Fig. 8

HIF-1α directly suppresses AFAP1L1 transcription under hypoxia. A AFAP1L1mRNA levels in HUVEC cells exposed to hypoxia (1% O2) for the indicated times. (n = 4 independent experiments). B Volcano plots showed the correlation between AFAP1L1 and HIF-1α or HIF-2α. C Western blot analysis of AFAP1L1, HIF-1α, HIF-2α protein levels in HUVEC cells exposed to hypoxia (1% O2) for the indicated times. Densitometric quantitation of western blot band intensity shown in C. Results are presented as mean ± SEM, statistical analyses were performed using one-way ANOVA with Bonferroni's post hoc test. (n = 4 independent experiments). D Western blot analysis of AFAP1L1, HIF-1α, HIF-2α and β-actin protein levels in HUVEC cells left untreated or exposed to hypoxia (1% O2) for 24 h as well as transfected with siNC, siHIF-1α or siHIF-2α. Densitometric quantitation of western blot band intensity shown in D. Results are presented as mean ± SEM, statistical analyses were performed using one-way ANOVA with Bonferroni's post hoc test. (n = 4 independent experiments). E Schematic diagram depicting the human AFAP1L1 promoter with the presence of hypoxia response element (HRE) sites from the JASPAR database and constructed mutant HRE sites (left panel) (TSS: transcription start site; ATG: initiating methionine codon). Luciferase reporter assay for AFAP1L1 promoter activity in HEK293T cells transfected with control vector, HIF-1α overexpression or HIF-2α overexpression plasmids (n = 4 independent experiments) (middle panel). Luciferase reporter assay for AFAP1L1 promoter activity in HEK293T cells following transfection of wHRE or mHRE luciferase reporter plasmids under HIF-1α overexpression (right panel). (n = 4 independent experiments). P values were calculated by one-way ANOVA with Bonferroni's post hoc test (A, C, F). Error bars represent the mean ± SD. HUVECs are transfected with shNC, shAFAP1L1 #1 or shAFAP1L1 #2 and then exposed to hypoxia (1% O2) for 24 h or left untreated. F Transwell assays of HUVECs under hypoxia (1% O2) or simultaneously transfected with shRNA. Scale bar: 50 µm. G Wound scratching assays of HUVECs under hypoxia (1% O2) or simultaneously transfected with shRNA. Scale bar: 200 µm. [rhodamine-conjugated phalloidin: red (to visualize the actin cytoskeleton); DAPI: blue (to visualize nuclei)]. H Tube formation Assay of HUVECs under hypoxia (1% O2) or simultaneously transfected with shRNA. Scale bar: 200 µm. I Rhodamine-phalloidin staining reveals the actin cytoskeleton and filopodia of HUVECs under hypoxia (1% O2) or simultaneously transfected with shRNA (rhodamine-conjugated phalloidin: red; DAPI: blue). Scale bar: 25 µm. J 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). K Quantification of wound area. 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). L 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 independent experiments). M, N 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 = 4 independent experiments). *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001; n.s.: no significance