rnascope target probe  (Addgene inc)

Journal: Molecular cell

Article Title: Profiling transcriptome composition and dynamics within nuclear compartments using SLAM-RT&Tag

doi: 10.1016/j.molcel.2025.02.012

Figure Lengend Snippet: A) Bar plot of IgG and SC35-targeted RT&Tag counts of CLK (left) and SRSF (right) transcripts in HEK293T cells. *p<0.05, BH-adjusted p-value. Data are represented as mean ± SEM. B-C) Genome track showing IgG and SC35-targeted RT&Tag signal over CLK2 (B, log scale) and SRSF5 (C) in HEK293T cells. The poison exon is marked with an asterisk and adjacent introns are highlighted. D) RNA-FISH targeting MALAT1 and SRSF11 intron (highlighted region in (F)) in K562 cells. Arrowheads mark overlap. Scale bar, 2μm. E-F) Genome track showing whole-cell RNA-sequencing (E) IgG and SC35-targeted RT&Tag signal (F) over SRSF11 in PladB-treated K562 cells. Poison exons are marked with asterisks and adjacent introns are highlighted. G) Bar plot of global (IgG) and localized (SC35) half-lives of SRSF11 in PladB-treated K562 cells. Fold change differences in localized/global half-lives are shown. See also .

Article Snippet: Subsequently, RNAscope probes targeting MALAT1 (Advanced Cell Diagnostics 578171-C3, 1:150 dilution) or retained intron sequences of MAN2C1, SRSF11, RPL3, RPS6KB2 (Advanced Cell Diagnostics custom, undiluted) were hybridized for 2 hours at 40°C.

Techniques: RNA Sequencing

Journal: Molecular cell

Article Title: Profiling transcriptome composition and dynamics within nuclear compartments using SLAM-RT&Tag

doi: 10.1016/j.molcel.2025.02.012

Figure Lengend Snippet: A) RT&Tag schematic: antibodies targeting a nuclear compartment epitope are used to tether pAG-Tn5 and an oligo(dT). B) Immunofluorescence of HEK293T nuclei stained for DAPI, H3K27me3 and SC35. Scale bar, 5μm. C) Principal component analysis of IgG, H3K27me3, and SC35-targeted RT&Tag in HEK293T cells. D) Volcano plot showing transcripts differentially enriched for H3K27me3 (left) and SC35 (right) over IgG-targeted RT&Tag in HEK293T cells (log 2 FC>2, FDR<0.05, n=3). E) Overlap of transcripts enriched for H3K27me3 and SC35-targeted RT&Tag in HEK293T cells. F) Genome tracks showing IgG, H3K27me3, and SC35-targeted RT&Tag signal over XIST (left) and MALAT1 (right) in HEK293T cells. See also .

Article Snippet: Subsequently, RNAscope probes targeting MALAT1 (Advanced Cell Diagnostics 578171-C3, 1:150 dilution) or retained intron sequences of MAN2C1, SRSF11, RPL3, RPS6KB2 (Advanced Cell Diagnostics custom, undiluted) were hybridized for 2 hours at 40°C.

Techniques: Immunofluorescence, Staining

Journal: Molecular cell

Article Title: Profiling transcriptome composition and dynamics within nuclear compartments using SLAM-RT&Tag

doi: 10.1016/j.molcel.2025.02.012

Figure Lengend Snippet: A) Violin plots of RNA-seq expression of speckle transcripts and top 5% expressed transcripts in K562 cells. *p<0.05, unpaired t-test. CPM-Counts per million. B) Scatter plot showing lack of correlation between gene expression (log 2 CPM) and distance to nuclear speckle (μm, TSA-seq) in K562 cells. C-D) Violin plots of number of exons per major isoform (C) and alternative isoforms (D) of speckle transcripts and all annotated mRNA transcripts in K562 cells. *p<0.05, unpaired t-test. E and F) Heatmaps of IgG and SC35-targeted RT&Tag signal 500bp upstream of the 3’end (E) and within introns (F) of speckle transcripts in K562 cells. G) Genome track showing IgG and SC35-targeted RT&Tag signal over MAN2C1 in K562 cells. H) RNA-FISH targeting MALAT1 and MAN2C1 intron (highlighted region in (F)) in K562 cells. Arrowheads mark overlap. Scale bars, 2μm. See also .

