Journal: Nature

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

doi: 10.1038/s41586-025-10052-8

Figure Lengend Snippet: a , Quantification of 10E4, Siglec-11, 9D5, cs-DDX21 and cs-hnRNP-U intensity per cell from three independent staining experiments on HUVECs. Data are mean ± s.e.m. a.u., arbitrary units. b , Western blot analysis of whole-cell lysates isolated from HUVECs after starvation and treatment with an RNase pool followed by 3 ng ml −1 VEGF-A 165 , VEGF-A 121 or EGF stimulation (top). Quantification of the ratio of pERK to total ERK was also calculated across the biological triplicates (bottom). Statistical assessment was performed with a two-sided Student’s t -test and P values are shown. Data are mean ± s.e.m. c , Representative images of starved HUVECs treated with an RNase pool, and subsequently with VEGF-A 165 or VEGF-A 121 , finally stained with anti-VEGF-A 165 (red) or anti-VEGF-A (red). Statistical assessment was performed with a Student’s t -test and P values are shown. Data are mean ± s.e.m. NS, not significant. d , Representative images of HUVECs treated with an RNase pool and stained with anti-VEGFR2 (red). Statistical assessment was performed with a Student’s t -test and P values are shown. Data are mean ± s.e.m. e , Representative images of starved HUVECs treated with VEGF-A 165 and then co-stained with anti-VEGF-A 165 (purple) and Siglec-11 (yellow). Three independent experiments were performed. f , Schematic of the microfluidic chip (top left) used to grow HUVECs without (top middle) and with (top right) RNase A for 6 days. Representative images of BFP expressed in the HUVECs. Statistical assessment of the total migration area was performed using an unpaired two-sided Student’s t -test (bottom). Data are mean ± s.e.m. Four independent experiments were performed. g , Representative image (maximum z -projection view (left) and z -projection slice view (right)) of sprouts from a +RNase A device for the cells (blue), F-actin (red) and PECAM1 (green). Four independent experiments were performed.

Article Snippet: Anti-VEGF-A 165 (AF293-NA, R&D Systems; immunoblot 1:1,000) and donkey anti-goat IgG secondary antibody (92632214, LI-COR Biosciences; immunoblot 1:1,000) were used as primary and secondary antibodies, separately.

Techniques: Staining, Western Blot, Isolation, Migration

Journal: Nature

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

doi: 10.1038/s41586-025-10052-8

Figure Lengend Snippet: a . Western blot analysis of whole cell lysate isolated from HUVECs after starvation and treatment with RNase pool followed by 25 ng/mL VEGF-A 165 stimulation. Quantification of the ratio of phosphorylated ERK (pERK) to total ERK 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. b . Western blot analysis as in (A) with HUVECs stimulated with 25 ng/mL of VEGF-A 121 . Statistical assessment and data are as in ( a ). c . Western blot analysis as in (A) with HUVECs stimulated with 25 ng/mL of EGF. Statistical assessment and data are as in ( a ). d . Western blot analysis as in (A) with HUVECs stimulated with 3 ng/mL of VEGF-A 165 . Quantification of the ratio of phosphorylated VEGFR2 (pVEGFR2) to total VEGFR2 is calculated across the biological triplicates. Statistical assessment and data are as in ( a ). e . MST assay of VEGF-A and small RNA binding. n = 3 biologically independent repeats. Data are mean ± s.e.m. f . Representative images of HUVECs after serum starvation and treated with or without VEGF-A 165 and then co-stained with anti-VEGF-A 165 (purple) and Siglec-11 (yellow). Zoomed region shown as an inset. Scale bar, 10 µm. 3 independent experiments were performed. g . Nearest neighbor distance analysis of the VEGF-A 165 and Siglec-11 in Figure S5E. For each pair, the nm distance from VEGF-A 165 to Siglec-11 was calculated across. These values were plotted in a density histogram. h . The four biological replicate microfluidic chip without (left) and with (right) 10 µM RNase A for 6 days. Scale bar, 200 µm. i . Immunofluorescence confocal image (maximum z-projection view) of HUVEC structures after 6 days of +RNase treatment on microfluidic chip. Cells (blue), F-actin (red), and PECAM1 (green). Scale bar, 100 µm. 4 independent experiments were performed.

Article Snippet: Anti-VEGF-A 165 (AF293-NA, R&D Systems; immunoblot 1:1,000) and donkey anti-goat IgG secondary antibody (92632214, LI-COR Biosciences; immunoblot 1:1,000) were used as primary and secondary antibodies, separately.

