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

Journal: bioRxiv

Article Title: DEAD box RNA helicases 5 and 17 are new host factors for Sindbis virus infection

doi: 10.1101/2023.09.21.558232

Figure Lengend Snippet: A ) Confocal microscopy analysis of SINV (+) RNA and DDX5 protein localization in mock and infected HCT116 cells at 24 hpi by RNA fluorescence in situ hybridization (FISH) (in magenta) combined with protein immunostaining (in green). DAPI staining (in blue) and merge of the different channels are shown. Magnification 40X, scale bar, 10µm. B ) RT-qPCR on SINV genomic (g) RNA upon DDX5 RIP or IgG RIP. Results are expressed as percentage of Input (total RNA) and represent the mean ± standard deviation (SD) of three biological replicates (n = 3). C ) Anti-dsRNA dot blot assay on serial dilutions of the total RNA (INPUT) and the undiluted RNA samples from DDX5-RIP or IgG-RIP, in mock and SINV infected conditions. J2 antibody was used to detect dsRNAs.

Article Snippet: Cells were fixed again with 3.7 % formaldehyde (Biosearch technologies) diluted in PBS 1X for10 min at room temperature and incubated over night at room temperature with the SINV genome specific LGC Biosearch Technologies’ Stellaris® RNA FISH Probe diluted in RNA FISH hybridization buffer (Stellaris, Biosearch technologies).

Techniques: Confocal Microscopy, Infection, Fluorescence, In Situ Hybridization, Immunostaining, Staining, Quantitative RT-PCR, Standard Deviation, Dot Blot

Journal: eLife

Article Title: HIV-1 nuclear import in macrophages is regulated by CPSF6-capsid interactions at the nuclear pore complex

doi: 10.7554/eLife.41800

Figure Lengend Snippet: ( A ) MDM from three donors were infected with HIV-1 NL4-3 4059 at an MOI of 8 for 72 hr. Viral DNA (green) was detected by FISH as described in Materials and Methods. CPSF6 (magenta) and CA (cyan) were detected by immunostaining and images were recorded by SDCM. The maximum projection of three focal planes acquired with an axial spacing of 0.2 µm is shown. Arrowheads indicate nuclear complexes. ( B ) MDM from three donors were infected with 100ng p24 (MOI 14.5) HIV-1 NL4-3 4059 (IN.eGFP) for 48 hr, fixed and click-labeled as in . CPSF5 (cyan) and CPSF6 (magenta) (top panel) or CPSF7 (white) and CPSF5 (cyan) (bottom panel) were detected by immunostaining. Images show a section through the nuclei of infected cells. Arrowheads indicate nuclear complexes. 112 cells were analyzed in total. ( C,D ) MDM from two donors in two independent experiments were infected with 100ng p24 (MOI 14.5) HIV-1 NL4-3ΔTat 4059 (IN.eGFP) ( C ) or HIV-1 NL4-3 4059 (IN.eGFP) ( D ). At 96 h p.i., a final concentration of 5 µM Flavopiridol ( D ) was added to the medium and infection was continued for 12 hr. Cells were fixed, click-labeled and CPSF6 (magenta) and laminA/C (white) were detected by immunostaining. Images show a section through the nucleus of representative infected cells. Arrowheads indicate nuclear complexes. 22 cells were analyzed in ( C ) and 37 cells in ( D ). ( E,F ) MDM from three donors, in two independent experiments, were infected at an MOI of 8 with HIV-1 NL4-3 4059 ( E ) or HIV-1 NL4-3ΔTat 4059 ( F ). At 108 h p.i. cells were fixed. CPSF6 (magenta) was detected by immunostaining and nucleus (blue) with Hoechst. Viral RNA (green) was detected by RNA FISH as explained in Materials and Methods. Arrowhead indicates nuclear complexes. 42 cells were analyzed in total in ( E ) and 49 cells in ( F ). ( G ) MDM from the same donors as in ( E,F ) were infected with HIV-1 NL4-3 4059 using the same conditions. At 96 h p.i. 5 µM Flavopiridol was added to the medium for 12 hr. Viral RNA (green) was detected by RNA FISH. CPSF6 (magenta) was detected by immunostaining and nucleus (blue) with Hoechst. Arrowheads indicate nuclear CPSF6 enrichments. 36 cells were analyzed in total. Scale bars in A-G: 5 µm.

Article Snippet: Stellaris probe for RNA FISH was synthesized by Biocat GmbH (Heidelberg, Germany) using HIV-1 NL4-3 proviral plasmid and labeled with CAL Fluor Red 610 dye.

Techniques: Infection, Immunostaining, Labeling, Concentration Assay

Journal: Developmental cell

Article Title: Xist Repeats A and B account for two distinct phases of X-inactivation establishment

doi: 10.1016/j.devcel.2020.05.021

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Stellaris FISH probes used for Atrx and Mecp2 nascent RNA FISH , LGC Biosearch Technologies , Custom.

Techniques: Recombinant, Isolation, Multiplex Assay, Software