Figure 8

Inhibition of HER2 restores lapatinib sensitivity in BT474 late stage lapatinib resistant cells. (A) BT474 parental and resistant cells were treated with pooled EGFR, HER2, HER3, ER siRNA, or non-targeting control siRNA, for 72 hours. Proliferation was measured using the Click-iT EdU (5-ethynyl-2'- deoxyuridine) Microplate Assay. Apoptosis was measured by detecting Annexin V expression. Signals were visualized and quantitated by the Celigo cytometer (Cyntellect, San Diego, CA, USA). (B) Down-regulation of EGFR, HER2, HER3, and ER in BT474 derivatives after siRNA treatment was detected by Western blot. Whole-cell extracts were analyzed with the indicated antibodies, including downstream signaling. (C) Growth fold change of double dosage (2 μM) lapatinib on BT474 early and late stage-lapatinib resistant cells for six-day treatment. Cell numbers were assessed using methylene blue and quantified by absorbance at 655 nm and normalized against Day 0. Significance between groups was determined by multiple comparisons using the Sidak method (*P < 0.0001, LLR + L versus LLR + 2L).