Article Snippet: Subsequently, RNAscope probes targeting MALAT1 (Advanced Cell Diagnostics 578171-C3, 1:150 dilution) or retained intron sequences of MAN2C1, SRSF11, RPL3, RPS6KB2 (Advanced Cell Diagnostics custom, undiluted) were hybridized for 2 hours at 40°C.

Techniques: RNA Sequencing, Expressing, Gene Expression

Journal: Molecular cell

Article Title: Profiling transcriptome composition and dynamics within nuclear compartments using SLAM-RT&Tag

doi: 10.1016/j.molcel.2025.02.012

Figure Lengend Snippet: A) Violin plots of distances from gene bodies of speckle transcripts and all mRNA transcripts to nuclear speckles (TSA-seq) in K562 cells. *p<0.05, unpaired t-test. B) Stacked bar plots of percentage of speckle transcripts and all mRNA transcripts transcribed within SPIN states (“speckle”, “active”, “repressive”, and “lamina”) in K562 cells. C-D) Heatmaps of IgG and SC35-targeted RT&Tag signal over introns (C) and 500bp upstream of the 3’end (D) of speckle transcripts transcribed from different SPIN states in K562 cells. E-F) Profile plots of IgG and SC35-targeted RT&Tag over retained introns (E) and 500bp upstream of the 3’end of transcripts that gain retained introns (F) in PladB-treated K562 cells. G) Genome tracks showing IgG and SC35-targeted RT&Tag signal over RPS6KB2 and RPL3 in PladB-treated K562 cells. Retained introns are highlighted. H) RNA-FISH targeting MALAT1 and introns (asterisk-marked regions in (F)) of RPS6KB2 (left) and RPL3 (right) in K562 cells treated with DMSO (top) or PladB (bottom) for 4h. Arrowheads mark overlap. Scale bars, 2μm. See also .

Article Snippet: Subsequently, RNAscope probes targeting MALAT1 (Advanced Cell Diagnostics 578171-C3, 1:150 dilution) or retained intron sequences of MAN2C1, SRSF11, RPL3, RPS6KB2 (Advanced Cell Diagnostics custom, undiluted) were hybridized for 2 hours at 40°C.

Techniques:

Journal: Molecular cell

Article Title: Profiling transcriptome composition and dynamics within nuclear compartments using SLAM-RT&Tag

doi: 10.1016/j.molcel.2025.02.012

Figure Lengend Snippet: A) Correlation plot of transcript levels within nuclear and cytoplasmic fractions in K562 cells. FPKM-Fragments Per Kilobase of transcript per Million mapped reads. B) Volcano plot showing differential kinetic analysis of speckle transcripts (k deg log 2 FC>2, FDR<0.05, n=3) in K562 cells (left). Bar plot of SC35 over IgG k deg for the top 15 persistent transcripts (right). C) Genome track showing IgG and SC35-targeted SLAM-RT&Tag signal over MALAT1 in K562 cells, half-lives (t 1/2 ) listed below. D) Violin plots of global (IgG) and localized (SC35) half-lives of speckle transcripts in K562 cells. *p<0.05, unpaired t-test. E) Violin plots of IgG over SC35 k deg of speckle transcripts in K562 cells treated with DMSO or PladB for 4h. *p<0.05, unpaired t-test. F) Genome track showing IgG and SC35-targeted SLAM-RT&Tag signal over FUS in PladB-treated K562 cells, half-lives (t 1/2 ) listed below. See also .