Techniques: Western Blot, Isolation, RNA Binding Assay, Staining, Immunofluorescence

Journal: Nature

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

doi: 10.1038/s41586-025-10052-8

Figure Lengend Snippet: a . Western blot analysis of the indicated amount of VEGF-A 165 . 2 independent experiments were performed. b . Lysates from HUVECs after serum starvation, treatment with or without 25 ng/mL VEGF-A 165 , and UV-crosslinking, were treated with or without RNase pool and immunoprecipitated (IP) with an anti-VEGF-A antibody (Proteintech). Immunoprecipitated samples were analyzed by Western blot using an anti-VEGF-A 165 antibody (R&D system). 3 independent experiments were performed. c . Principal component analysis (PCA) of VEGF-A 165 RIP-seq results. d . Enriched transcripts in VEGF-A 165 RIP-seq. Each dot represents a unique small non-coding RNA (ncRNA) transcript, colored by biotype as indicated in the legend. The red dotted line marks the filtering citeria: log 2 FoldChange (IP/Input) > 0.5 and adjusted p-value < 0.05. Statistical assessment was performed with a two-sided Wald test and P values are adjusted for multiple comparisons using the Benjamini-Hochbery procedure. Solid circles indicate true positive hits that were also significantly enriched relative to the IgG control (see for details). e . Abundance of enriched transcripts grouped by biotype. Color scheme matches ( d ). f . Overlap between enriched transcripts and glycoRNA defined by ManNAz-seq, grouped by ncRNA family. The p-value was calculated using a two-sided hypergeometric test with all human small ncRNAs as the background. g . Western blot analysis of the beads pre-conjugated with 5 µg of VEGF-A 165 . h . Representative images of the indicated HUVECs stained with anti-VEGF-A 165 (red). Scale bar, 10 µm. Quantification of the images with number of cells noted per biological triplicate. Statistical assessment was performed with a two-sided Student's t -test and P values are shown. Data are mean ± s.e.m. i . Western blot analysis of whole cell lysate isolated from the indicated HUVECs. Quantification of the ratio of phosphorylated VEGFR2 (pVEGFR2) to total VEGFR2 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. j . In vitro IP of VEGF-A 165 HS WT or HS(R/K) with small RNA and rPAL. 3 independent experiments were performed. k . MST assay of VEGF-A and small RNA treated with sialidase binding. n = 3 biologically independent repeats. Data are mean ± s.e.m. l . RNA-seq analysis of the sulfotransferases expressed from HUVECs and their FPKM values from ref. . m . EMSA analysis of the indicated VEGF-A proteins with or without the addition of rHS29.

Article Snippet: Anti-VEGF-A 165 (AF293-NA, R&D Systems; immunoblot 1:1,000) and donkey anti-goat IgG secondary antibody (92632214, LI-COR Biosciences; immunoblot 1:1,000) were used as primary and secondary antibodies, separately.

Techniques: Western Blot, Immunoprecipitation, Control, Staining, Isolation, In Vitro, Binding Assay, RNA Sequencing

Journal: Nature Communications

Article Title: Breast cancer remodels lymphatics in sentinel lymph nodes

doi: 10.1038/s41467-025-64981-z

Figure Lengend Snippet: a Expression of MGP in LECs after direct exposure to recombinant VEGF165 (5, 25, or 100 ng/mL), VEGF-C (5, 50, or 100 ng/mL), TGF-β (5, 25, or 100 ng/mL), or EGF (5, 25, or 100 ng/mL) are shown ( n = 4 HLECs). Data were depicted as Tukey box plots and analyzed using one-way ANOVA linear mixed models (Sidak correction) fitted separately for each parameter with group (recombinant vs control), dose and their interaction as fixed effects. b Gene expression changes in modified CM-exposed LECs are shown as determined by qPCR. CM was generated in the presence of antibodies against VEGFR3 (1 or 10 μg/mL, n = 4 HLECs), TGF-β (2 or 20 μg/mL, n = 8–10 HLECs), and EGF (1 or 10 μg/mL, n = 3–5 HLECs), compared to isotype control exposed samples and data were depicted as Tukey box plots showing relative gene changes and analyzed using a one-way ANOVA linear mixed models (Sidak) fitted separately for each parameter with group (antibody vs control) and dose and their interaction as fixed effects. c MGP expression determined by qPCR. CM generated with culture media devoid of VEGF supplement was used. Data were shown as Tukey box plots ( n = 9 HLECs). Data were analyzed using a one-way ANOVA linear mixed model (Sidak). The center line of the box plots represents the median, the box the 25th to 75th percentiles and the whiskers the inner fences. Statistics of group and dose effects are presented within the boxes; significant differences in comparison to the controls (defined as 1) are indicated by p values. Source data, non-significant p values and detailed experiment and n -numbers (biologically independent samples of cultured cells) are provided in the Source Data file.

Article Snippet: AACOCF3 (ab120350) was obtained from Abcam, fludarabine (S1491) from Seleckchem, antibodies targeting adrenomedullin (AF6108), EGF (AB-236NA), goat IgG control (AB-108-C), PDGF (AB-20-NA), rabbit IgG control (AB-105-C), and VEGF165 (AB-293-NA) from R&D Systems.

Techniques: Expressing, Recombinant, Control, Gene Expression, Modification, Generated, Comparison, Cell Culture