Journal: Cellular & Molecular Biology Letters

Article Title: YAP1 promoter-associated noncoding RNA affects Ewing sarcoma cell tumorigenicity by regulating YAP1 expression

doi: 10.1186/s11658-025-00736-4

Figure Lengend Snippet: pncRNA_YAP1-1 and pncRNA_YAP1-2 are associated with YAP1 promoter. A Schematic representation of YAP1 locus gene from the Genome Browser, where pncRNAs_YAP1 are annotated (green box). ENST00000614441.1 (AP0015271.2) is pncRNA_YAP1-1 and ENST00000566440.1 (AP0015271.1) is pncRNA_YAP1-2 . B Endogenous pncRNA_YAP1-1 and pncRNA_YAP1-2 levels were analysed in different ES cell lines. C Cytosolic and nuclear RNA was extracted from SK-N-MC cells and analysed by RT-qPCR. MALAT1 is used as nuclear marker and NORAD as cytosolic marker. Values are the mean ± SD of three independent experiments, expressed as % of total RNA. D FISH experiment was performed by using Stellaris™ RNA FISH. Fixed SK-N-MC cells were probed for pncRNA_YAP1-1 (probes labelled with Quasar 570, red) and DAPI (nuclear staining) and imaged with 40X objective. Scale bar: 75 µm

Article Snippet: Custom StellarisTM RNA FISH Probes were designed against pncRNA_YAP1-1 by utilising the Stellaris RNA FISH Probe Designer (LGC, Biosearch Technologies, Petaluma, CA, USA) available online at www.biosearchtech.com/stellarisdesigner .

Techniques: Quantitative RT-PCR, Marker, Staining

Journal: Cellular & Molecular Biology Letters

Article Title: YAP1 promoter-associated noncoding RNA affects Ewing sarcoma cell tumorigenicity by regulating YAP1 expression

doi: 10.1186/s11658-025-00736-4

Figure Lengend Snippet: pncRNA_YAP1-1 overexpression promotes YAP1 cytoplasmic accumulation. A SK-N-MC cells were transfected with empty vector (pcDNA3.1) or a plasmid overexpressing pncRNA_YAP1-1 , and cytosolic and nuclear protein extracts were obtained to monitor YAP1 localization. GAPDH was used as cytosolic marker, and H3 was used as nuclear marker. Histograms represent the mean ± SD of three independent experiments, expressed as %. B Cells were transfected as in A and fixed, and FISH experiment was performed by using Stellaris™ RNA FISH. Cells were probed for pncRNA_YAP1-1 (probes labelled with Quasar 570, red) and DAPI (nuclear staining) and imaged with 40X objective. Scale bar: 75 µm. C Cytosolic and nuclear RNA was extracted from SK-N-MC cells transfected as in A and analysed by RT-qPCR. MALAT1 is used as nuclear marker and NORAD as cytosolic marker. Values are the mean ± SD of three independent experiments, expressed as % of total RNA. Statistical analyses were performed using Student’s t -test, p -values: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001

Article Snippet: Custom StellarisTM RNA FISH Probes were designed against pncRNA_YAP1-1 by utilising the Stellaris RNA FISH Probe Designer (LGC, Biosearch Technologies, Petaluma, CA, USA) available online at www.biosearchtech.com/stellarisdesigner .

Techniques: Over Expression, Transfection, Plasmid Preparation, Marker, Staining, Quantitative RT-PCR

Journal: Methods (San Diego, Calif.)

Article Title: Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains

doi: 10.1016/j.ymeth.2017.06.025

Figure Lengend Snippet: Using smFISH and immunofluorescence to quantitate post-transcriptional regulation in whole-mount Drosophila brains. A. Schematic of the Drosophila larval brain, showing a cell lineage of neurons at different stages of differentiation. B. Magnified schematic of a single neuroblast illustrating how smFISH and immunofluorescence can be used to quantify the nascent transcripts (large, overlapping blue and purple circles in the nucleus), mature mRNA (small blue circles), and protein (green). C. Relative levels of the specific gene products in different cell types/stages as an indirect measure for transcription, the regulation of splicing, mRNA stability and translation.

