognosis and chemoresistance

doi: 10.1038/s41598-018-26468-4

Figure Lengend Snippet: GCNT3 overexpression reduces proliferation, invasion and changes metabolic capacities of CRC cells. (Panel A) xCELLigence proliferation assay of NoORF, GCNT3, Scramble and shGCNT3 7 cells. The rate of proliferation is determined by analyzing the slope of the proliferation line between the 12 and 60 h interval. Complete growth curve of NoORF and GCNT3 SW620 cells from 0 to 72 h after seeding is represented. Results were expressed as 12 h normalized cell index value. Data are represented as mean ± SEM of four independent experiments each performed in triplicate. Student’s t test was applied to assess statistically significant differences (*p < 0.05). (Panel B) Boyden chamber transwell assay of GCNT3 SW620 cell invasion through Matrigel. After 96 h, SW620 cells were fixed and stained with crystal violet (bottom panels) and counted under an optical microscope. Pictures were taken using an Olympus CKX41 microscope (Olympus, Tokyo, Japan), with a 20X LCAch objective and registered using analysis getIT software (Olympus). Scale bars 100 µm. Data are represented as mean ±SEM of three independent experiments each performed in triplicate. Student’s t test was applied to assess statistically significant differences (**p < 0.01). (Panel C) Oxygen consumption rate (OCR) of NoORF and GCNT3 SW620 cells. Bioenergetics parameters were obtained by adding 2 µM Oligomycin to block ATP-linked OCR, 0.2 µM FCCP to uncouple mitochondria for maximal OCR and 0.5 µM Rotenone/Antimycin A (Rot/AA) to shut down mitochondrial respiration. Right panel reflects the quantification of basal respiration (oxygen consumption used to meet cellular ATP demand, calculated by subtracting non-mitochondrial OCR obtained upon Rot/AA addition) and spare respiratory capacity (capability to respond to an energetic demand, calculated as the difference between maximal and basal OCR). Left panel represents OCR measurements over time for cells stably expressing NoORF or GCNT3. We show representative experiments of 6 measures (n = 3).

Article Snippet: In clinical samples, GCNT3 gene expression was analyzed using the specific TaqMan probe (Hs00953355_m1, Life Technologies, Carlsbad CA, USA).

Techniques: Over Expression, Proliferation Assay, Boyden Chamber Transwell Assay, Staining, Microscopy, Software, Blocking Assay, Stable Transfection, Expressing

Journal: Scientific Reports

Article Title: The role of glycosyltransferase enzyme GCNT3 in colon and ovarian cancer prognosis and chemoresistance

doi: 10.1038/s41598-018-26468-4

Figure Lengend Snippet: Genomic analysis of GCNT3 overexpression. (Panel A) Description of candidate genes selected for validation. (Panel B) Experimental validation of GCNT3 microarray results by qRT-PCR. Bar graph showing the correlation of microarray data with qRT-PCR transcript levels in three CRC cellular models. The X axis shows the selected panel of validated genes and the Y-axis represent the relative fold change by microarray or qRT-PCR. Data represent mean ± SEM of three independent experiments each performed in triplicate. Student’s t test was applied to assess statistically significant differences (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: In clinical samples, GCNT3 gene expression was analyzed using the specific TaqMan probe (Hs00953355_m1, Life Technologies, Carlsbad CA, USA).

Techniques: Over Expression, Biomarker Discovery, Microarray, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: The role of glycosyltransferase enzyme GCNT3 in colon and ovarian cancer prognosis and chemoresistance

doi: 10.1038/s41598-018-26468-4

Figure Lengend Snippet: Proteomic analysis of GCNT3 overexpression. (Panel A) Immunoprecipitation of NoORF and GCNT3 SW620 cell extracts using V5 antibody. V5-GCNT3 tag protein was detected by Western blot (Inp: Input, IP: Immunoprecipitate, FT; Flow through). Experiments performed in duplicate. Full-length blots/gels are presented in Supplementary Fig. . (Panel B) Protein-protein interaction network of GCNT3 using STRING. Statistically significant interactors of proteomic study were included in the analysis (p-value > 0.0005). Only top-10 statistically significant proteins included in nodes are named, as well as, PAM and CANX.

Article Snippet: In clinical samples, GCNT3 gene expression was analyzed using the specific TaqMan probe (Hs00953355_m1, Life Technologies, Carlsbad CA, USA).

