egfp smfish probes  (LGC Biosearch)

Journal: bioRxiv

Article Title: Destabilisation of bam transcripts terminates the mitotic phase of Drosophila female germline differentiation

doi: 10.1101/2024.07.24.604138

Figure Lengend Snippet: ( A ) Example ovarioles from mCherry RNAi or bam RNAi driven by bam-GAL4 , stained with Hoechst (DNA, blue). White arrows indicate 8 cell egg chambers. Scale bar 50 μm. ( B ) Quantitation of the proportion of ovarioles with one or more 8 cell egg chambers. n = 300 ovarioles for each genotype, from three replicate experiments. p < 0.003, t-test. ( C ) wild type germarium stained for bam mRNA (smFISH, magenta and gray-scale), f-actin (phalloidin, gray-scale), fusome (alpha-spectrin, yellow) and DNA (Hoechst, blue). Images are two z slices from a single stack. A single 2cc is outlined in cyan, and a 4cc is outlined in red. ( Di ) Cartoon depicting the different transgenes used. Staining for GFP protein (green), gfp smFISH (magenta), f-actin (phalloidin, gray-scale) and fusome (alpha-spectrin, yellow) in example germaria from ( ii ) BamFlyFOS ( Sarov et al , 2016 ) ( sfGFP probes), ( iii ) BamPGFP ( eGFP probes). Scale bars 15 μm.

Article Snippet: smFISH probes against bam, egfp, sfgfp and rbp9 were designed with the Stellaris Probe Designer (Biosearch Technologies) (Supplementary Table 1).

Techniques: Staining, Quantitation Assay

Journal: PLoS Biology

Article Title: Identification of 3′ UTR motifs required for mRNA localization to myelin sheaths in vivo

doi: 10.1371/journal.pbio.3001053

Figure Lengend Snippet: (A) Schematic of the MS2 system to visualize mRNA localization in oligodendrocytes. sox10 regulatory DNA drives expression of nuclear-localized MS2 coat protein, NLS-MCP-EGFP (orange crescent and green star). mbpa regulatory elements drive expression of mRNA encoding mScarlet-CAAX fluorescent protein with a repetitive sequence that creates 24 stem loops (24xMBS). When co-expressed, the mRNA–protein complex is exported from the nucleus and localized via the 3′ UTR. (B) Schematic of MS2 expression plasmids used for transient expression in oligodendrocytes with target sequences for Tol2 transposase to facilitate transgene integration. (C and D) Representative images of localization directed by the mbpa (C) or control sv40 3′ UTR (D). Asterisks mark cell bodies with high expression levels of the nuclear-localized MCP-EGFP. Boxed areas are enlarged to highlight sheath termini (arrows). (E) Average mRNA abundance per myelin sheath, measured by EGFP fluorescence intensity normalized to the average intensity of the sv40 control. sv40 : n = 5 larvae, 35 sheaths. mbpa : n = 6 larvae, 38 sheaths. (F) Average mRNA abundance per soma, measured by EGFP fluorescence intensity normalized to the average intensity of the sv40 control. sv40 : n = 11 larvae, 20 cell bodies. mbpa : n = 15 larvae, 21 cell bodies. (G and H) Representative images of 2 myelinating oligodendrocytes expressing mRNA lacking the 24xMBS . NLS-MCP-EGFP remains in the nucleus at 3 dpf (G) and 5 dpf (H). Scale bars, 10 μm. Statistical significance evaluated using Wilcoxon test. The underlying numerical data can be found in and Data. 3' UTR, 3' untranslated region; dpf, days post fertilization.

Article Snippet: The EGFP smFISH probes were purchased from Stellaris LGC Biosearch Technologies.

Techniques: Expressing, Sequencing, Control, Fluorescence

Journal: PLoS Biology

Article Title: Identification of 3′ UTR motifs required for mRNA localization to myelin sheaths in vivo

doi: 10.1371/journal.pbio.3001053

Figure Lengend Snippet: (A and B) Representative images of smFISH experiments using 4 dpf transgenic larva expressing EGFP-CAAX to mark oligodendrocytes. Images show sagittal sections of the hindbrain. DAPI stain labels nuclei. Sections were treated with smFISH probes designed to detect mbpa (A) or egfp (B) mRNA. Asterisks mark cell bodies and brackets mark myelin tracts. Scale bars, 10 μm. (C) Average mbpa mRNA density per cell body or equivalent volume of myelin from 3 to 5 dpf. Density was measured using the integrated density of fluorescence intensity in cell bodies and approximately equal volumes of myelin along the myelin tracts. A minimum (n) for each group was 3 larvae, 6 cell bodies, and 15 myelin regions. Statistical significance evaluated using Wilcoxon test. (D) Proportion of egfp or mbpa mRNA abundance in cell bodies compared to myelin tracts. A minimum (n) for each group was 3 larvae, 11 cell bodies, and 21 myelin regions. (E) Average mbpa mRNA density within individual sheaths plotted as a function of sheath length. Statistical significance evaluated using Spearman’s correlation coefficient. Shaded area represents 95% confidence interval. n = 7 embryos, 26 sheaths. The underlying numerical data can be found in – Data. dpf, days post fertilization; smFISH, single molecule fluorescent in situ hybridization.

