Figure 4.

TRAIL triggers caspase activation, but caspases are not involved in the induction of proliferation. A) SGBS preadipocytes were stimulated with growth medium alone or with TRAIL. At the indicated time points, proteins were isolated and subjected to Western blot analysis to assess the processing of caspase-8 and -3. β-actin was used as a loading control. One representative of 3 independent experiments performed is shown. B, C) SGBS preadipocytes were stimulated with growth medium alone or with TRAIL and/or zVAD.fmk. B) At the indicated time points, proteins were isolated and subjected to Western blot analysis to assess the inhibition of caspase-8 and -3 processing by zVAD.fmk. β-actin was used as a loading control. One representative of 3 independent experiments performed is shown. C) After 72 hours, [³H]-thymidine incorporation was measured. Data are expressed as means ± sem of six independent experiments performed. *P ≤ 0.05; ***P ≤ 0.001 compared with growth medium alone (directly above the bars). ns, not significant comparing the indicated treatments (above the connecting line). D, E) A stable, lentiviral-mediated knockdown of caspase-8 in SGBS preadipocytes was performed. Two shRNAs targeting caspase-8 (shC8.1 and shC8.2) were used. A hyper random sequence (shHRS) was used as a control. Subsequent experiments were performed with cells from bulk cultures. D) Western blot analysis to assess the extent of caspase-8 knockdown by shRNA expression. α-tubulin was used as a loading control. E) Stably modified SGBS preadipocytes were stimulated with growth medium alone or with increasing doses of TRAIL. After 72 hours, [³H]-thymidine incorporation was measured. Data are expressed as means ± sem of 3 independent experiments performed. **P ≤ 0.01; ***P ≤ 0.001 compared with growth medium alone.