Fig. 4: NRF2 overexpression is essential for adaptation of OE19-PT cells to lapatinib.

A NRF2 transcript levels were measured by qPCR to demonstrate decreased expression in OE19-PT cells stably transduced with doxycycline inducible shNRF2 (i-shNRF2) encoding lentiviral particles after incubation with 50 ng/mL doxycycline (Dox) for 24 h (n = 3). B OE19-PT cells expressing inducible shScr or shNRF2 were incubated with DMSO or with lapatinib (500 nM), without or with doxycycline (Dox; 50 ng/mL) for 12 days. Cell density was quantified by crystal violet staining. The data are expressed as fold relative to the day drugs were added (day 0) (n = 3). C Immunoblot analysis showing increased level of NRF2 expression in OE19-PT cells silenced with siKEAP1 (+) RNA for 24 h. OE19-PT cells transfected with scrambled siScr (−) RNA were used as control. GAPDH expression was used as loading control. Similar results were obtained in two independent experiments. D Quantification of cell density by crystal violet staining of OE19-PT cells silenced with siScr (−) or siKEAP1 (+), alone or in combination with lapatinib treatment (500 nM) for 3 days (n = 4). E Immunoblot analysis showing increased level of NRF2 expression in OE19-PT cells stably transduced with NRF2 encoding lentiviral particles. β-Tubulin expression was used as loading control. Similar results were obtained in three independent experiments. F Quantification of cell density by crystal violet staining of Ctrl (empty vector) and NRF2 over-expressing OE19-PT cells treated with lapatinib (500 nM) for 6 days. 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 (D) or unpaired t-test (A, B, F).