(A) Expression levels of HSF1 phospho-S326, HSF1 phospho-S303, total HSF1 and β-catenin was determined by Western blotting in CD44+/CD24− and in the depleted CD44−/CD24+ (Non-CSC) populations of MDA–MB-231 cells. Levels of β-actin expression were also measured as loading controls. Relative levels of phospho-S326 HSF1 and β-catenin, determined by densitometry are indicated. (B) Protein expression levels of HSF1 phospho-S303, HSF1 phospho-S326, total HSF1, β-catenin, S6K phospho-T389, total S6K was determined by Western blotting in MCF7 cells. GAPDH expression level was also measured as loading control. Relative levels of phospho-S326, phospho-S303, β-catenin and phospho-S6K was determined by densitometry as shown. (C) Protein expression levels (as in B) were measured in MCF10A cells. Relative levels of phospho-S326, phospho-S303, β-catenin and phosphor-S6K in the immunoblots were determined by densitometric analyses using Image J software, as shown. Experiments were performed twice with reproducible results.