Journal: Cell reports

Article Title: Tace/ADAM17 is a bi-directional regulator of axon guidance that coordinates distinct Frazzled and Dcc receptor signaling outputs

doi: 10.1016/j.celrep.2022.111785

Figure Lengend Snippet: (A–I) Tace RNA and protein expression in stage 13 and 14 embryos. (A–D″) RNA FISH for tace , labeled in green, and eagle neurons labeled in red. tace transcripts are enriched in the CNS and are expressed in eagle neurons (dotted white circles). (E–H″) Tace protein expression is detected in the brain and the ventral nerve cord by the endogenous GFP tag (green) and colocalizes with a large subset of the neurons, labeled by Elav (red). (I and I′) Tace protein colocalizes with the Islet-positive (red) EW neurons (yellow dotted circles). (J–R) tace mutant phenotypes in stage 15–16 embryos. GFP labels the EW and EG population of eagle neurons (yellow brackets in J). (K and L) Midline crossing of EW axons is disrupted in tace mutant embryos (asterisks indicate non-crossing segments). (K′ and L′) HRP-positive axon scaffolds show thinning of commissures (white triangles) and longitudinal tracks (yellow triangles). Red bracket indicates the rare occurrence of a segment with thickened commissures. (M) Summary of embryos analyzed. (N–Q′) tace mutants significantly enhance non-crossing defects in embryos that express FraΔC (indicated by asterisks). The region outlined in Q is magnified in Q′. (R) For embryos with indicated tace alleles, the percentage of non-crossing segments was compared with the corresponding FraΔC-overexpressing control group and quantified by Student’s t test. Number of embryos, n = 19, 27, 23, 24, 19, 18. (S and T) Compared with the control group, Tace overexpression significantly enhances the EW non-crossing phenotype (indicated by asterisks), which was quantified by Student’s t test in (T). Number of embryos, n = 23, 21. (U) Schematic describing the phenotypes observed in tace loss and gain of function. Scale bars represent 40 μm in (A), (B), (E), and (F) and 10 μm in the rest of the images. Anterior is up. Error bars indicate SEM; *p < 0.0332, **p < 0.0021.

Article Snippet: RNA FISH probe for Adam17 was generated by PCR amplifying the Adam17 sequence from pcDNA3.1-mADAM17 (Addgene) using the adam17_insitu _fwd and the Adam17_insitu_rev primer pair.

Techniques: Expressing, Labeling, Mutagenesis, Control, Over Expression

Journal: Cell reports

Article Title: Tace/ADAM17 is a bi-directional regulator of axon guidance that coordinates distinct Frazzled and Dcc receptor signaling outputs

doi: 10.1016/j.celrep.2022.111785

Figure Lengend Snippet: (A–G) Stage 16 Drosophila embryos, with GFP labeling eagle commissural neurons. (A and B) EW axons that defasciculate and project either ipsilaterally or away from the main EW axon bundles are observed in heterozygous or homozygous tace mutants (indicated by yellow triangles). (C–E) fra 3 , comm D e39 , and psn 12 alleles significantly enhance these phenotypes. Regions outlined by dashed lines are magnified in (A′), (B′), and (D′). (F) Quantification of the percentage of segments that have EW axon projection defects. Number of embryos, n = 17, 18, 17, 16, 24, 14, 14, 27. Statistical analysis was conducted with one-way ANOVA. (G) Schematic describing the phenotypes observed. (H–L) Stage 14 Drosophila embryos, with RNA FISH for comm shown in green and eagle neurons labeled in red. comm transcripts are detected at the midline (H′ and I′, white dotted lines) and in the cell bodies of eagle neurons (H–I‴, eagle neurons are outlined in solid yellow circles if expressing comm and in dotted yellow circles if not expressing comm ). (H″ and I″) Regions outlined in dashed lines are magnified in (H‴) and (I‴). (J) Quantification of the percentage of eagle neurons that express comm . Number of embryos, n = 24, 32. Statistical analysis was conducted with Student’s t test. (K) Quantification of the relative fluorescence intensity of comm RNA FISH signal normalized to the average fluorescence intensity of comm RNA FISH signal at the midline. Number of embryos, n = 9, 6. Statistical analysis was conducted with Student’s t test. (L) Schematic describing the phenotypes observed. In all micrographs, anterior is up and scale bars represent 10 μm. Error bars indicate SEM; **p < 0.0021, ***p < 0.0002, ****p < 0.0001.