Article Snippet: Subsequently, RNAscope probes targeting MALAT1 (Advanced Cell Diagnostics 578171-C3, 1:150 dilution) or retained intron sequences of MAN2C1, SRSF11, RPL3, RPS6KB2 (Advanced Cell Diagnostics custom, undiluted) were hybridized for 2 hours at 40°C.

Techniques:

Journal: Molecular cell

Article Title: Profiling transcriptome composition and dynamics within nuclear compartments using SLAM-RT&Tag

doi: 10.1016/j.molcel.2025.02.012

Figure Lengend Snippet: A) Bar plot of IgG and SC35-targeted RT&Tag counts of CLK (left) and SRSF (right) transcripts in HEK293T cells. *p<0.05, BH-adjusted p-value. Data are represented as mean ± SEM. B-C) Genome track showing IgG and SC35-targeted RT&Tag signal over CLK2 (B, log scale) and SRSF5 (C) in HEK293T cells. The poison exon is marked with an asterisk and adjacent introns are highlighted. D) RNA-FISH targeting MALAT1 and SRSF11 intron (highlighted region in (F)) in K562 cells. Arrowheads mark overlap. Scale bar, 2μm. E-F) Genome track showing whole-cell RNA-sequencing (E) IgG and SC35-targeted RT&Tag signal (F) over SRSF11 in PladB-treated K562 cells. Poison exons are marked with asterisks and adjacent introns are highlighted. G) Bar plot of global (IgG) and localized (SC35) half-lives of SRSF11 in PladB-treated K562 cells. Fold change differences in localized/global half-lives are shown. See also .

Article Snippet: Subsequently, RNAscope probes targeting MALAT1 (Advanced Cell Diagnostics 578171-C3, 1:150 dilution) or retained intron sequences of MAN2C1, SRSF11, RPL3, RPS6KB2 (Advanced Cell Diagnostics custom, undiluted) were hybridized for 2 hours at 40°C.

Techniques: RNA Sequencing

Journal: Clinical and Translational Medicine

Article Title: Integrated spatial multi‐omics profiling of Fusobacterium nucleatum in breast cancer unveils its role in tumour microenvironment modulation and cancer progression

doi: 10.1002/ctm2.70273

Figure Lengend Snippet: RNAscope FISH and CISH analyses demonstrate the heterogeneous distribution of F. nucleatum in breast tumour tissues. (A) RNAscope FISH analysis of tumour sections from BC patients. Inverse colour images are displayed to highlight bacteria vividly. Green box: F. nucleatum ‐negative region. Yellow box: F. nucleatum ‐positive region. Yellow arrows indicate F. nucleatum positivity. Red: F. nucleatum (ATCC25586) specific probe. Blue: DAPI. Scale bar: 200 µm (left), 50 µm (right). (B) RNAscope FISH analysis of BC tumour sections (as shown in A). Inverse colour images are displayed to highlight bacteria vividly. Yellow arrows indicate F. nucleatum positivity. Red: F. nucleatum (ATCC25586) specific probe. Blue: DAPI. Scale bar: 2 µm. (C) Upper left and right panels: RNAscope‐CISH images showing the spatial distribution of F. nucleatum in BC tumour tissue, with the F. nucleatum probe highlighted in red and the Eubacteria probe in green. Bottom left panel: Hematoxylin and eosin stain (H&E) of the RNAscope image. The scale bar is shown in the figure.

Article Snippet: Each slide was treated with 150 μL of RNAscope probe mixture targeting F. nucleatum (B‐Fusobacterium‐23S‐3zz‐C2; Advanced Cell Diagnostics).

