Journal: Nature

Article Title: GlycoRNA complexed with heparan sulfate regulates VEGF-A signalling

doi: 10.1038/s41586-025-10052-8

Figure Lengend Snippet: a , Representative images of U2OS cells co-stained with Siglec-11 (yellow) and 9D5 (magenta). An enlargement of the hatched box is shown, and a bright field (BF) is shown to represent the outline of the cell membrane. The nearest neighbour distance analysis of the Siglec pairs is also imaged (bottom). For each pair, the distance (µm) from that anchor (left side protein name in the figure key) to the other pair was calculated across the indicated number of cells. These values were plotted in a density histogram. b , Dot plot of genes identified in the genome-wide CRISPR-KO screen for the loss of Siglec-11 cell surface binding ranked by CRISPR score. The top 15 gene names are displayed, with cut-off shown. The inset illustrates how Siglec-11 could interact with a glycoRNA. c , Dot plot of genes identified in the genome-wide KO screen as in panel b , for the loss of 9D5 cell surface binding. The inset illustrates how 9D5 could interact with a glycoRNA. d , Upset plot analysis of genes with a score cut-off of −0.8 from the Siglec-11, 9D5 and MAA-I genome-wide KO screens. The common overlapping hits between 9D5 and Siglec-11 are highlighted in blue. The total number of hits for each intersection is noted. Statistical assessment was performed with a hypergeometric test and P values are shown without adjustment. e , Representative images of WT and KO U2OS cells stained live with 10E4, Siglec-11–Fc, 9D5 and Siglec-7 (all in red). Ab, antibody; dsRNA, double-stranded RNA; mE, mEmerald. f , Quantification of the indicated signal dot numbers and intensity per cell from panels e , g from three independent experiments. Mut, mutant. g , Representative images of the indicated cell lines stained live with anti-DDX21 and anti-hnRNP-U (both in red).

Article Snippet: For DDX21 and hnRNP-U staining, 2.5 μg ml −1 of anti-DDX21 (Novus Biological) and anti-hnRNP-U (Proteintech) were incubated with cells for 45 min on ice.

Techniques: Staining, Membrane, Genome Wide, CRISPR, Binding Assay, Mutagenesis

Journal: Nature

Article Title: GlycoRNA complexed with heparan sulfate regulates VEGF-A signalling

doi: 10.1038/s41586-025-10052-8

Figure Lengend Snippet: a . Sequence alignment of partial EXT2 coding sequences from WT and EXT2 knockout (KO) U2OS cells. Protospacer adjacent motif (PAM), green; sgRNA target, blue; mutated sequence, red. b . Western blot analysis of whole cell lysate. (left) from wild-type (WT) and (right) EXT2 KO U2OS cell lines. 2 independent experiments were performed. c . Representative images of WT and EXT2 KO U2OS cells stained with Siglec-9 (red). Scale bar, 10 µm. 3 independent experiments were performed. d . Number of cells quantified in Fig. , and EDF4C across 3 independent experiments. e . Representative images of WT and EXT2 KO U2OS cells fixed, permeabilized, and stained with 9D5. Scale bar, 10 µm. Quantification of 9D5 intensity counted from 3 independent experiments. Data are mean ± s.e.m. f . Western blot analysis of whole cell lysate from the indicated cell lines. 2 independent experiments were performed. g . Western blot analysis of whole cell lysate (right) and sequence alignment of partial EXT1 coding sequences (left) from WT and EXT1 KO U2OS cells. 2 independent experiments were performed. h . Sequence alignment of partial UXS1 coding sequences from WT and UXS1 KO U2OS cells. i . Representative images of WT, EXT1 KO, and UXS1 KO U2OS cells stained with 10E4, Siglec-11, 9D5, DDX21, and hnRNP-U (all in red), separately. Scale bar, 10 µm. 3 independent experiments were performed. j . Representative images of U2OS cells co-stained live with 9D5 (green) and anti-DDX21 (red) or anti-hnRNP-U (red). An enlargement of the hatched box is shown, and a bright field (BF) is shown to represent the outline of the cell membrane. Scale bar, 10 µm. 2 independent experiments were performed. k . Western blot analysis of whole cell lysate from WT and EXT2 KO U2OS cell lines. Quantification of the ratio of total DDX21 and hnRNP-U to GAPDH is calculated across the biological triplicates. Statistical assessment was performed with a two-sided Student's t -test and P values are shown. Data are mean ± s.e.m.

