Journal: International Journal of Molecular Sciences

Article Title: Aedes aegypti Piwi4 Structural Features Are Necessary for RNA Binding and Nuclear Localization

doi: 10.3390/ijms222312733

Figure Lengend Snippet: AePiwi4 harbors an NLS in the N-terminal region of the protein. Representative slices from image stacks of HEK293 cells transfected with ( A ) SV40NLS-eGFP, ( B ) eGFP, ( C ) AePiwi4NLS-eGFP, or ( D ) AePiwi4Nterminal-eGFP. Cells were stained with DAPI (blue) 24 h post-transfection. DAPI (left), eGFP (middle), and merged (right) channels are shown separately. Orthogonal views presented in merged channel. Scale bar = 15 μM. ( E ) Quantification of total eGFP fluorescence intensity sums subtracted from eGFP intensity sums in nuclear surfaces for each sample type, normalized by number of cells. Each black dot represents an individual picture. Red bars indicate SEM. ** = p ≤ 0.01, * = p ≤ 0.05, ns = non-significant.

Article Snippet: Nuclear surfaces were determined by DAPI display and eGFP pixel intensity values were extracted using Imaris software version 9.2.1.

Techniques: Transfection, Staining, Fluorescence

Journal: Journal of neuroscience research

Article Title: Spatial and Temporal Changes in Promoter Activity of the Astrocyte Glutamate Transporter GLT1 Following Traumatic Spinal Cord Injury

doi: 10.1002/jnr.22624

Figure Lengend Snippet: Generation of thoracic contusion SCI in BAC-GLT1-eGFP transgenic promoter reporter mice. In cresyl violet-stained sections of thoracic spinal cord from mice that had received a moderate T9 contusion, distinct differences can be observed among intact spinal cord immediately rostral to the injury site (A), the rostral periphery of the lesion site (B), and the center of the injury (C). For BAC-GLT1-eGFP analysis, spinal cord cross-sections were first subdivided into specific anatomical locations (D): dorsal column white matter (DC), dorsal horn gray matter (DH), intermediate gray matter (Int), and ventral horn gray matter (VH). In transgenic BAC-GLT1-eGFP reporter mice, GLT1 promoter activity drives expression of the eGFP reporter (E). In normal, uninjured animals (E–G), GLT1 is found in abundance in gray matter and to a lesser extent in white matter. To analyze GLT1-eGFP expression in astrocytes following SCI (examples, rostral intact cord: H; epicenter: I), individual eGFP+ cells were identified, and the region of fluorescence for each eGFP+ cell was outlined. eGFP average intensity, total intensity, and area for each cell were measured in Metamorph software (J). All images are from mice sacrificed at 14 days postsurgery. Scale bars = 50 μm.

Article Snippet: To analyze GLT1-eGFP expression in astrocytes following SCI (examples, rostral intact cord: H ; epicenter: I ), individual eGFP + cells were identified, and the region of fluorescence for each eGFP + cell was outlined. eGFP average intensity, total intensity, and area for each cell were measured in Metamorph software ( J ).

Techniques: Transgenic Assay, Staining, Activity Assay, Expressing, Fluorescence, Software

Journal: Journal of neuroscience research

Article Title: Spatial and Temporal Changes in Promoter Activity of the Astrocyte Glutamate Transporter GLT1 Following Traumatic Spinal Cord Injury

doi: 10.1002/jnr.22624

Figure Lengend Snippet: GLT1-eGFP expression was reduced in dorsal column white matter astrocytes following thoracic contusion in BAC-GLT1-eGFP mice. At 1, 4, and 14 days post-SCI, BAC-GLT1-eGFP mice were analyzed for average intensity (A) and area (B) of eGFP expression per cell (defined as cell expressing eGFP) in dorsal column white matter. The total intensity (C) of eGFP fluorescence per region (sum of the intensity of all individual eGFP-expressing cells in a region) was analyzed following injury. Total numbers of eGFP+ cells were also counted (D). *P < 0.05, **P < 0.001.

Article Snippet: To analyze GLT1-eGFP expression in astrocytes following SCI (examples, rostral intact cord: H ; epicenter: I ), individual eGFP + cells were identified, and the region of fluorescence for each eGFP + cell was outlined. eGFP average intensity, total intensity, and area for each cell were measured in Metamorph software ( J ).

Techniques: Expressing, Fluorescence

Journal: Journal of neuroscience research

Article Title: Spatial and Temporal Changes in Promoter Activity of the Astrocyte Glutamate Transporter GLT1 Following Traumatic Spinal Cord Injury

doi: 10.1002/jnr.22624

Figure Lengend Snippet: GLT1-eGFP expression in individual gray matter astrocytes increased following thoracic contusion in BAC-GLT1-eGFP mice. At 1, 4, and 14 days after thoracic contusion injury, gray matter at the lesion epicenter and in surrounding intact spinal cord was subdivided into dorsal horn, intermediate and ventral horn, as illustrated in Figure 1D. Each gray matter region was analyzed for average individual cell eGFP fluorescence intensity (A) and average individual eGFP+ cell area (B). *P < 0.05, **P < 0.001.