Article Snippet: RNA FISH probe for Adam17 was generated by PCR amplifying the Adam17 sequence from pcDNA3.1-mADAM17 (Addgene) using the adam17_insitu _fwd and the Adam17_insitu_rev primer pair.

Techniques: Labeling, Expressing, Fluorescence

Journal: Scientific Reports

Article Title: Development of encoded Broccoli RNA aptamers for live cell imaging of alphavirus genomic and subgenomic RNAs

doi: 10.1038/s41598-020-61573-3

Figure Lengend Snippet: Colocalization between SINV RNA FISH and Broccoli. The images (objective lens 20×) show infected BHK cells (MOI = 5), fixed with 3.7% formaldehyde in PBS 6 h after infection. After adding DFHBI-1T, RNA FISH E1 + E2 (red) colocalizes well with Broccoli-DFHBI-1T (green) signal for both TEUTR4Br and TEds10Br. The scatter plot (X – red, Y – green) shows strong colocalization (R colocalization = 0.9049). Scale bar = 100 μm.

Article Snippet: Cells in each well were then incubated with two custom Stellaris RNA FISH probe sets each containing 48 short Quasar 570-labeled non-overlapping oligonucleotides against SINV E1 and E2 genes (LGC Biosearch Technologies), 25 nM each, in hybridization buffer (working buffer plus 10% dextran) at 37 °C overnight.

Techniques: Infection

Journal: Scientific Reports

Article Title: Development of encoded Broccoli RNA aptamers for live cell imaging of alphavirus genomic and subgenomic RNAs

doi: 10.1038/s41598-020-61573-3

Figure Lengend Snippet: Rare discrepancy between RNA FISH and Broccoli signals. The images (objective lens 63×) show TEds10Br-infected BHK cells (MOI = 5), fixed with 3.7% formaldehyde in PBS at 6 h after infection. Although rare, some cells showed a discrepancy between FISH and Broccoli signals (white arrow), resulting in a separated cluster in the scatter plot (X – red, Y – green). Scale bar = 20 μm.

Article Snippet: Cells in each well were then incubated with two custom Stellaris RNA FISH probe sets each containing 48 short Quasar 570-labeled non-overlapping oligonucleotides against SINV E1 and E2 genes (LGC Biosearch Technologies), 25 nM each, in hybridization buffer (working buffer plus 10% dextran) at 37 °C overnight.

Techniques: Infection

Journal: Scientific Reports

Article Title: Development of encoded Broccoli RNA aptamers for live cell imaging of alphavirus genomic and subgenomic RNAs

doi: 10.1038/s41598-020-61573-3

Figure Lengend Snippet: Live cell imaging of infected cells in mouse brain slices incubated with DFHBI-1T. ( A ) Channel mode and ( B ) lambda mode imaging of fresh TEds10Br-infected brain or ( C ) TE-infected brain. Broccoli-DFHBI-1T signal (green) in cells of the corpus callosum. ( D ) RNA E1 FISH imaging (red) of infected neurons in formalin-fixed brain tissue counterstained with DAPI. ( E ) Image of the 250 μm slice (McIlwain tissue chopper) of TEds10Br-infected mouse brain (3 days after infection) in a 35-mm glass-bottom dish in imaging medium containing DFHBI-1T with area of imaging for A, B indicated in the red square. Scale bar = 50 μm.

Article Snippet: Cells in each well were then incubated with two custom Stellaris RNA FISH probe sets each containing 48 short Quasar 570-labeled non-overlapping oligonucleotides against SINV E1 and E2 genes (LGC Biosearch Technologies), 25 nM each, in hybridization buffer (working buffer plus 10% dextran) at 37 °C overnight.

Techniques: Live Cell Imaging, Infection, Incubation, Imaging