Effects of celastrol on gene expression. (A) Celastrol inhibits TNF-induced NF-κB–dependent reporter gene (SEAP) expression. A293 cells were transiently transfected with an NF-κB–containing plasmid linked to the SEAP gene and then treated with the indicated concentrations of celastrol. After 24 hours in culture with 1 nM TNF, cell supernatants were collected and assayed for SEAP activity as described in “Materials and methods.” Results are expressed as fold activity over the activity of the vector control. (B) Celastrol inhibits NF-κB–dependent reporter gene expression induced by TNFR, TRADD, TRAF, NIK, IKKβ, and p65. A293 cells were transiently transfected with the indicated plasmids along with an NF-κB–containing plasmid linked to the SEAP gene and then left either untreated or treated with 2.5 μM celastrol for 6 hours. Cell supernatants were assayed for secreted alkaline phosphatase activity as described in “Materials and methods.” Results are expressed as fold activity over the activity of the vector control. Bars indicate standard deviation. (C) Celastrol suppresses TAK1/TAB1-induced NF-κB activation. A293 cells were transiently transfected with a TAK1/TAB1 expression plasmid along with NF-κB–containing plasmid. After 24 hours, cells were treated with indicated concentrations of celastrol and incubated with the relevant plasmid for an additional 6 hours. The supernatants of the culture medium were assayed for SEAP activity as described in “Materials and methods.” The results shown are representative of 3 independent experiments. (D) A schematic diagram of the effect of celastrol on TNF-induced NF-κB activation and apoptosis.