Journal: eLife

Article Title: Sensory perception drives food avoidance through excitatory basal forebrain circuits

doi: 10.7554/eLife.44548

Figure Lengend Snippet:

Article Snippet: Strain, strain background (Adeno-Associated Virus Serotype 2/DJ8) , AAV-Ef1a-Flex-mRuby2 (DJ8); Flex-mRuby2 , This Paper; Neurocconnectivity Core at the Jan and Dan Duncan Neurological Research Institute , , Plasmid subcloned from Addgene:40260.

Techniques: Virus, Plasmid Preparation, Recombinant, Enzyme-linked Immunosorbent Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: Evaluation of Stable LifeAct-mRuby2- and LAMP1-NeonGreen Expressing A549 Cell Lines for Investigation of Aspergillus fumigatus Interaction with Pulmonary Cells

doi: 10.3390/ijms22115965

Figure Lengend Snippet: ( a ) Generation of A549 cell lines stably expressing LifeAct-mRuby2 (in red) and LAMP1-NG (NeonGreen in green). Scale bar 20 µm. ( b ) Quantification of corrected total cell fluorescence of LifeAct-Ruby2 cells. Example 1 illustrates the image and quantification of six individual cells, value for each cell is depicted. Example 2 shows measurements of 628 cells from a single experiment. The red line depicts a cut-off point for high CTCF values reflecting strongly expressing Ruby2-positive cells. ( c ) Expression of LAMP1, beta-actin, and NeonGreen in generated cell lines.

Article Snippet: LifeAct-mRuby2 was generated by fusing the coding sequence of the actin binding domain (aa 1–17) of yeast Abp140 [ ] to the 5′ end of the red fluorescent protein mRuby2 [ ] by PCR and subsequent subcloning into retroviral pQCXIN (Clontech Laboratories, Inc., Mountain View, CA, USA) using the Gateway technology.

Techniques: Stable Transfection, Expressing, Fluorescence, Generated

Journal: International Journal of Molecular Sciences

Article Title: Evaluation of Stable LifeAct-mRuby2- and LAMP1-NeonGreen Expressing A549 Cell Lines for Investigation of Aspergillus fumigatus Interaction with Pulmonary Cells

doi: 10.3390/ijms22115965

Figure Lengend Snippet: Infection of A549 and LifeAct-mRuby2 cells with A. fumigatus results in formation of actin rings around internalized fungal conidia. ( a ) Actin rings in A549 cells (white arrows). Immunofluorescence: Phalloidin-Alexa 555 (actin) in red, GFP-conidia in green, ( b ) actin rings in LifeAct-mRuby2-expressing cells (white arrows). GFP-conidia in green, ( c ) actin rings in LifeAct-mRuby2-expressing cells (white arrows). Immunofluorescence: Phalloidin-Alexa 350 (actin) in blue, GFP-conidia in green, ( d ) actin rings (white arrows) in A549 and LifeAct-mRuby2 cells visualized by phalloidin-Alexa 488 (green), Dapi in blue. ( e ) Expression and phosphorylation of cofilin in cells treated with gliotoxin. Red asterisk points on phosphorylation of cofilin after treatment with gliotoxin in control A549 cells. GT: Gliotoxin. Scale bar 10 µm. See also .

Article Snippet: LifeAct-mRuby2 was generated by fusing the coding sequence of the actin binding domain (aa 1–17) of yeast Abp140 [ ] to the 5′ end of the red fluorescent protein mRuby2 [ ] by PCR and subsequent subcloning into retroviral pQCXIN (Clontech Laboratories, Inc., Mountain View, CA, USA) using the Gateway technology.

Techniques: Infection, Immunofluorescence, Expressing

Journal: Nucleic Acids Research

Article Title: A supernumerary designer chromosome for modular in vivo pathway assembly in Saccharomyces cerevisiae

