Figure 3

PIDD promotes chemoresistance in NSCLC cells both in vitro and in vivo. H1299 cells were either infected with a PIDD overexpressing lentivirus (LV-PIDD) or were mock infected (LV-mock). (A) Western blotting showed increased levels of PIDD and NRF2 in LV-PIDD-infected cells as compared to LV-mock 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 higher levels of PIDD and NRF2 in lentivirus infected H1299 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 = 0.0011; ***P < 0.0001). (C) LV-PIDD infected H1299 cells showed increased viability as compared to the LV-mock H1299 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 < 0.01; ***P < 0.001; ns, P > 0.05). (D–F) Cisplatin sensitivity of LV-mock and LV-PIDD-infected H1299 tumors grown as xenografts in vivo. (D) Dissected tumors from the two experimental groups showed smaller size of tumors formed by LV-mock H1299 cells treated with cisplatin (Group 1), as compared to tumors formed by LV-PIDD-H1299 cells (Group 2) treated with cisplatin. (E) Quantification of tumor volume. The volume of LV-PIDD tumors were significantly larger than the mock group. Data are mean ± SEM for 5 mice per arm (Two way ANOVA; *P < 0.05; ***P < 0.001; ns, P > 0.05). (F) Average tumor weight at termination of the experiments. The weights of the LV-PIDD tumors (Group 2) were significantly larger than the mock group (Group 1). The data are means ± SEM reflecting 5 mice per arm. (Two tailed Student’s t-test; **P = 0.0081) compared with the control.