Figure 6.

Inhibition of Ca²⁺/calmodulin signaling reduces survival of NSCLC cells in response to TRAIL. (a) A549 cells were pretreated (1 h) with calmodulin inhibitor CMZ (10 μM) or FPZ (10 μM), followed by 2-h treatment with TRAIL (200 ng/ml). Processing of procaspase-8 was analyzed by immunoblot. (b) A549 cells were pretreated with calcium chelator BAPTA-AM (10 μM, 1 h), followed by 1-h treatment with biotinylated TRAIL (500 ng/ml). Protein complexes were precipitated using streptavidin agarose as described in Materials and Methods section. Biotinylated TRAIL was also added to untreated cell lysate at 500 ng/ml. DR4, FADD, c-FLIP_S, and procaspase-8 cleavage were detected by immunoblot using specific antibodies. (c) A549 cells were pretreated with calmodulin inhibitors FPZ and CMZ or calcium chelator BAPTA-AM (10 μM, 1 h). To avoid toxicity, cells were washed and treated for an additional 24 h with TRAIL (200 ng/ml). Cell death was analyzed by flow cytometry using Annexin V staining. (d) The effect of calcium chelator BAPTA-AM on clonogenic survival of A549 and H661 cells. Cells were either untreated (Untr) or pretreated with BAPTA-AM (10 μM, 1 h), followed by TRAIL treatment (200 ng/ml, 3 h). Then, cells were washed and incubated for an additional 14 days and a clonogenic assay was performed as described above. Error bars represent S.E. *P<0.05. Cl, cleaved; FADD, Fas-associated protein with death domain; IP, immunoprecipitation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase