Fig. S6.

Dyn1 activation by calcineurin blocks TRAIL-induced apoptosis in H1299 cells and influences TRAIL activity in several cancer cells. (A) TRAIL activity (100 ng/mL) in H1299 WT, Dyn1^KO, and H1299 Dyn1^KO cells reconstituted with Dyn1 WT (KO + WT) and Dyn1 S774A/S778A (KO + AA), in the presence or absence of calcineurin inhibitors. Cells were preincubated with CsA (20 µM) for 30 min, followed by treatment with 100 ng/mL of TRAIL and assayed for cell viability. Reduction in cell viability (%) was calculated relative to control wells containing no ligand. All of the results are mean values ± SD (n = 3). Two-tailed Student’s t tests were used to assess statistical significance. **P < 0.005; ns, nonsignificant. (B) TRAIL activity (100 ng/mL; overnight incubation) in the indicated cell lines, in both control (si-Ctrl) and Dyn1-depleted (si-Dyn1) cells, calculated relative to control wells containing no TRAIL. (C) Effects of calcineurin inhibition by CsA on TRAIL activity in several human cancer cell lines, including HCT116 colorectal carcinoma, SCC61 squamous carcinoma, HT1080 fibrosarcoma and MV3 melanoma cells, and caspase-3–deficient MCF-7 mammary carcinoma cells. Reduction in cell viability by TRAIL (%) was calculated relative to control wells incubated with 0 or 20 µM of CsA. All of the results are mean values ± SD (n = 3). (D) TRAIL-induced caspase-8 cleavage and activation is enhanced in CsA pretreated cells. MDA-MB-231 cells were treated with TRAIL (1 μg/mL) in the presence or absence of CsA for the time points indicated and analyzed by immunoblot for processing of caspase-8.