doi: 10.1093/nar/gkaa1167

Figure Lengend Snippet: Transformation efficiency and fidelity. ( A ) Transformation efficiencies for the in vivo assembly of 100 and 50 kb synthetic chromosomes. Both neochromosomes have been assembled with 2.5 kb (dark blue), 5 kb (lighter blue) and 10 kb (lightest blue) fragments. The number above each bar indicates the number of assembled fragments. Data were normalized to 10 8 cells using controls on YPD plates, performed in biological duplicates (replicate 1 and 2) in technical triplicates for replicate 1 and technical duplicates for replicate 2. The asterisks indicates that transformation with 5 kb fragments resulted in a significantly different transformation efficiency compared to both neighbouring efficiencies (two-tailed paired homoscedastic t -test P < 0.05). ( B ) Assembly fidelity. In vivo assembly of the 100 kb NeoChr12 from 43 fragments. Eleven colonies that grew on selective medium were tested for mRuby2 and mTurquoise2 fluorescence. Neochromosomes were subsequently screened based on their size on CHEF gel and lastly by Illumina sequencing. The percentage of correct colonies for each screening round is indicated.

Article Snippet: Deletion of mRuby2 (IMF37) or Venus (IMF38), or both mRuby2 and Venus (IMF39) from the 100 kb neochromosome in IMF6 did not increase the growth rate ( ).

Techniques: Transformation Assay, In Vivo, Two Tailed Test, Fluorescence, Illumina Sequencing

Journal: Nucleic Acids Research

Article Title: A supernumerary designer chromosome for modular in vivo pathway assembly in Saccharomyces cerevisiae

doi: 10.1093/nar/gkaa1167

Figure Lengend Snippet: Quantification of neochromosome copy number. ( A ) neochromosome copy number as determined by whole genome sequencing. ( B ) neochromosome copy number estimation based on mRuby2 fluorescence. CEN.PK113-7D with no copies of mRuby2 was used as negative control and IMX2224 with a single copy of mRuby2 integrated in the genome as positive control. ( C ) Schematic overview of the potential segregation of circular neochromosome upon cell division.

Article Snippet: Deletion of mRuby2 (IMF37) or Venus (IMF38), or both mRuby2 and Venus (IMF39) from the 100 kb neochromosome in IMF6 did not increase the growth rate ( ).

Techniques: Sequencing, Fluorescence, Negative Control, Positive Control

Journal: The Plant journal : for cell and molecular biology

Article Title: Live-cell CRISPR imaging in plants reveals dynamic telomere movements.

doi: 10.1111/tpj.13601

Figure Lengend Snippet: Figure 5. Simultaneous visualization of telomeric DNA by CRISPR–dCas9 and the GFP-tagged telom- eric repeat binding protein 1 (TRB1). (a) Immunoflu- orescence staining against Sp-dCas9-mRuby2. (b) Immunofluorescence staining against TRB1-GFP. (c) Overlay showing almost complete co-localization, except for putative blunt-ended telomeres (indi- cated by arrows, nucleus is counterstained with DAPI (in blue). Scale bars: 2 lm.

Article Snippet: Plasmids encoding for eGFP (pSiM24-eGFP) and mRuby2 (pcDNA3-mRuby2) were obtained from Addgene (http:// www.addgene.com). dCas9 and fluorescence protein (FP) sequences were generated with primers containing homologous flanks for subsequent Gibson Assembly cloning into the pCAS9TP backbone (for a full list of primers, see Appendix S8).

Techniques: CRISPR, Binding Assay, Staining

Journal: The Plant journal : for cell and molecular biology

Article Title: Live-cell CRISPR imaging in plants reveals dynamic telomere movements.

doi: 10.1111/tpj.13601

Figure Lengend Snippet: Figure 6. Comparison of Sa-dCas9 and Sp-dCas9. Telomeres were visualized by the simultaneous application of two dCas9 orthologues (Sa-dCas9 and Sp-dCas9). (a) Immunofluorescence staining against Sa-dCas9-eGFP. (b) Immunofluorescence staining against Sp-dCas9-mRuby2. (c) Overlay showing complete co-localization. Nucleus is coun- terstained with DAPI (in blue). (d) Quantification of the number of telomere signals observed by two different dCas9 orthologues (n = 18). Scale bars: 10 lm.

Article Snippet: Plasmids encoding for eGFP (pSiM24-eGFP) and mRuby2 (pcDNA3-mRuby2) were obtained from Addgene (http:// www.addgene.com). dCas9 and fluorescence protein (FP) sequences were generated with primers containing homologous flanks for subsequent Gibson Assembly cloning into the pCAS9TP backbone (for a full list of primers, see Appendix S8).

Techniques: Comparison, Staining