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. 2017 Mar 20;8:14179. doi: 10.1038/ncomms14179

Figure 4. Amplification of TRAIL apoptotic effect via polymeric particles conjugation is caspase dependent and increases death receptor expression.

Figure 4

(a) Brightfield micrographs of particle (diameter: 500 nm)-functionalized COLO 205 tumour cells treated with 0.1 μg ml−1 TRAIL under fluid shear stress exposure (shear stress: 4.0 dyn cm−2) for 1 h in the absence and presence of 50 μM pan caspase inhibitor Z-VAD-FMK. (b) Viability of particle-functionalized COLO 205 tumour cells treated with 0.1 μg ml−1 TRAIL under fluid shear stress exposure for 1 h in the presence of 50 μM pan caspase inhibitor Z-VAD-FMK, 50 μM caspase-8 inhibitor Z-IETD-FMK or 50 μM caspase negative control inhibitor Z-FA-FMK. N=4 for all treatments. (c,d) Annexin-V/propidium iodide (PI) flow cytometry plots of particle-functionalized COLO 205 tumour cells treated with 0.1 μg ml−1 TRAIL in the presence of a fluid shear stress for 1 h without and with Z-VAD-FMK treatment. N=4 for all treatments. (e) Annexin-V quantification of particle-functionalized COLO 205 tumour cells treated with 0.1 μg ml−1 TRAIL under fluid shear stress exposure for 1 h in the presence of 50 μM pan caspase inhibitor Z-VAD-FMK, 50 μM caspase-8 inhibitor Z-IETD-FMK or 50 μM caspase negative control inhibitor Z-FA-FMK. N=4 for all treatments. (f) Caspase-8 activity of particle-functionalized COLO 205 tumour cells after treatment with TRAIL (0.1 μg ml−1) in the presence and absence of fluid shear stress for 1 h. N=4 for all treatments. (g) TRAIL death receptor (DR) 4 and 5 expression after treatment of particle-functionalized COLO 205 tumour cells with TRAIL (0.1 μg ml−1) in the presence and absence of fluid shear stress for 1 h. N=5 for all treatments. Data are reported as mean±s.e. Different treatment groups were compared for statistical significance using Student's two-tailed t-test for two conditions and one-way analysis of variance (ANOVA) for multiple comparisons. Percent of max represents the number of events normalized according to FlowJo algorithms. *P<0.05 and **P<0.01. NS, not significant.