Techniques: Over Expression, Immunoprecipitation, Western Blot

Journal: Scientific Reports

Article Title: The role of glycosyltransferase enzyme GCNT3 in colon and ovarian cancer prognosis and chemoresistance

doi: 10.1038/s41598-018-26468-4

Figure Lengend Snippet: Biological processes enriched by GCNT3 overexpression in CRC cells. REViGO Scatterplot of GO categories enriched in GCNT3 genomic (Panel A) and proteomic (Panel B) analysis. GO enrichment analysis for statistically significant transcripts and proteins. The remaining terms after the redundancy reduction were plotted in a two dimensional space. Bubble sizes indicates the p-value (log10 p-value). Semantic space is based on the semantic similarity, which is the degree of relatedness between two entities by measuring the similarity of their annotation meanings. The list of enriched GO terms is subjected to redundancy reduction, based on the “most informative common ancestor” approach in REVIGO and is represented by cluster representatives in a scatterplot. The x- and y-axes of the scatterplot represent the distance between the cluster representatives.

Article Snippet: In clinical samples, GCNT3 gene expression was analyzed using the specific TaqMan probe (Hs00953355_m1, Life Technologies, Carlsbad CA, USA).

Techniques: Over Expression

Journal: Scientific Reports

Article Title: The role of glycosyltransferase enzyme GCNT3 in colon and ovarian cancer prognosis and chemoresistance

doi: 10.1038/s41598-018-26468-4

Figure Lengend Snippet: Clinical relevance of GCNT3 expression in epithelial ovarian cancer (EOC). (Panel A) Association between GCNT3 expression and time to treatment failure (TTF) in EOC. Kaplan–Meier plots for GCNT3 expression in 56 EOC patients. (Panel B) Association of GCNT3 gene expression profiles with response. The expression levels of GCNT3 in non-responders and responders groups are shown. The median value of GCNT3 expression is indicated by the horizontal bar on the graph (Man-Whitney U-test for P values). (Panel C) ROC analysis of the GCNT3 signature in EOC patients. The AUC was 0.667. (Panel D) Forest plot showing the meta-analysis of hazard ratio (HR) and 95% confidence interval (CI) estimates for TTF for the prognostic significance of GCNT3 expression in EOC patients from six different studies. (Panel E) Protein and mRNA expression levels of GCNT3 in non-infected EOC cells and Caov3 stable cell lines overexpressing GCNT3. Proteins were detected by western blot using specific antibodies against GCNT3 and β-Actin. mRNA expression levels of GCNT3 and VEGFA were measured by RT-QPCR. Data represent mean ± SEM of three independent experiments. Student’s t test was applied to assess statistically significant differences (***p < 0.001). Full-length blots/gels are presented in Supplementary Fig. . (Panel F) Boyden chamber transwell assay of GCNT3 Caov3 invasion through Matrigel. After 96 h, Caov3 cells were fixed and stained with crystal violet (bottom panels) and counted under an optical microscope. Pictures were taken using an Olympus CKX41 microscope (Olympus, Tokyo, Japan), with a 20X LCAch objective and registered using analysis getIT software (Olympus). Scale bars 100 µm. On the left, xCELLigence proliferation assay of NoORF and GCNT3 Caov3 cells. The rate of proliferation is determined by analyzing the slope of the proliferation line between the 12 and 60 h interval. Data are represented as mean ± SEM of three independent experiments each performed in triplicate. Student’s t test was applied to assess statistically significant differences (**p < 0.01).

Article Snippet: In clinical samples, GCNT3 gene expression was analyzed using the specific TaqMan probe (Hs00953355_m1, Life Technologies, Carlsbad CA, USA).

Techniques: Expressing, Gene Expression, Infection, Stable Transfection, Western Blot, Quantitative RT-PCR, Boyden Chamber Transwell Assay, Staining, Microscopy, Software, Proliferation Assay

Journal: Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism

Article Title: Xenon preconditioning reduces brain damage from neonatal asphyxia in rats.

doi: 10.1038/sj.jcbfm.9600184

Figure Lengend Snippet: Figure 7 Putative modulation of xenon on CREB transcriptional pathway associated with survival signals. Xenon activates protein kinase C (PKC) which triggers phosphorylation of mitogen-activated protein kinases (MAPK) including p38 MAPK and extracellular signal-regulated kinase (ERK) (Weber et al, 2005). Consequently, cAMP-response element-binding protein (CREB) is transformed into the phospho-CREB (pCREB) form (Roberson et al, 1999). The CREB and CREB-binding protein (CBP) interaction can then induce transcription of a battery of CREB-dependent survival genes. The transcribed cAMP (cyclic adenosine 30,50-monophosphate)-response element binding (CREB)-dependent proteins, including brain-derived neuro- trophic factor (BDNF) and prosurvival protein Bcl-2, can be enhanced, which promotes cells to tolerate hypoxia–ischaemic insult. The activation of Ras (a small guanine nucleotide- binding protein that is essential for the proliferation, differentia- tion, and survival of cells) by neurotrophic factors and prosurvival proteins can in turn initiate more MAPK phosphor- ylation and by so doing it will enhance the survival signals.