Techniques: RNAscope, Bacteria, H&E Stain

Journal: Clinical and Translational Medicine

Article Title: Integrated spatial multi‐omics profiling of Fusobacterium nucleatum in breast cancer unveils its role in tumour microenvironment modulation and cancer progression

doi: 10.1002/ctm2.70273

Figure Lengend Snippet: GeoMx digital spatial profiler (DSP) experimental protocols. GeoMx DSP was used to assess F. nucleatum ‐related niches in BC tumours. 5 µm continuous FFPE sections were prepared from the tissue samples, three consecutive sections were selected for distinct analyses: one for H&E staining to assess tissue morphology, one for RNAscope CISH staining to identify the spatial distribution of F. nucleatum , and one for DSP analysis after the immunohistochemical (IHC) staining targeting immune (CD45+) and epithelial (PanCK+) compartments. Based on H&E and RNAscope CISH imaging of continuous tissue sections, regions of interest (ROIs) were delineated and categorized as either F. nucleatum ‐positive or F. nucleatum ‐negative. Samples were analyzed for F. nucleatum ‐positive AOIs and F. nucleatum ‐negative AOIs in the CD45+ and PanCK+ redions, involving the release of photocleavable barcode oligonucleotides for sequencing. The sequenced oligonucleotides provided spatial information about the respective RNA and protein targets in F. nucleatum ‐positive or F. nucleatum ‐negative regions.

Article Snippet: Each slide was treated with 150 μL of RNAscope probe mixture targeting F. nucleatum (B‐Fusobacterium‐23S‐3zz‐C2; Advanced Cell Diagnostics).

Techniques: Staining, RNAscope, Immunohistochemical staining, Immunohistochemistry, Imaging, Sequencing

Journal: Clinical and Translational Medicine

Article Title: Integrated spatial multi‐omics profiling of Fusobacterium nucleatum in breast cancer unveils its role in tumour microenvironment modulation and cancer progression

doi: 10.1002/ctm2.70273

Figure Lengend Snippet: Spatial transcriptomic and proteomic analysis. (A) RNAscope‐CISH images showing the distribution of F. nucleatum (red) in BC tissue, with serial IHC images depicting the distribution of CD45+ (red) and PanCK+ (green) cells, identifying immune and epithelial compartments in BC tissue, respectively. The inset highlights representative AOIs for F. nucleatum‐ positive and F. nucleatum ‐negative areas with corresponding UV‐exposed regions. (B) Top: Gene overlap quantified at RNA and protein levels using OmicStudio tools ( https://www.omicstudio.cn/tool ). Bottom: Percentage of differentially expressed transcripts and proteins, calculated by significance ( p < .05). (C, D) Principal component analysis of all quantified RNAs (C) and proteins (D). Data were analyzed using R (version 4.1.3). (E, F) Heatmaps showing differentially expressed transcripts (E) and proteins (F). Data were analyzed using R (version 4.1.3) and visualized with the ComplexHeatmap package.

Article Snippet: Each slide was treated with 150 μL of RNAscope probe mixture targeting F. nucleatum (B‐Fusobacterium‐23S‐3zz‐C2; Advanced Cell Diagnostics).

Techniques: RNAscope

Journal: Clinical and Translational Medicine

Article Title: Integrated spatial multi‐omics profiling of Fusobacterium nucleatum in breast cancer unveils its role in tumour microenvironment modulation and cancer progression

doi: 10.1002/ctm2.70273

Figure Lengend Snippet: Differential and functional enrichment analysis of transcriptomics and proteomics. (A, B) Volcano plots showing differentially expressed transcripts (A) and proteins (B). Bar charts highlight significant changes in RNAs and proteins ( p < .05, FC > 1.2). (C, D) KEGG pathway analysis of significantly different RNAs (C) and proteins (D). (E, F) KEGG pathway analysis of significantly different RNAs in MCF‐7 and MDA‐MB‐231 cells co‐cultured with F. nucleatum . Enrichment was performed using OmicStudio tools at https://www.omicstudio.cn/tool . Data were analyzed with R version 4.1.3 (ggplot2 package: 3.3.3).

Article Snippet: Each slide was treated with 150 μL of RNAscope probe mixture targeting F. nucleatum (B‐Fusobacterium‐23S‐3zz‐C2; Advanced Cell Diagnostics).