Article Snippet: Here StellarisTM smFISH probes were purchased from LGC BioSearch Technologies (California, USA).

Techniques: Immunofluorescence

Journal: Methods (San Diego, Calif.)

Article Title: Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains

doi: 10.1016/j.ymeth.2017.06.025

Figure Lengend Snippet: Simultaneous detection of multiple RNA species using smFISH. A. Schematic of the 3rd instar Drosophila larva brain illustrating the position of a neuroblast lineage . B-B’’’. Exemplar image of smFISH in the third instar larval brain generated using two sets of probes; one targets the exon region of the transcripts encoding RNA binding protein brain tumor ( brat ) (magenta) and the other targets the 3′UTR of the same gene (cyan) . Neuroblasts and progeny are labeled by driving the expression of membrane-tethered GFP using pros-gal4. C. Schematic of the 3rd instar Drosophila brain illustrating the position of mushroom body lobes, calyx, and Kenyon cell nuclei. D-D’’’. Detection of msp-300 mRNA in the calyx of larval mushroom bodies. The mushroom body is identified by the expression of Dlg1 protein coupled to GFP, the axon bundle, and cell nuclei are labeled with HRP and DAPI, respectively. Scale bars represent 50 µm. E. Schematic of the adult Drosophila brain illustrating the position of mushroom body lobes, calyx, and Kenyon cell nuclei. F-F’’’. Detection of CaMKII::YFP mRNA with a smFISH probe targeting the YFP mRNA sequence, and CaMKII::YFP protein in the adult mushroom body calyx (dotted line). G. Representative line profile comparing intensity values of single foci in YFP mRNA positive samples (blue line, F’) versus wild type control (yellow line, H’). H-H’’’. Negative control showing the YFP smFISH probe in a wild-type adult brain. Abbreviations: NB-neuroblast, ML-medial lobe, VL- ventral lobe, wt- wild type.

Article Snippet: Here StellarisTM smFISH probes were purchased from LGC BioSearch Technologies (California, USA).

Techniques: Generated, RNA Binding Assay, Labeling, Expressing, Membrane, Sequencing, Control, Negative Control

Journal: Methods (San Diego, Calif.)

Article Title: Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains

doi: 10.1016/j.ymeth.2017.06.025

Figure Lengend Snippet: Overview of smFISH, larval brain dissection, and orientation. A. Overview of the smFISH protocol for brain tissue. After brains are dissected from third instar larvae or adult flies, samples are fixed in 4% formaldehyde. After series of washes, brains are incubated in hybridization buffer containing the probe mixture targeting the gene of interest. Hybridization step takes place at 37 °C for between 8–15 h in a light protected environment. Following hybridization, samples undergo a further three washes and can then be mounted for imaging with the anti-fade mounting medium. B. Scissor dissection method for larval brains. Immobilize larva by gently holding the tip of the larval head using a pair of dull tweezers. Then cut the larva in half at approximately the position of the second bend of the trachea tubes. The brain should be exposed from the remaining tissue and can be isolated using scissors. This dissection method reduces tissue damage in comparison to isolating the brain by pulling the larvae apart using two pairs of tweezers. C. Distribution of different types of neuroblasts in the larval brain. Brain should be mounted in the orientation that is suitable for the purpose of the experiment.

Article Snippet: Here StellarisTM smFISH probes were purchased from LGC BioSearch Technologies (California, USA).

Techniques: Dissection, Incubation, Hybridization, Imaging, Isolation, Comparison

Journal: Methods (San Diego, Calif.)