Article Snippet: The EGFP smFISH probes were purchased from Stellaris LGC Biosearch Technologies.

Techniques: Transgenic Assay, Expressing, Staining, Fluorescence, In Situ Hybridization

Journal: PLoS Biology

Article Title: Identification of 3′ UTR motifs required for mRNA localization to myelin sheaths in vivo

doi: 10.1371/journal.pbio.3001053

Figure Lengend Snippet: (A) smFISH images of a single optical section of a myelin sheath in a 3-dpf larva spinal cord. mbpa transcripts line the myelin sheath. Arrows highlight clusters of mbpa mRNA transcripts. (B) smFISH images of a single optical section of myelin tracts in the hindbrain of a 5-dpf larva. Boxed area magnified to highlight sheath termini (arrows). (C) smFISH images of a single optical section in transverse plane of myelin sheaths in a 5-dpf larva midbrain. Scale bars (A, B, and D), 5 μm; (C, boxed enlargements), 1 μm. (D) Representative images from MS2 system showing colocalization of mRNA containing mbpa 3′ UTR and F-actin in a myelinating oligodendrocyte. Asterisk marks the cell body, and boxes are magnified to highlight sheath termini. Arrows highlight sheaths with mRNA, and arrowheads highlight sheaths lacking mRNA. (E) Proportion of sheaths with mRNA enriched in sheath termini at 4 dpf using the MS2 system. Proportion measured as (sheaths with enrichment / number of sheaths) = 10/35 sv40 , 18/38 mbpa . (F) Average fluorescence intensity of MS2 mRNA reporter containing the sv40 or mbpa 3′ UTRs across a 7-μm distance, at 0.2-μm intervals, from myelin sheath termini at 4 dpf. Each line scan was normalized to the average fluorescent intensity per sheath. All normalized values for each distance were then averaged. Shaded area represents 95% confidence interval. Statistical significance was evaluated every 0.2 μm using Wilcoxon test, and the distance between 0.8–1.0 μm was statistically significant (blue line). sv40 3′ UTR n = 5 larvae, 35 sheaths. mbpa 3′ UTR n = 6 larvae, 38 sheaths. The underlying numerical data can be found in and Data. 3' UTR, 3' untranslated region; dpf, days post fertilization; F-actin, filamentous actin; smFISH, single molecule fluorescent in situ hybridization.

Article Snippet: The EGFP smFISH probes were purchased from Stellaris LGC Biosearch Technologies.

Techniques: Fluorescence, In Situ Hybridization

Journal: PLoS Biology

Article Title: Identification of 3′ UTR motifs required for mRNA localization to myelin sheaths in vivo

doi: 10.1371/journal.pbio.3001053

Figure Lengend Snippet: (A) Work flow to identify 3′ UTR candidates from RNA-seq data [ , , ]. (B) Representative images from MS2 system showing localization of mRNAs containing different 3′ UTR sequences in oligodendrocytes. Asterisks mark cell bodies. Scale bars, 10 μm. (C) Table listing candidate 3′ UTRs incorporated into the MS2 system, 3′ UTR length, and the percentage of sequence that was cloned based on the annotated genome (GRCz11). (D) Average mRNA abundance, measured by average EGFP fluorescent intensity, per myelin sheath for each 3′ UTR. Normalized to sv40 control, statistical significance evaluated using Wilcoxon test. A minimum (n) of 5 larvae and 18 sheaths were used in each condition at 4 dpf. The underlying numerical data can be found in and Data. 3' UTR, 3' untranslated region; dpf, days post fertilization; RNA-seq, RNA sequencing.

Article Snippet: The EGFP smFISH probes were purchased from Stellaris LGC Biosearch Technologies.

Techniques: RNA Sequencing, Sequencing, Clone Assay, Control

Journal: PLoS Biology

Article Title: Identification of 3′ UTR motifs required for mRNA localization to myelin sheaths in vivo

doi: 10.1371/journal.pbio.3001053

Figure Lengend Snippet: Representative images of smFISH experiments to visualize egfp , eif4ebp2 , or fmr1 mRNA localization at 4 dpf (A and B) and 5 dpf (C and D) in sagittal sections of hindbrain (A, C) or transverse sections of the Mauthner axon in the spinal cord (B, D). Dashed lines outline cell bodies marked by EGFP-CAAX. Scale bars, 5 μm (A, C) or 1 μm (B, D). dpf, days post fertilization; smFISH, single molecule fluorescent in situ hybridization.

