Figure 2.

HIF-1α and HIF-2α are required for hypoxia-induced ITGA5 expression. A, Immunoblot assays were performed using lysates prepared from MCF-7, ZR.75.1, MDA-MB-231 (231) and MCF10A cells exposed to 20% or 1% O₂ for 48 h (left panel) or 1mM of DMOG or an equal volume of DMSO (right panel). The fold increase of ITGA5 (normalized by actin) protein under 1% O₂ compared to 20% O₂ conditions is denoted beneath the immunoblot. Note: 10× lower exposure times for ITGA5 were used for 231 and MCF10A cells as compared to MCF-7 and ZR75.1 cells. B, Immunohistochemical staining for HIF-1α and ITGA5 on sections of MDA-MB-231 orthotopic breast tumors. Images were deconvoluted and pseudo-colored for ITGA5 (red) and HIF-1α (blue) staining to assess colocalization (right). C-D, ITGA5 (C) and ITGB1 (D) mRNA levels were analyzed by qPCR in MDA-MB-231 subclones, which were stably transfected with a non-targeting control shRNA vector (NTC) or vector encoding either HIF-1α shRNA (sh1α) or HIF-2α shRNA (sh2α) and exposed to 20% or 1% O₂ for 24 h (mean ± SEM, n = 3-5); **P < 0.01 versus NTC at 1% O₂ or ^### P < 0.001 versus NTC at 20% O₂ (twoway ANOVA with Bonferroni posttest). E, Immunoblot assays were performed using lysates prepared from MDA-MB-231 subclones exposed to 20% or 1% O₂ for 48 hours. ITGA5 densitometry measurements normalized by actin levels are denoted for each sample. F-G, ITGA5 (F) and ITGB1 (G) mRNA levels were analyzed in tumors established with MDA-MB-231 subclones (mean ± SEM, n = 5); *P < 0.05, **P < 0.01 versus NTC (one-way ANOVA with Bonferroni posttest). (H) Flow cytometry analysis to assess the fluorescent intensity of ITGA5 antibody binding to MDA-MB-231 cells was conducted for subclones exposed to 20% or 1% O₂ for 48 h (mean ± SEM, n = 3); **P < 0.01 versus NTC at 1% O₂; ## P < 0.01 versus NTC at 20% O₂ (two-way ANOVA with Bonferroni posttest for multiple comparisons).