Fig. 4

FSCN1 shRNA could reverse the upregulation of tube formation, migration and sprouting capabilities of HRMECs under hypoxia. HRMECs were transfected with non-specific sequences shRNA (shNC), FSCN1 shRNA, or left untreated and then exposed to CoCl₂ (200 μmol/L) for 24 h. The group without any treatment is taken as the control group. A Transwell assays of HRMECs under hypoxia factors (CoCl₂) stimulation or simultaneously transfected with shFSCN1. Scale bar: 50 µm. (n = 4 per group, data pooled from 4 independent experiments). B Wound scratching assays of HRMECs under hypoxia factors (CoCl₂) stimulation or simultaneously transfected with shFSCN1. Scale bar: 200 µm. [rhodamine-conjugated phalloidin: red (to visualize the actin cytoskeleton); DAPI: blue (to visualize nuclei)]. (n = 4 per group, data pooled from 4 independent experiments). C Tube formation Assay of HRMECs under hypoxia factors (CoCl₂) stimulation or simultaneously transfected with shFSCN1. Scale bar: 200 µm. (n = 4 per group, data pooled from 4 independent experiments). D Three-dimensional (3D) Bead Sprouting Assay reveals the in vitro sprouting capabilities of HRMECs under hypoxia factors (CoCl₂) stimulation or simultaneously transfected with shFSCN1. Scale bar: 100 µm. (n = 10 per group, data pooled from 4 independent experiments). E Rhodamine-phalloidin staining (top panels) reveals the actin cytoskeleton and filopodia of HRMECs under hypoxia factors (CoCl₂) stimulation or simultaneously transfected with shFSCN1 (rhodamine-conjugated phalloidin: red; DAPI: blue). Scale bar: 25 µm. The down panels represent a partial magnification to see filamentous pseudopodia in more detail. (n = 12 per group, data pooled from 4 independent experiments). F Quantification of migrated cells. Results are presented as mean ± SEM, statistical analyses were performed using One-way ANOVA with Bonferroni's post hoc test. (n = 4 per group, data pooled from 4 independent experiments). G Quantification of wound area. The top panels represent the initial state of the wound area at 0 h, and the down panels represent the area after a period of 24 h of hypoxic migration. Results are presented as mean ± SEM, statistical analyses were performed using One-way ANOVA with Bonferroni's post hoc test. (n = 4 per group, data pooled from 4 independent experiments). H Quantification of Tube formation length. Results are presented as mean ± SEM, statistical analyses were performed using One-way ANOVA with Bonferroni's post hoc test. (n = 4 per group, data pooled from 4 independent experiments). I Quantification of length and number of sprouts per bead. Results are presented as mean ± SEM, statistical analyses were performed using Kruskal–Wallis with Bonferroni's post hoc test. (n = 10 per group, data pooled from 4 independent experiments). J Quantification of length and number of filopodia per cell. Results are presented as mean ± SEM, statistical analyses were performed using Kruskal–Wallis with Bonferroni's post hoc test. (n = 12 per group, data pooled from 4 independent experiments)