Article Snippet: For DDX21 and hnRNP-U staining, 2.5 μg ml −1 of anti-DDX21 (Novus Biological) and anti-hnRNP-U (Proteintech) were incubated with cells for 45 min on ice.

Techniques: Sequencing, Knock-Out, Western Blot, Staining, Membrane

Journal: Nature

Article Title: GlycoRNA complexed with heparan sulfate regulates VEGF-A signalling

doi: 10.1038/s41586-025-10052-8

Figure Lengend Snippet: a . Representative images of U2OS cells treated with heparinase pool for 30 min, and stained live with 10E4, Siglec-11, 9D5, anti-DDX21, anti-hnRNP-U, Siglec-7-Fc, and Siglec-9-Fc (all in red), separately. Scale bar, 10 µm. b . Quantification of data in A with the number of cells per condition from 3 independent experiments is shown. Statistical assessment was performed with a two-sided Student's t -test and P values are shown. Data are mean ± s.e.m. c . Nearest neighbor distance analysis of the 10E4 and Siglec-11 in Fig. . For each pair, the nm distance from 10E4 to Siglec-11 was calculated across each time point. These values were plotted in a density histogram. d . Quantification fraction of 10E4-occupied, Siglec-11 dots in Fig. from 3 independent experiments. e . Representative images of U2OS cells co-stained with the indicated 10E4 (Cyan), Siglec-11 (yellow), and DDX21 (magenta). An enlargement of the hatched box is shown, and a bright field (BF) is shown to represent the outline of the cell membrane. Scale bar, 10 µm. 2 independent experiments were performed.

Article Snippet: For DDX21 and hnRNP-U staining, 2.5 μg ml −1 of anti-DDX21 (Novus Biological) and anti-hnRNP-U (Proteintech) were incubated with cells for 45 min on ice.

Techniques: Staining, Membrane

Journal: Nature

Article Title: GlycoRNA complexed with heparan sulfate regulates VEGF-A signalling

doi: 10.1038/s41586-025-10052-8

Figure Lengend Snippet: a , Representative images of U2OS cells treated with a heparinase pool for 30 min, recovering for the indicated times and co-stained with 10E4 (cyan), Siglec-11 (yellow) and 9D5 (magenta). An enlargement of the hatched box is shown, and a bright field is shown to represent the outline of the cell membrane. Three independent experiments were performed. b , Schematic of a HSPG with the regions of activity for the various enzymes and HS oligos perturbed in the following experiments. 2S, 2- O -sulfation; 6S, 6- O -sulfation; NS, N -sulfation. c , Representative images of WT, NDST1 -KO-treated, HS6ST1 -KO-treated, HS2ST1 -KO-treated, Sulf1 stably overexpressing (OE)-treated, Sulf2 stably OE-treated, Tega HS #09-treated and Tega HS #37-treated U2OS cells stained with 10E4, Siglec-11, 9D5, anti-DDX21 and anti-hnRNP-U (all in red) separately. d , Quantification of data in panel c with the number of cells counted from three independent experiments. Data are mean ± s.e.m.

Article Snippet: For DDX21 and hnRNP-U staining, 2.5 μg ml −1 of anti-DDX21 (Novus Biological) and anti-hnRNP-U (Proteintech) were incubated with cells for 45 min on ice.

