Fig. 5: Continuous lapatinib treatment gives rise to NCI-N87 persistent cells.

A Immunoblot analysis showing the level of HER2 expression in breast and oesophageal adenocarcinoma cell lines. MDA-MB-231 cells are triple negative breast cancer cells that lack HER2 expression. β-Tubulin expression was used as loading control. Similar results were obtained in two independent experiments. B Oesophageal adenocarcinoma cell lines were treated with various concentrations of lapatinib for 3 days as indicated, followed by crystal violet staining and quantification (n = 3). C NCI-N87 cells were treated with lapatinib (50 nM) for 42 days. Cell density was measured by crystal violet staining. The data are expressed as fold relative to the day lapatinib was added (day 0) (n = 3). D NCI-N87 parental (PT) cells were treated with lapatinib (50 nM) for 21 days and released from therapy for 1 day (NCI-N87-PS) or 14 days (OE19-PT*) prior to being mock treated with DMSO or exposed to lapatinib (50 nM) for 3 days. Cell density was measured by crystal violet staining (n = 3). E The level of NRF2 expression in NCI-N87 cell lines was quantified by immunoblot analysis (n = 4). β-Tubulin expression was used as loading control. F Immunoblot analysis showing increased level of NRF2 expression in NCI-N87 cells stably transduced with doxycycline inducible NRF2 (i-NRF2) encoding lentiviral particles after incubation with 50 ng/mL doxycycline (Dox) for 24 h. β-Tubulin expression was used as loading control. Similar results were obtained in two independent experiments. G NCI-N87 cells expressing an empty lentiviral construct (i-Ctrl) or a construct encoding inducible NRF2 (i-NRF2) were treated with lapatinib (50 nM) in the presence of doxycycline (Dox; 50 ng/mL) for 6 days. Cell density was quantified by crystal violet staining. The data are expressed as fold relative to the day lapatinib was added (day 0) (n = 3). Statistical analyses were performed by one-way ANOVA (E) or unpaired t-test (D, G).