Figure 5.
Celastrol inhibits constitutively active NF-κB in MM cells. Celastrol suppressed NF-κB in a time-dependent dependent manner in U266, and RPMI 8826 cells. (A, left panel) U266 cells (2 × 106 mL−1) were treated with 2.5 µM celastrol for 0, 30, 60, 120 and 240 min; nuclear extracts were prepared, and 20 µg of the nuclear extract protein was used for elisa-based DNA binding assay as described in Methods. (A, right panel) RPMI 8826 cells (2 × 106 mL−1) were treated with 2.5 µM celastrol for 0, 30, 60, 120 and 240 min; DNA binding assay for NF-κB was performed. The results shown are representative of three independent experiments. (B, left panel) U266 cells were treated with 2.5 µM celastrol for 0, 30, 60, 120 and 240 min. Cytoplasmic extracts were prepared and Western blotting was done using phospho-IκBα and anti-IκBα antibodies. Equal protein loading and purity was evaluated by Western blotting against GAPDH. (B, right panel) U266 cells were treated with 2.5 µM celastrol for 0, 30, 60, 120 and 240 min. Nuclear extracts were prepared and Western blotting was done using antibodies for phospho-p65 and p65. Equal protein loading and purity was evaluated by Western blotting against lamin B. (C) U266 cells were incubated with medium or with 2.5 µM celastrol for 4 h and then analysed for the intracellular distribution of p65 by immunocytochemistry. Red indicates p65, and blue indicates nuclei (original magnification × 200). The results shown are representative of three independent experiments. (D) U266 cells were treated with 2.5 µM celastrol for 0, 30, 60, 120 and 240 min. Whole cell extracts were prepared and probed for phosphor-specific IKKα/β protein. Equal protein loading was evaluated by IKKα.