Article Snippet: The membrane was treated with blocking solution (5% nonfat dry milk in Tween-containing Tris-buffered saline (10 mmol/L Tris, 150 mmol/L NaCl, 0.1% Tween, pH 8.0)) and incubated with rabbit monoclonal antibody directed against phosphorylated cAMP (cyclic adenosine 30,50-monophosphate)-response element binding protein (pCREB), CREB, Bcl-2 (1:1000, New England Biolab, Hitchin, UK) brain-derived neurotrophic factor (BDNF) (1:250, Santa Cruz Biotech, Wembley, UK), or a-tubulin (1:2000, Sigma, Poole, UK) overnight at 41C.

Techniques: Phospho-proteomics, Binding Assay, Transformation Assay, Battery, Derivative Assay, Activation Assay

Journal: Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism

Article Title: Xenon preconditioning reduces brain damage from neonatal asphyxia in rats.

doi: 10.1038/sj.jcbfm.9600184

Figure Lengend Snippet: Figure 6 Phosphorylated cAMP (cyclic adenosine 30,50-mono- phosphate)-response element binding protein (pCREB) immu- noreactivity in pups’ cerebral cortex after exposure to 70% xenon (A) or 70% N2O (B). (C) Expression of Bcl-2 and brain- derived neurotrophic factor (BDNF) after exposure to 70% xenon. Each band is a representative image from one of three independent experiments. NC ¼ negative control of pCREB (cell extracts from SK-N-MC cells); PC ¼ positive control of pCREB (SK-N-MC cells/forskolin þ IBMX).

Article Snippet: The membrane was treated with blocking solution (5% nonfat dry milk in Tween-containing Tris-buffered saline (10 mmol/L Tris, 150 mmol/L NaCl, 0.1% Tween, pH 8.0)) and incubated with rabbit monoclonal antibody directed against phosphorylated cAMP (cyclic adenosine 30,50-monophosphate)-response element binding protein (pCREB), CREB, Bcl-2 (1:1000, New England Biolab, Hitchin, UK) brain-derived neurotrophic factor (BDNF) (1:250, Santa Cruz Biotech, Wembley, UK), or a-tubulin (1:2000, Sigma, Poole, UK) overnight at 41C.

Techniques: Binding Assay, Expressing, Derivative Assay, Negative Control, Positive Control

Journal: Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism

Article Title: Xenon preconditioning reduces brain damage from neonatal asphyxia in rats.

doi: 10.1038/sj.jcbfm.9600184

Figure Lengend Snippet: Figure 7 Putative modulation of xenon on CREB transcriptional pathway associated with survival signals. Xenon activates protein kinase C (PKC) which triggers phosphorylation of mitogen-activated protein kinases (MAPK) including p38 MAPK and extracellular signal-regulated kinase (ERK) (Weber et al, 2005). Consequently, cAMP-response element-binding protein (CREB) is transformed into the phospho-CREB (pCREB) form (Roberson et al, 1999). The CREB and CREB-binding protein (CBP) interaction can then induce transcription of a battery of CREB-dependent survival genes. The transcribed cAMP (cyclic adenosine 30,50-monophosphate)-response element binding (CREB)-dependent proteins, including brain-derived neuro- trophic factor (BDNF) and prosurvival protein Bcl-2, can be enhanced, which promotes cells to tolerate hypoxia–ischaemic insult. The activation of Ras (a small guanine nucleotide- binding protein that is essential for the proliferation, differentia- tion, and survival of cells) by neurotrophic factors and prosurvival proteins can in turn initiate more MAPK phosphor- ylation and by so doing it will enhance the survival signals.

Article Snippet: The membrane was treated with blocking solution (5% nonfat dry milk in Tween-containing Tris-buffered saline (10 mmol/L Tris, 150 mmol/L NaCl, 0.1% Tween, pH 8.0)) and incubated with rabbit monoclonal antibody directed against phosphorylated cAMP (cyclic adenosine 30,50-monophosphate)-response element binding protein (pCREB), CREB, Bcl-2 (1:1000, New England Biolab, Hitchin, UK) brain-derived neurotrophic factor (BDNF) (1:250, Santa Cruz Biotech, Wembley, UK), or a-tubulin (1:2000, Sigma, Poole, UK) overnight at 41C.

Techniques: Phospho-proteomics, Binding Assay, Transformation Assay, Battery, Derivative Assay, Activation Assay

Journal: Journal of visualized experiments : JoVE

Article Title: The Evoked Potential Operant Conditioning System (EPOCS): a research tool and an emerging therapy for chronic neuromuscular disorders

doi: 10.3791/63736

Figure Lengend Snippet: List of equipment and materials for an example EPOCS setup

Article Snippet: Constant-current stimulator , Digitimer Ltd. , DS8R , The DS8R enjoys EPOCS automation support. If controlled manually, other constant-current stimulators may be used provided they have an external TTL trigger and can achieve a pulse duration of 1 ms or more..

Techniques: Blocking Assay, Transmission Assay, Battery