Figure 4. KEAP1-KO reduces trametinib-induced ROS through increased glutathione synthesis.
(A) HCC364 or CALU1 cells were treated with DMSO or trametinib for 72 hr. ROS was measured by DCFDA fluorescence. Error bars represent SD of the mean of two biological replicates. (B, D) CALU1 or HCC364 cells were treated with DMSO or trametinib for 72 hr and metabolites were measured by liquid chromatography tandem mass spectrometry. Error bars represent SD of eight replicates. (C) Glutathione synthesis pathway. (E) Expression of GPX1 and GSR in HCC364 and CALU1 cells treated with DMSO or trametinib for 72 hr. Error bars represent the SD of the mean of three biological replicates. ***p<0.005, **p<0.01, *p<0.02.
Figure 4—figure supplement 1. KEAP1-KO reduces drug-induced ROS.
(A) HCC827 or MGH065 cells or (B) CALU1 cells were treated with DMSO or drug for 72 hr. ROS was measured by DCFDA fluorescence. Error bars represent SD of the mean of two biological replicates.
Figure 4—figure supplement 2. Glutathione levels in HCC364 and CALU1 cells.
Oxidized glutathione (glutathione disulfide, GSSG) is increased in KEAP1-KO cells, but the ratio of reduced to oxidized glutathione (GSH/GSSG) is higher in KEAP1-KO cells. CALU1 and HCC364 cells were treated with DMSO or trametinib for 72 hr, and metabolites were measured by liquid chromatography tandem mass spectrometry. Error bars represent SD of eight replicates.
Figure 4—figure supplement 3. KEAP1-KO alters expression of SHMT1/2 in some cell lines.
(A) Expression of SHMT1/2 in HCC364 and CALU1 cells treated with DMSO or trametinib for 72 hr. (B) Expression of SHMT1/2 in HCC827 or MGH-065 cells treated with erlotinib or LDK378 for 72 hr. Error bars represent SD of the mean of three biological replicates.
Figure 4—figure supplement 4. KEAP1-KO alters expression of GPX1 and GSR.
Expression of GPX1 and GSR in HCC827 or MGH-065 cells treated with erlotinib or LDK378 for 72 hr. Error bars represent SD of the mean of three biological replicates.