Y-box binding protein 1 (YB-1) regulates the expression of epidermal growth factor receptor (EGFR) in basal-like breast cancer cells. (a) The levels of YB-1 and EGFR proteins were compared between immortalized breast epithelial cells, 184 htert, SUM149 and HCC1937 basal-like breast cancer cells. Actin was evaluated as a control for equal protein input. (b) 184 htert cells were transfected with an EGFR promoter (1 kb) luciferase construct (pER1), a control renilla plasmid (pRL-TK) and either flag-EV or flag-YB-1 or flag-YB-1(A102). Luciferase and renilla activity were measured after 24 hours. YB-1 induced EGFR promoter activity by 1.5-fold (P = 0.04, N = 6), whereas the A102 mutant did not. (c) SUM149 cells were treated with YB-1 small interfering RNA (siRNA) (5 nM) for 48 h. The cells were then transfected with the EGFR reporter for 24 h and compared with the empty vector. Loss of YB-1 expression resulted in a 78% decrease in EGFR reporter activity (P = 4.53 × 10-5, N = 6). Inset: evidence that siRNA targeting YB-1 causes a decrease in expression of the protein. Actin was used as a loading control. (d) The same experiment was repeated using HCC1937 cells treated with 20 nM YB-1 siRNA for 48 h. Loss of YB-1 expression resulted in a 77% reduction in EGFR promoter activity (P = 5.98 × 10-7, N = 6).