Journal: bioRxiv

Article Title: Xenium In Situ Profiling Uncovers HSPG-Dependent SULF1/VEGFR2 Signaling Mediating Vascular Remodeling in Moyamoya Disease

doi: 10.64898/2026.04.28.721514

Figure Lengend Snippet: HBMECs were infected with LV-RNAi-NC or LV-SULF1-RNAi and cultured overnight in medium containing 1% FBS, followed by stimulation with VEGF-A165 (50 ng/mL) for 0, 10, and 30 min. A , Representative western blots of VEGFR2, p-VEGFR2, ERK1/2, p-ERK1/2, AKT, p-AKT, SULF1, and β-actin. B through H , Quantitative analysis of VEGFR2 ( B ), p-VEGFR2 ( C ), ERK1/2 ( D ), p-ERK1/2 ( E ), AKT ( F ), p-AKT ( G ), and SULF1 ( H ). Results were mean ± SD for three individual experiments. n =3, ** P <0.01.

Article Snippet: For signaling assays, HBMECs were cultured overnight in medium containing 1% fetal bovine serum (FBS) and then stimulated with vascular endothelial growth factor A165 (VEGF-A165) protein (HY-P70458A; MedChemExpress [MCE], New Jersey, USA) for 0, 10, and 30 min. For heparinase III pretreatment experiments, HBMECs were cultured in medium containing 1% FBS for 24 h, pretreated with heparinase III (HY-P2953; MCE, New Jersey, USA) at 20 mU for 2 h, and then stimulated with VEGF-A165 (50 ng/mL) for 0, 10, and 30 min. For functional assays and qRT-PCR, transfected HBMECs were treated with VEGF-A165 at a final concentration of 10 ng/mL.

Techniques: Infection, Cell Culture, Western Blot

Journal: bioRxiv

Article Title: Xenium In Situ Profiling Uncovers HSPG-Dependent SULF1/VEGFR2 Signaling Mediating Vascular Remodeling in Moyamoya Disease

doi: 10.64898/2026.04.28.721514

Figure Lengend Snippet: HBMECs were processed as follows: LV-oe-vector group, cells were transfected with the control lentiviral plasmid; LV-oe-vector+heparinase III group, cells were transfected with the control lentiviral plasmid and pretreated with heparinase III; LV-oe-SULF1 group, cells were transfected with the LV-SULF1 lentiviral plasmid; LV-oe-SULF1+heparinase III group, cells were transfected with the LV-SULF1 lentiviral plasmid and pretreated with heparinase III. All groups were stimulated with VEGF-A165 (50 ng/mL) for 0, 10, and 30 min. A , Representative western blots of VEGFR2, p-VEGFR2, ERK1/2, p-ERK1/2, AKT, p-AKT, SULF1, and β-actin. B through H , Quantitative analysis of p-VEGFR2 ( B ), VEGFR2 ( C ), ERK1/2 ( D ), p-ERK1/2 ( E ), AKT ( F ), p-AKT ( G ), and SULF1 ( H ). Results were mean ± SD for three individual experiments. n =3, ** P <0.01.

Article Snippet: For signaling assays, HBMECs were cultured overnight in medium containing 1% fetal bovine serum (FBS) and then stimulated with vascular endothelial growth factor A165 (VEGF-A165) protein (HY-P70458A; MedChemExpress [MCE], New Jersey, USA) for 0, 10, and 30 min. For heparinase III pretreatment experiments, HBMECs were cultured in medium containing 1% FBS for 24 h, pretreated with heparinase III (HY-P2953; MCE, New Jersey, USA) at 20 mU for 2 h, and then stimulated with VEGF-A165 (50 ng/mL) for 0, 10, and 30 min. For functional assays and qRT-PCR, transfected HBMECs were treated with VEGF-A165 at a final concentration of 10 ng/mL.

Techniques: Plasmid Preparation, Transfection, Control, Western Blot

Journal: bioRxiv

Article Title: Xenium In Situ Profiling Uncovers HSPG-Dependent SULF1/VEGFR2 Signaling Mediating Vascular Remodeling in Moyamoya Disease

doi: 10.64898/2026.04.28.721514

Figure Lengend Snippet: HBMECs were transfected with LV-RNAi-NC, LV-SULF1-RNAi, LV-oe-vector, and LV-oe-SULF1 lentiviral plasmids, respectively. After transfection, cells were treated with VEGF-A165 (10 ng/mL). A , Representative images of tube formation in each group after 6 h. Scale bars are shown in the panels. B and C , Quantitative analysis of branch points ( B ) and total segments length ( C ). D , Representative microscopic images of migrating HBMECs in the Transwell assay after 24 h. Scale bars are shown in the panels. E , Quantitative analysis of the number of migrating cells. Results were mean ± SD for three individual experiments. n =3, * P <0.05, ** P <0.01.

