Fig 1: Influence of SDF1 on the migration, invasion and angiogenesis of VECs. (A) Expression levels of SDF1 in the cultured supernatants of NPCs were detected by ELISA following 24 h of treatment with various concentrations (0, 10, 50, 100, 200 and 500 µg/ml) of anti-SDF1 antibody. (B) Expression levels of SDF1 in the cultured supernatants of NPCs were detected by ELISA following 0, 12, 24 and 48 h treatment with 100 µg/ml of anti-SDF1 antibody. (C) Tube formation ability of VECs was analyzed by Matrigel tube formation assay, and the total branch length was calculated with ImageJ (magnification, ×100) after VECs were treated with the conditioned media from OE-SDF1 or the control group transfected-NPCs along with anti-SDF1 antibody (100 µg/ml, 24 h) or not. (D and E) Cell migration and invasion abilities were analyzed by Transwell assay with SDF1 overexpressed or non-treated NPCs as a chemokine (magnification, ×100). n=3, *P<0.05, **P<0.01, ***P<0.001. SDF1, stromal cell-derived factor 1; NPCs, nucleus pulposus cells; OE-, overexpression; VECs, vascular endothelial cells.
Fig 2: Influence of PTEN inhibition on the angiogenesis of VECs. Cultural supernatant derived from NPCs with SDF1 overexpression or not were used to stimulate VECs which were pretreated with SF1670 (10 µM, 30 min), and then the following assays were performed. (A) Western blotting was performed to detect the protein expression levels of p-AKT and AKT in VECs. (B) VEC viability was detected by Cell Counting Kit-8 assay. (C) Tube formation ability was analyzed by Matrigel tube formation assay, and the total branch length was calculated by ImageJ (magnification, ×100). (D and E) VEC migration and invasion were analyzed by Transwell assay with SDF1 overexpressed or non-treated NPCs as a chemokine (magnification, ×100). n=3, *P<0.05, **P<0.01, ***P<0.001. AKT, AKT serine/threonine kinase 1; NPCs, nucleus pulposus cells; OE-, overexpression; p-, phosphorylated; PTEN, phosphatase and tensin homolog; SDF1, stromal cell-derived factor 1; VECs, vascular endothelial cells.
Fig 3: Influence of the SDF1/C-X-C receptor 4 axis on the proliferation, migration, invasion and angiogenesis of VECs. VECs were first co-treated with AMD3100 (10 µM, 30 min) and the conditioned media from NPCs with OE-SDF1 or controls, and then the following assays were carried out. (A) Western blot analysis was performed to detect the expression levels of p-AKT and AKT in VECs. (B) VEC viability was detected by Cell Counting Kit-8 assay. (C) The tube formation ability of VECs was analyzed by Matrigel tube formation assay, and the total branch length was calculated by ImageJ (magnification, ×100). (D and E) The migration and invasion of VECs was analyzed by Transwell assay with SDF1 overexpressed or non-treated NPCs as a chemokine (magnification, ×100). n=3, *P<0.05, **P<0.01, ***P<0.001. SDF1, stromal cell-derived factor 1; NPCs, nucleus pulposus cells; OE-, overexpression; p-, phosphorylated; AKT, AKT serine/threonine kinase 1; VECs, vascular endothelial cells.
Fig 4: Upregulation of SDF1 in NPCs increases the activation of phosphatidylinositol-3-kinase/AKT signaling in VECs. (A) Primary NPCs were identified by immunofluorescence. The cells simultaneously expressed green (aggrecan) and red (type II collagen) fluorescence, demonstrated a chondrocyte-like phenotype and were identified as NPCs. (B and C) After transfection of OE-SDF1 adenovirus, the mRNA and protein expression of SDF1 in NPCs was detected by reverse transcription-quantitative PCR and western blotting. (D) Immunofluorescence of NPCs revealed visible SDF1 expression. (E) VECs were stimulated with the conditioned media from NPCs with OE-SDF1 or control, and a western blot was used to assess the protein expression levels of p-AKT, AKT and PTEN in VECs. n=3, *P<0.05; ***P<0.001. Magnification, ×200. SDF1, stromal cell-derived factor 1; NPCs, nucleus pulposus cells; OE-, overexpression; p-, phosphorylated; AKT, AKT serine/threonine kinase 1; PTEN, phosphatase and tensin homolog; NC, negative control; VECs, vascular endothelial cells.
Fig 5: Influence of AKT inhibition on the angiogenesis of VECs. VECs with MK-2206 (10 µM, 30 min) treatment or non-treated were cultured in the cultural supernatant derived from NPCs with OE-SDF1 or controls, and then the following assays were performed. (A) Western blotting was performed to detect the expression levels of p-AKT and AKT in VECs. (B) VEC viability was detected by Cell Counting Kit-8. (C) Tube formation ability in VECs was analyzed by Matrigel tube formation assay, and the total branch length was calculated by ImageJ (magnification, ×100). (D and E) Migration and invasion abilities of VECs were analyzed by Transwell assay, with SDF1 overexpressed or non-treated NPCs as a chemokine (magnification, ×100). n=3, **P<0.01, ***P<0.001. SDF1, stromal cell-derived factor 1; NPCs, nucleus pulposus cells; OE-, overexpression; p-, phosphorylated; AKT, AKT serine/threonine kinase 1; VECs, vascular endothelial cells.
Supplier Page from Abcam for Anti-SDF1 antibody [EPR1216]