HER2 activation is maintained in surviving SKBR3 cells with reactivation of HER3 and downstream signalling pathways via the autocrine ligand release.

A, SKBR3 cells were assessed for HER2 phosphorylation by FRET (see Methods) after the cells were treated with 1 µM Iressa for different durations. B, In the upper panel, SKBR3 and MCF-7 cells were lysed after treatment with either DMSO or 1 µM Iressa for the durations shown and the phosphorylation state of HER3 on Tyr1289 was determined using phosphospecific antibody. In the lower panels, SKBR3 cells were lysed after treatment with either DMSO, 3 µM AG 1478 or 1 µM Iressa for the durations illustrated. Phospho-PKB, phospho-MAPK and the total levels of PKB and Erk1/Erk2 were assessed using appropriate the antibodies. C, Cell viability experiments with SKBR3 cells were performed after treatment with DMSO or 1 µM Iressa with or without growth factors for 4 days. In the first condition DMSO was used as a vehicle control. In other conditions 1 µM Iressa was utilised alone or together with 100 ng/ml TGF, 100 ng/ml heregulin β, 100 ng/ml heregulin β-1 or 20 ng/ml betacellulin. The viable cells were counted in a cell viability analyzer after 4 days using Trypan Blue to stain dead cells. D, SKBR3 cells were treated with 20 µg/ml of anti-betacellulin, Iressa alone or Iressa in combination with 20 µg/ml of anti-betacellulin for 4 days before the cells were counted in a cell viability analyzer. DMSO was used as a vehicle control.