Article Snippet: For signaling assays, HBMECs were cultured overnight in medium containing 1% fetal bovine serum (FBS) and then stimulated with vascular endothelial growth factor A165 (VEGF-A165) protein (HY-P70458A; MedChemExpress [MCE], New Jersey, USA) for 0, 10, and 30 min. For heparinase III pretreatment experiments, HBMECs were cultured in medium containing 1% FBS for 24 h, pretreated with heparinase III (HY-P2953; MCE, New Jersey, USA) at 20 mU for 2 h, and then stimulated with VEGF-A165 (50 ng/mL) for 0, 10, and 30 min. For functional assays and qRT-PCR, transfected HBMECs were treated with VEGF-A165 at a final concentration of 10 ng/mL.

Techniques: Transfection, Plasmid Preparation, Transwell Assay

Journal: bioRxiv

Article Title: Xenium In Situ Profiling Uncovers HSPG-Dependent SULF1/VEGFR2 Signaling Mediating Vascular Remodeling in Moyamoya Disease

doi: 10.64898/2026.04.28.721514

Figure Lengend Snippet: HBMECs were transfected with LV-RNAi-NC, LV-SULF1-RNAi, LV-oe-vector, and LV-oe-SULF1 lentiviral plasmids, respectively. After transfection, cells were treated with VEGF-A165 (10 ng/mL) for 24 h. A , Representative cell scratch images of HBMECs at 0 and 24 h in each group. Dashed lines indicate the wound margins. Scale bars are shown in the panels. B , Quantitative analysis of relative migration ability. Results were mean ± SD for three individual experiments. n =3, ** P <0.01.

Article Snippet: For signaling assays, HBMECs were cultured overnight in medium containing 1% fetal bovine serum (FBS) and then stimulated with vascular endothelial growth factor A165 (VEGF-A165) protein (HY-P70458A; MedChemExpress [MCE], New Jersey, USA) for 0, 10, and 30 min. For heparinase III pretreatment experiments, HBMECs were cultured in medium containing 1% FBS for 24 h, pretreated with heparinase III (HY-P2953; MCE, New Jersey, USA) at 20 mU for 2 h, and then stimulated with VEGF-A165 (50 ng/mL) for 0, 10, and 30 min. For functional assays and qRT-PCR, transfected HBMECs were treated with VEGF-A165 at a final concentration of 10 ng/mL.

Techniques: Transfection, Plasmid Preparation, Migration

Journal: bioRxiv

Article Title: Xenium In Situ Profiling Uncovers HSPG-Dependent SULF1/VEGFR2 Signaling Mediating Vascular Remodeling in Moyamoya Disease

doi: 10.64898/2026.04.28.721514

Figure Lengend Snippet: HBMECs were transfected with LV-RNAi-NC, LV-SULF1-RNAi, LV-oe-vector, and LV-oe-SULF1 lentiviral plasmids, respectively. After transfection, cells were treated with VEGF-A165 (10 ng/mL) for 24 h. The mRNA levels of ANGPT2 ( A ), ESM1 ( B ), KDR ( C ), ICAM1 ( D ), and VCAM1 ( E ) were detected by qRT-PCR. Results were mean ± SD for three individual experiments. n =3, * P <0.05, ** P <0.01.

Article Snippet: For signaling assays, HBMECs were cultured overnight in medium containing 1% fetal bovine serum (FBS) and then stimulated with vascular endothelial growth factor A165 (VEGF-A165) protein (HY-P70458A; MedChemExpress [MCE], New Jersey, USA) for 0, 10, and 30 min. For heparinase III pretreatment experiments, HBMECs were cultured in medium containing 1% FBS for 24 h, pretreated with heparinase III (HY-P2953; MCE, New Jersey, USA) at 20 mU for 2 h, and then stimulated with VEGF-A165 (50 ng/mL) for 0, 10, and 30 min. For functional assays and qRT-PCR, transfected HBMECs were treated with VEGF-A165 at a final concentration of 10 ng/mL.

Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR