Figure 2. KEAP1-KO alters NRF2 levels.

(A) KEAP1-KO does not affect p-ERK. Whole cell lysates of HCC364-Cas9 cells with the indicated sgRNAs treated with DMSO or trametinib for 48 hr. (B) KEAP1-KO increases NRF2 levels. Nuclear and cytoplasmic fractions of HCC364 cells. (C) Immunoblot showing NRF2 expression in CALU1 and HCC364. (D) Crystal violet colony formation assays. 10,000 CALU1 cells expressing the indicated ORFs were treated with DMSO for 8 days or trametinib for 10 Days. 10,000 HCC364 cells expressing the indicated ORFs were treated with DMSO for 10 days or trametinib/vemurafenib for 21 days. Error bars represent the SD of the mean of triplicate wells. (E) Immunoblot showing expression of wildtype KEAP1 or KEAP1 G333C in A549 cells. (F) Expression of wildtype KEAP1 resensitized A549 cells to trametinib. 5000 cells were treated with 25 nM trametinib for 12 days. Error bars represent the SD of six wells. (A–F) Experiments were performed two independent times, and one representative experiment is shown. ***p=0.0001, **p=0.0005, *p<0.002.

DOI: http://dx.doi.org/10.7554/eLife.18970.009

Figure 2—figure supplement 1. KEAP1-KO alters NRF2 levels and does not activate ERK.

(A) Immunoblot showing p-ERK suppression in 72 hr erlotinib-treated sgKEAP1 HCC827 cells. (B) Immunoblot showing p-ERK suppression and BIM expression in 14 day trametinib-treated HCC364 and CALU1 cells. (C) Immunoblot showing increase in NRF2 upon KEAP1 knockout in CALU1, MGH-065, and HCC827. For MGH-065 and HCC827, loading controls are the same as in Figure 1—figure supplement 4.

DOI: http://dx.doi.org/10.7554/eLife.18970.010

Figure 2—figure supplement 2. NRF2 is necessary and sufficient for resistance.

10,000 HCC827 cells were treated with 100 nM erlotinib for 10 days. Error bars represent the SD of the mean of triplicate wells. ***p<0.0001, **p<0.005.

DOI: http://dx.doi.org/10.7554/eLife.18970.011