Figure 2. Hypoxia-inducible POSTN secretion by glioma cells promotes the recruitment of macrophages.
A. Immunofluorescence staining for POSTN (green) and HIF-1α (red). Scale bars, 100 μm. B. A graphical analysis of figure (A) showing that both HIF-1α and POSTN expression increased as the grade of the glioma increased. HIF-1α and POSTN expression was analyzed with ImageJ. *, P <0.05; **, P <0.01 (n =5 tumors; mean ± s.e.m.; two-tailed unpaired t-test). C. Western blot analysis of POSTN and HIF-1α levels in U87 and U251 cells that were exposed to hypoxia showing that they increased over the time-course. D. Representative images of THP-1 macrophage-like cells migrating towards different concentrations of rPOSTN in transwell assays. Scale bar, 100 μm. Graphical analysis of (D)showing that THP-1 induced macrophages to migrate towards rPOSTN in a dose-dependent manner. E and F. Representative images of THP-1 macrophage-like cells migrating towards normoxia- or hypoxia- treated U87 or U251 cell culture supernatants pre-incubated with or without anti-POSTN antibodies. A graphical analysis of (E) and (F) shows that the increased macrophages migration toward hypoxia-treated glioma cell supernatants was attenuated by the application of anti-POSTN antibodies. Scale bar, 100 μm. G and H. Representative images of THP-1 macrophage-like cells migrating towards different conditioned culture supernatants that were obtained from U87 and U251 cells. I. A graphical analysis of (E) and (F) showing that the increased migration observed in THP-1 induced macrophages toward hypoxia-treated U87 and U251 cell supernatants was reversed by transfecting the cells with siPOSTN before the induction of hypoxia. Moreover, pretreatment of U87 or U251 cells with ACF during exposure to hypoxia also eliminated this trend. J. Western blot analysis of POSTN levels in U87 or U251 cells that were transfected with siPOSTN (1205 or 876) or vector. K. A graphical analysis of (J) showing that there was a significant decrease in POSTN expression in the siPOSTN groups in both U87 and U251 cells. The relative POSTN expression level was determined using ImageJ software. The results are shown as the means ± s.e.m. (n = 3; *, P <0.05; two-tailed unpaired t-test). L. Representative images of THP-1 macrophages migrating toward complete PRIM-1640 medium in the presence of PBS or ACF (3 μM). M. A graphical analysis of (L) showing that ACF had no direct effect on macrophage migration. V. Representative images of THP-1 macrophages migrating towards the conditioned medium of U87/U251 cells when exposed to a normoxic or hypoxic environment. O. A graphical analysis of (V) showing that the migratory activity of macrophages was impaired by hypoxia. All transwell assays were repeated three times. *, P <0.05; **, P <0.01; NS, P >0.05 (n = 5 fields, mean ± s.e.m., two-tailed unpaired t-test).