Cell culture
MOLM-13 cells were cultured in Roswell Park Memorial Institute 1640 Medium (Thermo Fisher Scientific). U2OS (American Type Culture Collection (ATCC)) were cultured in McCoy’s 5A Medium (Thermo Fisher Scientific). HEK293T cells were cultured in DMEM media (Thermo Fisher Scientific). MOLM-13, U2OS and HEK293T cells were supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 1× penicillin–streptomycin (Thermo Fisher Scientific). Primary umbilical vein endothelial cells (HUVECs; ATCC) were cultured in vascular cell basal medium (ATCC) supplemented with endothelial cell growth kit-VEGF (ATCC). Keratinocyte cells (ATCC) were cultured in dermal cell basal medium (ATCC) supplemented with keratinocyte growth kit (ATCC). All the experiments on HUVECs are performed before passage 10. All cells were cultured at 37 °C with 5% CO 2 and maintained as mycoplasma negative. U2OS cells were transfected with Avalanche-Omni Transfection Reagent (EZ Biosystems). Immortalized HUVECs expressing BFP (Im-HUVEC-BFP; a gift from R. Kamm at MIT)79 were cultured in VascuLife VEGF-Mv endothelial medium (Lifeline Cell Technology) and passage p15 was used for experiments.
Live-cell enzyme and chemical treatments
For RNase treatment, RNase A (Sigma) and ShortCut RNase III (New England Biolabs (NEB)) were added directly to the cell culture at a final concentration of 18 µM and 100 U ml−1, respectively. MOLM-13, U2OS, HUVECs and keratinocytes were all treated for 45 min.
For heparinase treatment, heparinase I (NEB), heparinase II (NEB) and heparinase III (NEB) were added directly to the cell culture at a final concentration of 4 units per millilitre (each) for 30 min.
For sodium chloride (NaCl) and sodium chlorate (NaClO 3 ) treatment, NaCl and NaClO 3 were added to the cell culture at a final concentration of 50 mM for 24 h.
For exogenous Tega HS chain treatment, rHS09 (TEGA Therapeutics) and rHS37 (TEGA Therapeutics) were added directly to the cell culture at a final concentration of 4 µM for 60 min.
For serum starvation, HUVECs were cultured in vascular cell basal medium (PCS100030, Bioresource Center) without endothelial cell growth kit-VEGF (Bioresource Center) after being washed briefly three times with phosphate buffer saline (PBS). VEGF-A 165 (Thermo Fisher Scientific), VEGF-A 121 (Thermo Fisher Scientific), EGF (Thermo Fisher Scientific), VEGF-A 165 HS WT (Supplementary Table 3, cell-free Escherichia coli protein production by Liberum Biotech) and VEGF-A 165 HS(R/K) (Supplementary Table 3, cell-free E. coli protein production by Liberum Biotech) were then added to starved HUVECs at a final concentration of 3 ng ml−1 and 25 ng ml−1 for 5 min, separately. The WT and R/K proteoforms differ by approximately 400 Da (or approximately 1% total mass) so equal mass was added in each experiment, which is approximately equal moles.
Live-cell labelling and microscopy analysis
Adherent cells were cultured on glass coverslips #1.5 (Bioscience Tools) 24 h before labelling. MOLM-13 cells were counted and then blocked as per the manufacturer’s protocol with Human TruStain FcX (Fc block, BioLegend) for 15 min on ice before labelling. For Siglecs staining in live cells, 1 µg ml−1 of recombinant human IgG1 Fc (R&D Systems), Siglec-1–Fc chimera protein (R&D Systems), Siglec-2–Fc chimera protein (R&D Systems), Siglec-3–Fc chimera protein (R&D Systems), Siglec-4–Fc chimera protein (R&D Systems), Siglec-5–Fc chimera protein (R&D Systems), Siglec-6–Fc chimera protein (R&D Systems), Siglec-7–Fc chimera protein (R&D Systems), Siglec-8–Fc chimera protein (R&D Systems), Siglec-9–Fc chimera protein (R&D Systems), Siglec-10–Fc chimera protein (R&D Systems), Siglec-11–Fc chimera protein (R&D Systems), Siglec-14–Fc chimera protein (R&D Systems) and Siglec-15–Fc chimera protein (R&D Systems) were precomplexed with 0.5 µg ml−1 of donkey anti-human IgG AF647 (ImmunoResearch) secondary antibody in FACS buffer (0.5% BSA (Sigma) in 1× PBS) for 45 min on ice. For 9D5, 10E4 and 3G10 staining, 2.5 µg ml−1 9D5 (Absolute Antibody), 1 µg ml−1 anti-HS 10E4 (Amsbio), 1 µg ml−1 anti-HS 3G10 (Amsbio) and anti-VEGF (R&D Systems) were precomplexed with 1.25 µg ml−1 of goat anti-rabbit AF647 secondary antibody (Thermo Fisher Scientific), 0.5 µg ml−1 of goat anti-mouse AF647 secondary antibody (Thermo Fisher Scientific) or donkey anti-goat IgG AF647 (Thermo Fisher Scientific) secondary antibodies in FACS buffer for 45 min on ice, separately. For VEGF-A 165 , VEGF-A 121 and VEGFR2 staining, 1 µg ml−1 of anti-VEGF-A 165 (R&D Systems), 1 µg ml−1 of anti-VEGF-A (Proteintech), and 1 µg ml−1 anti-VEGFR2 (R&D Systems) were precomplexed with 0.5 µg ml−1 of goat anti-rabbit AF647 secondary antibody and donkey anti-goat AF647 secondary antibody (Thermo Fisher Scientific). Precomplexed antibodies were then incubated with cells for 45 min on ice. 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. Cells were gently washed twice by FACS buffer and then stained with 2.5 µg ml−1 of goat anti-rabbit AF647 for 30 min on ice.
... continue reading