Article Title: Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains

doi: 10.1016/j.ymeth.2017.06.025

Figure Lengend Snippet: Simultaneous detection of RNA and protein using smFISH in conjunction with antibody staining. A. The smFISH protocol is compatible with conventional antibody staining. An exemplary image showing simultaneous detection of Prospero RNA and Miranda protein. B-D Image without nuclear DAPI stain showing Prospero RNA and Miranda protein (B) and the respective greyscale images (C–D). Scale bar represents 10 µm (A) and 5 µm (B–D).

Article Snippet: Here StellarisTM smFISH probes were purchased from LGC BioSearch Technologies (California, USA).

Techniques: Staining

Journal: Methods (San Diego, Calif.)

Article Title: Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains

doi: 10.1016/j.ymeth.2017.06.025

Figure Lengend Snippet: Distinguishing transcriptional versus post-transcriptional regulation with smFISH. A. Location of the probe targeting the exon (magenta) and intron (blue) region of brat transcripts. B-B”. Quantitative analysis of brat transcription level in neuroblast (red outline) and neuroblast progeny (yellow outline). The smFISH signal showing primary transcription foci in B and B’ and automatic detection of foci using the “Spots” tool in the Imaris image analysis software (B”). C. Average intensity of transcription foci in neuroblast and progeny is not significantly different, suggesting brat RNA is transcribed at approximately the same level in both cell types (neuroblast: n = 6 neuroblasts/brain, 3 brains; neuroblast progeny: n = 100 foci, 3 brains). D-D”. Quantitative analysis of total brat RNA in neuroblast and neuroblast progeny. The smFISH signals of brat RNA detected by the exon probe is shown in D-D’ and the region of interest selected for average intensity analysis is shown in D”. Statistical analysis shows the level of total brat transcripts is significantly increased in neuroblast progeny (neuroblast: 6 neuroblast/brain, 3 brains; progeny: 6 clusters/brain, 3 brains). Scale bar represents 10 µm.

Article Snippet: Here StellarisTM smFISH probes were purchased from LGC BioSearch Technologies (California, USA).

Techniques: Software

Journal: Methods (San Diego, Calif.)

Article Title: Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains

doi: 10.1016/j.ymeth.2017.06.025

Figure Lengend Snippet: Quantitative analysis of sparse transcripts and RNA/protein expression in neuroblasts and progeny. A-A”. imp RNA is detected in Imp::GFP larval brains using an smFISH probe targeting the gfp sequence (magenta), with simultaneous detection of Imp::GFP protein (green). Individual foci of imp RNA are detected using the “Spots” tool in Imaris (yellow). B-C. Imp protein is expressed at a low level in third instar larval neuroblasts and is selectively up-regulated in a sub-population of neuroblast progeny. Quantitative analysis (one-way ANOVA with Tukey posthoc analysis) shows Imp protein level is significantly higher in selected neuroblast progeny (C). D-E. Up-regulation of imp RNA is found in the cell population that also expresses a high level of Imp protein (D-D’: yellow dotted line region). Quantitative analysis of foci density reveals that neuroblast progeny with higher levels of Imp protein also exhibit a significantly increased level of imp mRNA (E). neuroblast: red; Imp + progeny: yellow Imp − progeny: white. n = 3 cells or cell clusters/brain , 3 brains total. Scale bar represents 10 µm.

Article Snippet: Here StellarisTM smFISH probes were purchased from LGC BioSearch Technologies (California, USA).

Techniques: Expressing, Sequencing

Journal: Methods (San Diego, Calif.)

