Figure 3
HECTD3 depletion resulted in increase of caspase-8 activation and apoptosis induced by TRAIL, Fas Ab, and TNFα (a) HECTD3 depletion in HeLa by shRNA led to increase of caspase-8, -3, and PARP cleavage (cl-) after treatment with different dosages of TRAIL (25–50 ng/ml), anti-CD95 Ab (100–400 ng/ml with 10 μg/ml cycloheximide (CHX, blocking NF-κB pro-survival target gene expression)), and TNFα (2.5–10 ng/ml with 10 μg/ml CHX) for 16 h. Lush means control shRNA against the luciferase gene. Hsh means shRNA against HECTD3. (b) HECTD3 depletion in MDA-MB-231 by shRNA resulted in increase of caspase-8, -3, and PARP cleavage after treatment with different dosages of TRAIL (25–50 ng/ml), anti-CD95 Ab (100–400 ng/ml with 10 μg/ml CHX), and TNFα (2.5–5 ng/ml with 10 μg/ml CHX) for 16 h. (c) HECTD3 depletion in MDA-MB-231 significantly increased Annexin V-positive apoptotic cells. Stable cells were treated with 50 ng/ml TRAIL for 16 h and stained with FITC-Annexin V and 7-AAD. The stained cells were analyzed by flow cytometry. The quantitative results are shown on the right panel. Error bars are S.D. from three independent experiments. **P<0.01, t-test. (d) HECTD3 depletion decreased MDA-MB-231 cell viability in the presence of different dosages of TRAIL for 2 days, as determined by the SRB assay. Error bars are S.D. from three independent experiments. **P<0.01, t-test. Lusi means control siRNA against the luciferase gene. Hsi means siRNA against HECTD3. (e) HECTD3 depletion in MDA-MB-231 significantly decreased the colony formation in the presence of TRAIL. MDA-MB-231 cells were seeded in 12-well plates at 100/well. LusiRNA and HECTD3 siRNA were transfected on the second day. Two days later, the cells were left untreated or treated with 10 ng/ml TRAIL for 2 weeks. The quantitative results are shown on the right panel. Error bars are S.D. from three independent experiments. **P<0.01; t-test
