Fig. 3.
Stable knockdown of Nrf2 by overexpression of Keap1 increases the susceptibility of A549 lung carcinoma cells to chemotherapeutic agents. (A) Two A549-derived cell lines stably expressing the control vector or Keap1–CBD were established using a lentivirus-based approach. Cell lysates from these two cell lines were subjected to immunoblot analysis with antibodies against CBD, Nrf2, NQO1, HO-1 and tubulin (left panel). The NQO1 enzymatic activity was measured by reduction of DCPIP (middle panel). The intracellular glutathione level of the two cell lines was determined with QuantiChrom glutathione assay kit. (B) Cell viability was assessed by the MTT assay following 48 h treatment with the indicated doses of cisplatin, doxorubicin and etoposide. (C) Cells growing on glass slide were treated with different doses of cisplatin for 48 h and labeled with fluorescein-deoxyuridine triphosphate using the In Situ Cell Death Detection kit. Pictures were taken under fluorescent microscope (left panel). Thirteen fields of each slide were counted to get the total number of apoptotic cells (table). (D) The two cell lines were treated with doxorubicin for 48 h; apoptotic cell death was measured using Cell Death Detection ELISAPLUS kit. (E) Cells were treated with cisplatin, doxorubicin and etoposide for 48 h and stained with Annexin V–fluorescein isothiocyanate and propidium iodide using the Annexin V–Fluorescein Isothiocyanate Apoptosis Detection Kit, followed by flow cytometric analysis. The data are presented as means ± SDs. *P < 0.05.