Article Snippet: The EGFP smFISH probes were purchased from Stellaris LGC Biosearch Technologies.

Techniques: In Situ Hybridization

Journal: Molecular cell

Article Title: RNase L reprograms translation by widespread mRNA turnover escaped by antiviral mRNAs

doi: 10.1016/j.molcel.2019.07.029

Figure Lengend Snippet: (A) TapeStation analysis of the 28S and 18S rRNAs. (B) Immunoblot analysis of eIF2α, eIF2α-P51S (p-eIF2α), GADD34, and GAPDH in indicated A549 cell lines. (C) Quantification of the p-eIF2α:eIF2α ratio as represented in (B). Bars represent the average ratio +/− SEM from independent replicates (n=5-9). (D) S-35 metabolic in A549 cells transfected with poly(I:C) or treated with 250 uM sodium arsenite then treated with or without ISRIB (50 nM). (E) Quantification of IFN-β secretion from WT and RL-KO A549 via ELISA. Limit of quantification was 50 pg/ml. Bars represent Average +/− S.D. from three independent experiments. *** indicates p-value <0.001 as determined by student’s t-test. (F) Diagram of the eGFP expression vector in which eGFP ORF containing the IFN-β UTRs is driven by the IFN-β promoter. (G) smFISH for GAPDH and eGFP mRNAs in A549-WT cells with the IFN-β promoter-eGFP vector stably incorporated. GFP fluorescence is shown. (H) Similar to (G) but in A549- RL-KO cells. (I) Quantification of mean eGFP intensity from at least fifteen WT and RL-KO cells as represented in (G) and (H).

Article Snippet: Custom IFNB1, eGFP, and IL-6 smFISH probes ( Data S6 ) were designed using Stellaris smFISH probe designer (Biosearch Technologies) available online at http://www.biosearchtech.com/stellaris-designer .

Techniques: Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Expressing, Plasmid Preparation, Stable Transfection, Fluorescence

Journal: Molecular cell

Article Title: RNase L reprograms translation by widespread mRNA turnover escaped by antiviral mRNAs

doi: 10.1016/j.molcel.2019.07.029

Figure Lengend Snippet: (A) Diagrams of endogenous IFN-β gene and eGFP constructs driven by the IFN-β promoter. (B) smFISH for IFN-β (top images) and eGFP (lower images) mRNAs from expression constructs depicted directly to the left in (A) eighth hours post-poly(I:C). IFN-β smFISH was performed in WT and RL-KO A549 cells with the IFN-βpI5,GORFI3 construct stably incorporated. (C) Quantification of smFISH foci per cell as represented in the images in (B) directly to the left. (D) Diagram of CMV promoter-driven eGFP. (E) smFISH for eGFP mRNA driven by the CMV promoter in WT cells with or without poly(I:C) transfection. smFISH for GAPDH mRNA and GFP fluorescence in WT and RL-KO cells is shown in Figure S7A,B. (F) Quantification of eGFP smFISH represented in (E). (G) Immunoblot analysis of p-IRF3 (rep 1). Normalized p-IRF3 band intensity from two experiments are shown below. (H) IFN-β smFISH from non-deconvolved images. Arrows mark high-intensity RNase A-and Actinomycin D-sensitive foci consistent with nascent transcripts at IFN-β loci (Figure S7D,E,F). (I) Quantification of the relative intensity of IFN-β TSS in WT and RL-KO cells represented in (H). Between 111-138 foci were analyzed from three independent experiments. (J) Model of RNase L-mediated regulation of translation via mRNA degradation escaped by antiviral mRNAs.

Article Snippet: Custom IFNB1, eGFP, and IL-6 smFISH probes ( Data S6 ) were designed using Stellaris smFISH probe designer (Biosearch Technologies) available online at http://www.biosearchtech.com/stellaris-designer .

Techniques: Construct, Expressing, Stable Transfection, Transfection, Fluorescence, Western Blot

Journal: Molecular cell

Article Title: RNase L reprograms translation by widespread mRNA turnover escaped by antiviral mRNAs

doi: 10.1016/j.molcel.2019.07.029

Figure Lengend Snippet: Key Resources Table

Article Snippet: Custom IFNB1, eGFP, and IL-6 smFISH probes ( Data S6 ) were designed using Stellaris smFISH probe designer (Biosearch Technologies) available online at http://www.biosearchtech.com/stellaris-designer .

Techniques: Recombinant, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Plasmid Preparation, Software