Fig 4. Differential protein levels and phenotypes are exhibited among MCF10A cells following selection through IH.

(A) Analysis of protein levels was performed using the Reverse Phase Protein Array (RPPA) on the parental MCF10A line, passaged control clone 7, IH-selected clone 4, and IH-selected clone 9. Each clone was submitted in duplicate. Hierarchical clustering of significant (FDR ≤ 0.05) protein markers in control versus IH-selected samples is shown. The clustering was carried out with Euclidean distance metric with average linkage, and the data was median normalized and log2 transformed. The clustering was arbitrarily stopped at 2 and 5 groups for the samples and features respectively. (B) Western blotting analysis of p53 and E-cadherin protein. p53 protein is reduced by in IH-selected clones. E-cadherin is completely lost following selection by IH, and N-cadherin protein is detectable, indicating EMT in IH-selected clones. (C) Quantitative RT-PCR was performed on DNA levels of the P53 and E-CAD loci. A 0.5 fold reduction in the number of copies of P53 and E-CAD indicates a loss of one allele of each. (D) Western blotting against HIF-1α and GLUT-1 indicates constitutive stabilization of HIF-1α and its downstream effector GLUT-1 in IH-selected cells. MCF10A cells cultured in hypoxia are used as a positive control.