FIGURE 3.

Celastrol potentiates the apoptotic effect of bortezomib in various MM cell lines. (A) U266 cells were treated with CSL (0.5 μM), bortezomib (10 nM) and a combination of both for 24 h at 37°C. Whole-cell extracts were prepared, separated on SDS-PAGE, and subjected to Western blot analysis using antibody against pro- and cleaved caspase-3. The same blots were stripped and reprobed with β-actin antibody to show equal protein loading. (B) U266 cells were treated with CSL (0.5 μM), bortezomib (10 nM) and a combination of both for 24 h at 37°C. Whole-cell extracts were prepared, separated on SDS-PAGE, and subjected to Western blot analysis using antibody against PARP. The same blots were stripped and reprobed with β-actin antibody to show equal protein loading. Results typical of two independent experiments are shown. (C) U266 cells were treated with CSL (0.5 μM), bortezomib (10 nM), and a combination of both for 24 h at 37°C. Caspase-3 activity/luminescence of each sample was measured in a plate-reading luminometer. Columns, mean; bars, SD. ^∗p < 0.05, ^∗∗p < 0.01. (D) H929 cells were treated with CSL (0.5 μM), bortezomib (10 nM), and a combination of both for 24 h at 37°C. Apoptosis was determined by measuring the degree of DNA fragmentation in the cytoplasm of cells. Columns, mean; bars, SD. ^∗p < 0.05. (E) KMS-11 cells were treated with CSL (0.5 μM), bortezomib (10 nM), and a combination of both for 24 h at 37°C. Apoptosis was determined by measuring the degree of DNA fragmentation in the cytoplasm of cells. Columns, mean; bars, SD. ^∗p < 0.05. (F) U266 cells were treated with CSL (0.5 μM), bortezomib (10 nM), and a combination of both for 24 h at 37°C. Twenty micrograms of the nuclear protein was used for DNA-binding assay as described in the section “Materials and Methods.” Columns, mean; bars, SD. ^∗p < 0.05.