Techniques: Staining, Membrane, Activity Assay, Stable Transfection

Journal: Nature

Article Title: GlycoRNA complexed with heparan sulfate regulates VEGF-A signalling

doi: 10.1038/s41586-025-10052-8

Figure Lengend Snippet: a . Representative zoom-out images of Fig. . Scale bar, 10 µm. 3 independent experiments were performed. b . Sequence alignment of partial NDST1 , HS6ST1 , and HS2ST1 coding sequences from wild-type (WT) and the indicated knock-out (KO) U2OS cell lines. Protospacer adjacent motif (PAM), green; sgRNA target, blue; mutated sequence, red. c . Western blot analysis of whole cell lysate from WT, NDST1 , HS6ST1 , and HS2ST1 KO U2OS cells. 2 independent experiments were performed. d . Representative images of WT and NaCl or NaClO 3 treated U2OS cells stained with 10E4, Siglec-11, 9D5, anti-DDX21, and anti-hnRNP-U (all in red). Scale bar, 10 µm. 3 independent experiments were performed. e . Representative images of WT and NaCl or NaClO 3 treated U2OS cells stained with 3G10 (red). Scale bar, 10 µm. 1 independent experiment was performed. f . Representative images of mEmerald-Sufl1 and mEmerald-Sulf2 stably overexpressing U2OS cells. Scale bar, 10 µm. 2 independent experiments were performed. g . Western blot analysis of whole cell lysate isolated from mEmerald-Sufl1 and mEmerald-Sulf2 stably overexpressing U2OS cells. 2 independent experiments were performed.

Article Snippet: For DDX21 and hnRNP-U staining, 2.5 μg ml −1 of anti-DDX21 (Novus Biological) and anti-hnRNP-U (Proteintech) were incubated with cells for 45 min on ice.

Techniques: Sequencing, Knock-Out, Western Blot, Staining, Stable Transfection, Isolation

Journal: Nature

Article Title: GlycoRNA complexed with heparan sulfate regulates VEGF-A signalling

doi: 10.1038/s41586-025-10052-8

Figure Lengend Snippet: a . Representative images of HUVECs treated with the RNase pool or heparinase pool, and stained live with 10E4, Siglec-11, 9D5, anti-DDX21, and anti-hnRNP-U (all in red). Scale bar, 10 µm. b . Representative images of keratinocytes with the RNase pool or heparinase pool, and stained live with 10E4, Siglec-11, 9D5, anti-DDX21, and anti-hnRNP-U (all in red). Scale bar, 10 µm. c . Quantification of 10E4, Siglec-11, 9D5, cs-DDX21, and cs-hnRNP-U intensity per cell from 3 independent staining experiments on keratinocytes. Data are mean ± s.e.m.

Article Snippet: For DDX21 and hnRNP-U staining, 2.5 μg ml −1 of anti-DDX21 (Novus Biological) and anti-hnRNP-U (Proteintech) were incubated with cells for 45 min on ice.

Techniques: Staining

Journal: Advanced Science

Article Title: EIF1AX Nucleolar Condensates Enhance Susceptibilities for the Management of Endometrial Cancer

doi: 10.1002/advs.202504238

Figure Lengend Snippet: Identification of proteins interacting with EIF1AX within nucleolar condensates. a) Workflow of the mass spectrometry‐based strategy used to identify proteins interacting with EIF1AX‐WT (WT), EIF1AX‐NLS (NLS), and EIF1AX‐NLS N11A/D142A (N11A/D142A). b) Venn diagram showing common and unique interacting proteins among the WT, NLS, and N11A/D142A groups. c) Chord diagrams illustrating enriched pathways among common interactors (left) and group‐specific interactors (right). d,e) Candidate EIF1AX‐binding proteins identified through yeast two‐hybrid screening (d) and co‐immunoprecipitation (Co‐IP) assays (e). f) Immunofluorescence staining of UBTF and EIF1AX in HEC‐1A cells. Scale bars: 10 µm (overview); 5 µm (zoom). Fluorescence intensity profiles along the indicated arrow directions are shown. g) Co‐IP analysis of interactions among EIF1AX‐WT, EIF1AX‐NLS, and EIF1AX‐NLS N11A/D142A in HEC‐1A cells. h) Immunofluorescence staining of DDX21 in HEC‐1A cells expressing WT, NLS, or N11A/D142A EIF1AX. Scale bar: 10 µm. Fluorescence intensity tracings along the indicated arrows are quantified. i) Denaturing GST pull‐down assays showing interactions between His‐DDX21 and GST‐tagged EIF1AX‐WT, IDR1, IDR2, RBP, or IDR1+IDR2 fragments. j) Proposed model: EIF1AX forms phase‐separated condensates with DDX21 via its IDR region, while binding to DDX21 through its RBP domain.

