Figure 4

PIDD-induced chemoresistance in NSCLC depends on NRF2. MGH7 or H1299-LV-PIDD cells were infected with shRNA lentiviruses targeting NRF2 (NRF2-shRNA1 and NRF2-shRNA2) or a non-targeting lacZ shRNA control (NC). (A) Western blotting was performed to determine the expression levels of NRF2 and NQO1 in both NC and NRF2-shRNA infected cells. The upper and lower panels were from the same gel. The gel was transferred to the same membrane, which was cut to probe with different antibodies. The entire image of each exposed membrane is shown. (B) Quantification of NRF2 and NQO1 protein levels in lentivirus infected MGH7 cells. Data are presented as the densitometric means of three immunoblotting replicates and are normalized to expression of GAPDH. Error bars represent standard error of the mean (SEM) (Two tailed Student’s t-test, *P = 0.0302; **P = 0.0055; ***P < 0.0001). (C) Viability of MGH7 cells infected with lentiviruses expressing either NC or shNRF2 (NRF2-shRNA2) after treatment with different doses of cisplatin for 72 hours. Viability was quantified by the MTT assay. (D) Western blotting was performed to determine the expression levels of NRF2, NQO1 and PIDD in both NC and NRF2-shRNA infected cells. The upper and lower panels were from the same gel. The gel was transferred to the same membrane, which was cut to probe with different antibodies. The entire image of each exposed membrane is shown. (E) Quantification of NRF2, NQO1 and PIDD protein levels in lentivirus infected H1299-LV-PIDD cells. Data are presented as the densitometric means of three immunoblotting replicates and are normalized to expression of ß-actin. Error bars represent standard error of the mean (SEM) (Two tailed Student’s t-test, **P = 0.0019 for NRF2 in shRNA2; **P = 0.0037 for NQO1 in shRNA2). (F) NRF2 shRNA2-infected H1299-LV-PIDD cells showed decreased viability as compared to the negative control cells after treatment with different doses of cisplatin for 24 hours. Viability was quantified by the MTT assay. Data are mean ± SEM for three experiments (Two way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001; ns, P > 0.05).