Journal: Nature Communications

Article Title: Modular fluorescence complementation sensors for live cell detection of epigenetic signals at endogenous genomic sites

doi: 10.1038/s41467-017-00457-z

Figure Lengend Snippet: Development and validation of the BiAD sensor 1. a Representative fluorescence microscopy image of the BiFC signal ( yellow channel ) generated upon transfection of the BiAD sensor 1 in iMEF cells. A plasmid encoding NLS-mRuby2 was used to identify transfected cells ( red channel ). b Representative fluorescence microscopy images documenting co-localization of the BiFC signal with DAPI stained major satellite DNA ( upper panel ) and 5mC marks detected by co-transfection of the sensor modules with MBD-Cerulean ( lower panel ). c Representative fluorescence microscopy images documenting the 5mC specificity of the BiAD sensor. The BiFC signal was lost with the MBD R44Q 5mC-binding pocket mutant ( left ) and in cells with globally reduced DNA methylation levels ( right ). A plasmid encoding NLS-mRuby2 was used to identify transfected cells ( red channel ). The transfection, imaging and display settings of the images shown in panels a , c are identical. d Quantification of the experiments representatively shown in panels a – c . The error bars represent the s.e.m. for two biological repeats (for details cf . Methods and Supplementary Tables , , and ). All cells were fixed at 48 h after transfection. Scale bar for all images is 10 µm

Article Snippet: This was followed by Gibson assembly with PCR fragments encoding for mRuby2 (Dr Michael Davidson, Addgene plasmid no. 54768) , VenN or VenC.

Techniques: Biomarker Discovery, Fluorescence, Microscopy, Generated, Transfection, Plasmid Preparation, Staining, Cotransfection, Binding Assay, Mutagenesis, DNA Methylation Assay, Imaging

Journal: Nature Communications

Article Title: Modular fluorescence complementation sensors for live cell detection of epigenetic signals at endogenous genomic sites

doi: 10.1038/s41467-017-00457-z

Figure Lengend Snippet: Development and validation of the BiAD sensor 2. a Representative fluorescence microscopy image showing that BiAD sensor 2 gives rise to distinct BiFC signals in HEK293 cells. NLS-mRuby2 was used as transfection control. b Co-localization of the BiFC signal with the TALE protein fused to mRuby2 documents the high-DNA specificity of the BiAD sensor. c Loss of the BiFC signal when the R44Q MBD variant was used as a detector module confirms the 5mC specificity of the BiAD sensor. d Quantification of the experiments representatively shown in panels a – c . The error bars represent the s.e.m. for two biological repeats (for details cf. Methods and Supplementary Tables , , and ). All cells were fixed at 48 h after transfection. Scale bar for all images is 10 µm

Article Snippet: This was followed by Gibson assembly with PCR fragments encoding for mRuby2 (Dr Michael Davidson, Addgene plasmid no. 54768) , VenN or VenC.

Techniques: Biomarker Discovery, Fluorescence, Microscopy, Transfection, Control, Variant Assay

Journal: Nature Communications

Article Title: Modular fluorescence complementation sensors for live cell detection of epigenetic signals at endogenous genomic sites

doi: 10.1038/s41467-017-00457-z

Figure Lengend Snippet: Visualization of DNA methylation changes using BiAD sensor 2. a Representative fluorescence microscopy images documenting that strong BiFC signals are formed upon transfection of the BiAD sensor 2 into HCT116 WT cells ( top ). In contrast, very low fluorescence reconstitution was observed in DNMT1 hypomorphic HTC116 cells that have globally reduced methylation levels ( bottom ). A plasmid encoding for NLS-mRuby2 was used to identify transfected cells. b The BiFC signals were rescued in the DNMT1 hypomorphic HTC116 cells by the exogenous expression of active DNMT1 ( top ) but not of catalytically inactive DNMT1 ( bottom ). c Quantification of the experiments representatively shown in panels a , b . d Time course of DNA demethylation by treatment of HEK293 cells with 5-aza-dC and recovery. In each sample, the BiAD sensor was transfected 2 days before the cells were fixed for imaging. e Quantification of the experiments representatively shown in panel d . Error bars in all images represent the s.e.m. for two biological replicates (for details cf . Supplementary Tables , , and ). Scale bar for all images is 10 µm. The imaging and display setting of the BiFC images shown in panels a , b , and d are identical. In panel d the contrast of the mRuby2 channel was increased to enhance the visibility of transfected cells. This was done with the same settings for all time points

Article Snippet: This was followed by Gibson assembly with PCR fragments encoding for mRuby2 (Dr Michael Davidson, Addgene plasmid no. 54768) , VenN or VenC.

Techniques: DNA Methylation Assay, Fluorescence, Microscopy, Transfection, Methylation, Plasmid Preparation, Expressing, Imaging

Journal: Light, Science & Applications

Article Title: Excitation spectral microscopy for highly multiplexed fluorescence imaging and quantitative biosensing

doi: 10.1038/s41377-021-00536-3

Figure Lengend Snippet: a , b Color-coded Mito-pHRed absolute pH maps of live HeLa cells after the application of 20 µM CCCP for 6 h, for cells without ( a ) and with ( b ) the co-expression of fluorescently untagged Parkin. c Distribution of the measured pH values in each mitochondrion for a , b . d Excitation spectra on our setup for the five fluorescent species unmixed to enable absolute pH imaging with three additional FP markers. e Unmixed images of color-coded Mito-pHRed absolute pH map, mOrange2-Parkin, PhiYFP-LC3, and LAMP1-Clover for two Parkin-expressing live HeLa cells after the application of 20 µM CCCP for 4 h. f Overlay of the images in e . g Zoom-in of the white box in f , as well as its separation into different channels. Cyan, magenta, and red arrowheads point to individual mitochondria only marked by Parkin, LC3, and LAMP1, with respective matrix pH values quantified from the enclosed Mito-pHRed signals indicated. Orange arrowhead points to a mitochondrion marked by both Parkin and LC3. h Distribution of the Mito-pHRed-quantified matrix pH of individual mitochondria in the sample labeled by the different markers (color diamonds), compared to that in untreated HeLa cells (black circles). Shaded band: typical range of cytoplasmic pH. Experiments were performed with 8-wavelength excitation at 10 fps (0.8 s acquisition time for each spectral image). Scale bars: 5 µm ( a , b ); 10 µm ( e , f ); 2 µm ( g )

Article Snippet: LAMP1-mOrange2 was constructed by replacing mRuby2 in the LAMP1-mRuby2 (Addgene #55902; a gift from Michael Davidson) with mOrange2 between the AgeI and NotI sites.

Techniques: Expressing, Imaging, Labeling