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. 2012 Jul;97(7):1092–1100. doi: 10.3324/haematol.2011.053421

Figure 3.

Figure 3.

Thiostrepton-induced mitochondrial apoptotic pathway in DLBCL cells. (A) SUDHL4 and OCI-LY19 cells were treated with 5 and 10 μM thiostrepton for 48 h and cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against p-AKT, p-Bad and Beta-actin. (B) SUDHL4 and OCI-LY19 cells were transfected with either 100 nM scrambled siRNA or 50 and 100 nM specific siRNA targeted against FoxM1 for 48 h. After incubation, cells were lysed and immunoblotted with antibodies against p-AKT, p-Bad and Beta-actin. (C) After treating with 10 μM thiostrepton for indicated time periods, SUDHL4 cells were lysed in 1% Chaps lysis buffer and subjected to immunoprecipitation with anti-Bax 6A7 monoclonal antibody and probed with specific polyclonal anti-Bax antibody for detection of conformationally changed Bax protein. In addition, the total cell lysates were applied directly to SDS–PAGE, transferred to immobilon membrane and immunoblotted with specific anti-Bax polyclonal antibody. (D) Loss of mitochondrial membrane potential by thiostrepton treatment of DLBCL cells. DLBCL cells were treated with and without 5 and 10 μM thiostrepton for 48 h. Live cells with intact mitochondrial membrane potential and dead cells with lost mitochondrial membrane potential were measured by JC-1 staining and analyzed by flow cytometry as described in Design and Methods section. The graph displays the mean ± SD (standard deviation) of 3 independent experiments. (E) Thiostrepton-induced release of cytochrome c. SUDHL4 and OCI-LY19 cells were treated with and without 5 and 10 μM thiostrepton for 48 h. Mitochondrial free, cytosolic fractions were isolated as described in Design and Methods section. Cell extracts were separated on SDS-PAGE, transferred to PVDF membrane, and immunoblotted with an antibody against cytochrome c. The blots were stripped and re-probed with an antibody against actin for equal loading. (F) SUDHL4, OCI-LY19 and HBL-1 cells were treated with and without 5 and 10 μM thiostrepton for 48 h. Cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against caspase-9, caspase-3, cleaved caspase-3, PARP and beta-actin.