Article Snippet: The following primary antibodies and dilutions were used: DDX21 (Cell Signaling Technology, Danvers, MA, USA, #59278; 1:1000), PCNA (Cell Signaling Technology, #2586; 1:1000), Cleaved Caspase‐3 (Cell Signaling Technology, #9664; 1:1000), p16 (Sigma‐Aldrich, St. Louis, MO, USA, #SAB56000308; 1:1000), EIF1AX (Sigma‐Aldrich, #HPA002561; 1:1000), and γH2AX (Sigma‐Aldrich, #SAB5700688; 1:1000).

Techniques: Mass Spectrometry, Binding Assay, Two Hybrid Screening, Immunoprecipitation, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Fluorescence, Expressing

Journal: Advanced Science

Article Title: EIF1AX Nucleolar Condensates Enhance Susceptibilities for the Management of Endometrial Cancer

doi: 10.1002/advs.202504238

Figure Lengend Snippet: EIF1AX interacts with DDX21 to suppress rDNAs transcription. a) Schematic representation of rDNA organization and transcription. b) Levels of pre‐rRNA, 28S rRNA, and 18S rRNA in HEC‐1A cells expressing WT, NLS, or N11A/D142A EIF1AX. c) Sucrose gradient fractionation (10–45%) of cell lysates monitored by UV absorbance at 254 nm. Peaks correspond to 40S, 60S, and 80S ribosomal complexes. d) Pre‐rRNA levels in HEC‐1A cells expressing WT, NLS, N11A/D142A, or NLS with DDX21 knockdown (NLS+siDDX21). e,f) metaplots (e) and Heatmap (f) and showing enrichment of EIF1AX and DDX21 around transcription start sites (TSSs) of all annotated genes in HEC‐1A cells. g) CUT&Tag‐seq profiles of EIF1AX and DDX21 binding at the rDNA promoter in HEC‐1A cells. The EIF1AX‐binding motif identified at peak summits is shown. h,i) Electrophoretic mobility shift assay (EMSA). j) Luciferase reporter assay. k) CUT&Tag‐qPCR analysis. l) Nascent pre‐rRNA transcription in EIF1AX‐NLS‐expressing cells. m) Proposed model of EIF1AX binding to the 28S/18S promoter regions, as predicted by AlphaFold. Data are presented as mean ± SD; ns: not significant, * p < 0.05, as determined by one‐way ANOVA.

Article Snippet: The following primary antibodies and dilutions were used: DDX21 (Cell Signaling Technology, Danvers, MA, USA, #59278; 1:1000), PCNA (Cell Signaling Technology, #2586; 1:1000), Cleaved Caspase‐3 (Cell Signaling Technology, #9664; 1:1000), p16 (Sigma‐Aldrich, St. Louis, MO, USA, #SAB56000308; 1:1000), EIF1AX (Sigma‐Aldrich, #HPA002561; 1:1000), and γH2AX (Sigma‐Aldrich, #SAB5700688; 1:1000).

Techniques: Expressing, Fractionation, Knockdown, Binding Assay, Electrophoretic Mobility Shift Assay, Luciferase, Reporter Assay