Fig. 1.

Itga6 expression is down-regulated in response to deletion of HIF-1 in PyMT+ cultured cells and in tumors. a HIF-1 WT or KO cells were cultured at normoxia or hypoxia (6 h, 0.5 % O₂) and the relative expression of Itga6 mRNA levels compared after normalization to Ints3. The mean fold-change in Itga6 levels was compared to KO cells cultured at hypoxia (set to 1.0) (*p < 0.01, ANOVA with Bonferroni post-test; n = 3 biological replicates). b Western blotting was performed for ITGA6 protein using whole cell extracts (WCE) prepared from near confluent HIF-1 WT and KO cells cultured at either normoxia (N) or hypoxia (H). Tata-binding protein (TBP) is shown as a loading control. c HIF-1 WT and KO adherent cells were exposed to normoxia or hypoxia (6 h, 0.5 % O₂), fixed, immunostained with CD49f-FITC and counterstained with DAPI; the scale bar represents 20 μm. d The relative abundance of Itga6 mRNA after normalization to Ints3 in the same set of HIF-1α WT and KO tumors as subjected to FACS analysis in panel (e) (p < 0.01, Student’s t-test). e Representative histograms derived from FACS analysis of live, CD49-FITC stained cells isolated from HIF-1 WT (black histogram) and KO (blue histogram) tumors relative to the isotype antibody control (red histogram). To normalize the histogram height between samples, the y-axis shows the % Max (the number of cells in each bin divided by the number of cells in the bin that contains the largest number of cells). Data shown are representative of n ≥5 tumors/genotype. The percentage of tumor cells positive for CD49f-FITC was determined based on the live, singlet, Lin^neg parent population using FlowJo and the gating strategy presented in Additional file 4: Figure S4. f The mean ± SEM in the fold change of CD49f-FITC median fluorescence intensity (MFI) between WT and KO PyMT tumors; all data are expressed relative to each genotype’s corresponding isotype control MFI (p < 0.01, Student’s t-test; n ≥5 tumors/genotype)