Figure 2

Transcriptional and posttranslational mechnisms lead to an in crease in EGFR expression after BRCA1 inhibition. (A) EGFR mRNA levels were determined in control MECs (light gray bars) and in mammary epithelial cells (MECs) expressing small hairpin RNA directed against BRCA1 (dark bars). RNA levels were normalized for GAPDH (glyceraldehyde 3-phosphate dehydrogenase) expression. RT-PCR, real-time reverse transcriptase-polymerase chain reaction; HMLE, human mammary epithelial cells. (B) Decreased EGFR promoter activity as a result of short-term BRCA1 suppression in MECs and MCF-7 cancer cells. BRCA1 control and small interfering RNA (siRNA) and the full-length EGFR promoter were transfected as indicated, and luciferase activity was normalized for Renilla thymidine kinase expression. (C and D) EGFR half-life increases from less than 30 minutes to over 70 minutes after BRCA1 inhibition. Control and BRCA1 sh2-expressing MCF-10A cells were transfected with hemagglutinin-tagged EGFR 48 hours prior to the time course, serum-deprived for 8 hours, then treated with cycloheximide at 100 μg/ml for 2 hours, and stimulated with epidermal growth factor at point 0. Lysates were prepared and immunoblotted at the indicated time points. The chemiluminescence signal was quantified as in Figure 1B. Similar results were obtained with BRCA1 sh1-expressing cells. In D the ratio of the optical density (OD) for EGFR to Tubulin is plotted against time.