Techniques: Functional Assay, Cell Culture

Journal: Clinical and Translational Medicine

Article Title: Integrated spatial multi‐omics profiling of Fusobacterium nucleatum in breast cancer unveils its role in tumour microenvironment modulation and cancer progression

doi: 10.1002/ctm2.70273

Figure Lengend Snippet: Key proteins and their role in signalling pathways. (A) Venn diagram showing intersecting genes of the MAPK and focal adhesion signalling pathways at the transcriptomic and proteomic levels. (B) Pathway network analysis of the MAPK signalling, focal adhesion, and necroptosis pathways at the transcriptomic and proteomic levels. (C) Differential expression of VEGFD and PAK1 in F. nucleatum‐ positive and F. nucleatum ‐negative tumour regions (* p < .05; ** p < .01). (D) Protein–protein interaction (PPI) network displaying associations among the top 20 ranked proteins. (E) Network heatmap showing the association of VEGFD and PAK1 with the top 20 important proteins. (F) Changes in significantly different proteins in the MAPK signalling pathway. Red indicates upregulation; green indicates downregulation. The statistical significance was calculated using the Student's t ‐test.

Article Snippet: Each slide was treated with 150 μL of RNAscope probe mixture targeting F. nucleatum (B‐Fusobacterium‐23S‐3zz‐C2; Advanced Cell Diagnostics).

Techniques: Quantitative Proteomics

Journal: Clinical and Translational Medicine

Article Title: Integrated spatial multi‐omics profiling of Fusobacterium nucleatum in breast cancer unveils its role in tumour microenvironment modulation and cancer progression

doi: 10.1002/ctm2.70273

Figure Lengend Snippet: Mechanism of F. nucleatum promoting proliferation and migration of breast cancer cells. (A) Confocal microscopy showing spatial interaction between breast cancer cells (green: cytoskeleton; blue: nuclei) and F. nucleatum (red). Scale bar: 5 µm. (B) Line graph of CCK‐8 cell proliferation assay ( n = 5; Student's t ‐test). Statistical significance was determined by Student's t ‐test (* p < .05; ** p < .01; *** p < .001). (C) The wound‐healing assay was initiated with a uniform scratch width of .5 mm. (D) Migration distance quantification. Scratch width was measured at four predefined equidistant points per well at 0 and 24h using ImageJ, and distance was normalized to the initial width (0 h) ( n = 4; Student's t ‐test). (E, F) Line graph of CCK‐8 cell proliferation assay ( n = 5; Student's t ‐test). * p < .05; ** p < .01; *** p < .001. (G) Western blot analysis of VEGFD, PAK1, and MAPK pathway proteins in MDA‐MB‐231 and MCF‐7 cells co‐cultured with F. nucleatum . Blots are representative of three biological replicates. (H, I) EdU proliferation assay: EdU staining (red: proliferating cells; blue: DAPI) (H) and quantification of EdU‐positive cells (I). Three independent replicates were analyzed ( n = 3; Student's t ‐test). (J) Line graph of CCK‐8 proliferation assay after siRNA interference ( n = 5; Student's t ‐test). * p < .05; ** p < .01; *** p < .001. (K, M) Wound‐healing assay: Representative scratch images (K), schematic of the plate insert (L), and migration distance quantification (M). Scratch closure was measured at four equidistant positions per well ( n = 4; Student's t‐test). * p < .05; ** p < .01; *** p < .001. (N, O) Transwell migration assay: Crystal violet‐stained migrated cells (N) and quantification (O). Four independent experiments were performed ( n = 4; Student's t ‐test). * p < .05; ** p < .01; *** p < .001.

Article Snippet: Each slide was treated with 150 μL of RNAscope probe mixture targeting F. nucleatum (B‐Fusobacterium‐23S‐3zz‐C2; Advanced Cell Diagnostics).

Techniques: Migration, Confocal Microscopy, CCK-8 Assay, Proliferation Assay, Wound Healing Assay, Western Blot, Cell Culture, Staining, Transwell Migration Assay