Article Snippet: To analyze GLT1-eGFP expression in astrocytes following SCI (examples, rostral intact cord: H ; epicenter: I ), individual eGFP + cells were identified, and the region of fluorescence for each eGFP + cell was outlined. eGFP average intensity, total intensity, and area for each cell were measured in Metamorph software ( J ).

Techniques: Expressing, Fluorescence

Journal:

Article Title: Modulation of RNA editing by functional nucleolar sequestration of ADAR2

doi: 10.1073/pnas.2336131100

Figure Lengend Snippet: rRNA is required for ADAR2 nucleolar localization. (a) NIH 3T3 cells stably expressing EGFP-ADAR2 were permeabilized with Triton X-100 in the presence or absence of RNase A, and the localization of EGFP-ADAR2 was monitored by epifluorescence microscopy over time. The time course (0–4 min) for three representative cells is presented for the control (–RNase A) and treated (+RNase A) groups. (b) NIH 3T3 cells were treated for 2 h with actinomycin D (0.05 μg/ml) to selectively inhibit rRNA synthesis and assessed for overall cellular morphology by differential interference contrast microscopy, where a decrease in the size of the nucleoli (35%, n = 20 nucleoli, P < 0.05) was observed. The subcellular localization of endogenous ADAR2 was determined by using fluorescence microscopy with an anti-ADAR2 antibody.

Article Snippet: For mutant localization studies, average EGFP fluorescence intensity ( metamorph , Universal Imaging, Media, PA) was measured in all areas containing nucleolin staining and in the extranucleolar space within the nucleus.

Techniques: Stable Transfection, Expressing, Epifluorescence Microscopy, Microscopy, Fluorescence

Journal:

Article Title: Modulation of RNA editing by functional nucleolar sequestration of ADAR2

doi: 10.1073/pnas.2336131100

Figure Lengend Snippet: The DRBM domains of ADAR2 are required for nucleolar localization. (a) A schematic diagram indicating the structure of EGFP as well as wild-type (EGFP-ADAR2) and mutant fusion proteins is presented showing deletion (EGFP-Δ76–281) or mutation [EGFP-KA (127, 281)] of the DRBM domains. The sequences surrounding conserved lysine residues at positions 127 and 281 in DRBM1 and DRBM2, respectively, are indicated by the one-letter amino acid code, and introduced alanine residues at these positions are indicated in red. NLS, nuclear localization signal. (b) Subcellular localization of ADAR2 and nucleoli was determined by fluorescence microscopy for EGFP (green) or by using an antinucleolin antibody (red), respectively. (c) Average EGFP fluorescence overlapping with nucleolin localization was quantified by using metamorph image analysis software and defined as nucleolar localization. The ratio of average fluorescence intensity for all nucleoli in a cell (Fn) to the average nucleoplasmic (nonnucleolar) fluorescence intensity (Fo) is shown (n = 30 cells; mean ± SD).

Article Snippet: For mutant localization studies, average EGFP fluorescence intensity ( metamorph , Universal Imaging, Media, PA) was measured in all areas containing nucleolin staining and in the extranucleolar space within the nucleus.

Techniques: Mutagenesis, Fluorescence, Microscopy, Software

Journal:

Article Title: Modulation of RNA editing by functional nucleolar sequestration of ADAR2

doi: 10.1073/pnas.2336131100

Figure Lengend Snippet: ADAR2 can rapidly shuttle between nucleolar and nucleoplasmic compartments. (a) A single nucleolus was bleached (arrow) in NIH 3T3 cells stably expressing EGFP-ADAR2, and nuclei were monitored by using confocal microscopy; the time interval (seconds) after the start of fluorescence monitoring is indicated. (b) Quantification of fluorescence intensity by using metamorph image analysis software is presented as the ratio of the fluorescence at time t (Ft) and initial fluorescence (F0) for bleached nucleoli (▪), unbleached nucleoli within the same cell (▵), and the nucleoli in adjacent cells (▾) (mean ± SD; n = 9 pairs of cells). The time at which the single nucleolus was bleached (5 s) is indicated with an arrow.

Article Snippet: For mutant localization studies, average EGFP fluorescence intensity ( metamorph , Universal Imaging, Media, PA) was measured in all areas containing nucleolin staining and in the extranucleolar space within the nucleus.

Techniques: Stable Transfection, Expressing, Confocal Microscopy, Fluorescence, Software