Article Title: Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains

doi: 10.1016/j.ymeth.2017.06.025

Figure Lengend Snippet: Using smFISH to identify neuroblasts, GMCs and immature neurons. A-A’’’. Conventional antibody labelling for neuroblast (Deadpan antibody; green) and ganglion mother cells (GMCs) (Deadpan − Asense ± ; magenta). B-B’’’. Immature neurons are distinguished from the newly born neuroblast progeny by the up-regulation of Prospero protein (bright yellow cells: B-B’; yellow outlined region: B”-B’”). C-E’. Labelling neuroblasts, GMCs and immature neurons using smFISH. Neuroblasts, GMCs and immature neurons are labeled with cyclin B , castor and prospero , respectively. F. Schematics showing labelling of neuroblast and neuroblast progeny using the conventional antibody staining method or the new smFISH method. neuroblast: green outlined region; GMCs: magenta outlined region; immature neuron: yellow outlined region. Scale bar represents 10 µm.

Article Snippet: Here StellarisTM smFISH probes were purchased from LGC BioSearch Technologies (California, USA).

Techniques: Labeling, Staining

Journal: Cellular & Molecular Biology Letters

Article Title: YAP1 promoter-associated noncoding RNA affects Ewing sarcoma cell tumorigenicity by regulating YAP1 expression

doi: 10.1186/s11658-025-00736-4

Figure Lengend Snippet: pncRNA_YAP1-1 and pncRNA_YAP1-2 are associated with YAP1 promoter. A Schematic representation of YAP1 locus gene from the Genome Browser, where pncRNAs_YAP1 are annotated (green box). ENST00000614441.1 (AP0015271.2) is pncRNA_YAP1-1 and ENST00000566440.1 (AP0015271.1) is pncRNA_YAP1-2 . B Endogenous pncRNA_YAP1-1 and pncRNA_YAP1-2 levels were analysed in different ES cell lines. C Cytosolic and nuclear RNA was extracted from SK-N-MC cells and analysed by RT-qPCR. MALAT1 is used as nuclear marker and NORAD as cytosolic marker. Values are the mean ± SD of three independent experiments, expressed as % of total RNA. D FISH experiment was performed by using Stellaris™ RNA FISH. Fixed SK-N-MC cells were probed for pncRNA_YAP1-1 (probes labelled with Quasar 570, red) and DAPI (nuclear staining) and imaged with 40X objective. Scale bar: 75 µm

Article Snippet: The Custom StellarisTM RNA FISH Probes against pncRNA_YAP1-1 were labelled with Quasar 570 Dye (LGC, Biosearch Technologies).

Techniques: Quantitative RT-PCR, Marker, Staining

Journal: Cellular & Molecular Biology Letters

Article Title: YAP1 promoter-associated noncoding RNA affects Ewing sarcoma cell tumorigenicity by regulating YAP1 expression

doi: 10.1186/s11658-025-00736-4

Figure Lengend Snippet: pncRNA_YAP1-1 overexpression promotes YAP1 cytoplasmic accumulation. A SK-N-MC cells were transfected with empty vector (pcDNA3.1) or a plasmid overexpressing pncRNA_YAP1-1 , and cytosolic and nuclear protein extracts were obtained to monitor YAP1 localization. GAPDH was used as cytosolic marker, and H3 was used as nuclear marker. Histograms represent the mean ± SD of three independent experiments, expressed as %. B Cells were transfected as in A and fixed, and FISH experiment was performed by using Stellaris™ RNA FISH. Cells were probed for pncRNA_YAP1-1 (probes labelled with Quasar 570, red) and DAPI (nuclear staining) and imaged with 40X objective. Scale bar: 75 µm. C Cytosolic and nuclear RNA was extracted from SK-N-MC cells transfected as in A and analysed by RT-qPCR. MALAT1 is used as nuclear marker and NORAD as cytosolic marker. Values are the mean ± SD of three independent experiments, expressed as % of total RNA. Statistical analyses were performed using Student’s t -test, p -values: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001

Article Snippet: The Custom StellarisTM RNA FISH Probes against pncRNA_YAP1-1 were labelled with Quasar 570 Dye (LGC, Biosearch Technologies).

Techniques: Over Expression, Transfection, Plasmid Preparation, Marker, Staining, Quantitative RT-PCR