Figure 2.

DR4 mediates apoptosis of NSCLC cells in response to TRAIL treatment. (a) DR4 and DR5 in A549 cells were blocked using specific antagonizing antibodies for 30 min before TRAIL treatment (3 h, 200 ng/ml). Processing of procaspase-8 and cleavage (Cl) of PARP were analyzed by immunoblot. (b) DR5 was silenced in A549 cells using specific small interfering RNA (siRNA), and 48 h post-siRNA transfection, cells were treated with TRAIL (3 h, 200 ng/ml). (c) Expression of surface DR4 assessed by flow cytometry as described in Materials and Methods section. Filled histograms represent antibody control and green histograms represent staining with DR4 antibody. (d) Surface DR4 in a panel of NSCLC cells was blocked using a neutralizing antibody for 30 min prior TRAIL treatment (3 h, 200 ng/ml). Cell death was analyzed by Annexin V staining using flow cytometry. (e) Expression of procaspase-8 and DR4 in lung adenocarcinomas and adjacent non-neoplastic tissues was analyzed by immunoblot. Error bars represent S.E. *P<0.05. Ctr, control; a-DR, antagonizing DR4, or DR5 antibody; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; N, normal tissue; T, tumor tissue; scr, scrambled; Untr, untreated