Fig. 2

ITGA6/CD49f expression is reduced in response to loss of HIF activity in MDA-MB-231 cells and tumors. a Mean ± SEM of the expression of ITGA6 mRNA relative to PPIA, in adherent, cultured MDA-MB-231 cells (n = 3 independent biological experiments/genotype). mRNA levels were evaluated after 0, 6 or 24 h of hypoxic exposure. All data are expressed relative to shControl cells cultured at normoxia (set to 1.0); *p-value <0.05 by ANOVA. b Western blotting was performed for ITGA6 protein using whole cell extracts (WCE) prepared from near confluent MDA-MB-231 shControl or shHIF1A/shHIF2A cells cultured at either normoxia (N) or chronic hypoxia (H). Tata-binding protein (TBP) is shown as a loading control. c MDA-MB-231 shControl, shHIF1A, shHIF2A and shHIF1A/shHIF2A cells were harvested from normoxic cell culture at 90 % confluence, stained with CD49f-FITC in suspension, replated onto chamber well slides and stained with Hoechst 33342; scale bar represents 20 μM. d MDA-MB-231 shControl, shHIF1A, shHIF2A and shHIF1A/shHIF2A cells were harvested from normoxic or chronic hypoxic cell culture at 90 % confluence, stained with CD49f-FITC in suspension in flow buffer, and subjected to FACS analysis. Plots represent the % Max of stained cells (red histogram) versus the corresponding genotype’s isotype control (blue histogram); data presented are representative of 4 biological replicate experiments. e The average ± SEM in the fold change (FC) in the CD49f MFI among all four genotypes of cultured cells (n = 4 independent experiments/genotype; shControl set to 1.0; p <0.05.). f The average ± SEM in the fold change (FC) of ITGA6 mRNA levels expressed in tumors among all four genotypes (n = 4 tumors/genotype; shControl set to 1.0; p <0.05.); total RNA was prepared from homogenized whole tumors