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. 2010 Aug 18;285(43):33520–33528. doi: 10.1074/jbc.M110.158378

FIGURE 5.

FIGURE 5.

γ-T3 suppresses STAT3-regulated antiapoptotic gene products, induces apoptosis, and potentiates chemotherapeutic agents. A, γ-T3 suppresses STAT3-regulated antiapoptotic gene products. U266 cells (2 × 106/ml) were treated with 25 μm γ-T3 for the indicated time intervals, after which whole-cell extracts were prepared, and 40-μg portions of those extracts were resolved on 10% SDS-PAGE; the membrane was sliced according to molecular weight, and the gel was probed using antibodies against cyclin D1, Bcl-2, Bcl-xL, survivin, and VEGF. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. B, γ-T3 induces caspase-3-dependent PARP cleavage. U266 cells were treated with 60 μm γ-T3 for the indicated times, and whole-cell extracts were prepared, separated on SDS-PAGE, and subjected to Western blotting against caspase-3 antibody and PARP antibody. The same blots were stripped and reprobed with β-actin antibody to show equal protein loading. C, γ-T3 causes significant accumulation of cells in the sub-G1 phase. U266 cells (2 × 106/ml) were synchronized by incubation overnight in the absence of serum and then treated with 25 μm γ-T3 for the indicated times, after which the cells were washed, fixed, stained with propidium iodide, and analyzed for DNA content by flow cytometry. D, effects of γ-T3 on the proliferation of multiple myeloma cells. MM cells were plated in triplicate, treated with the indicated concentrations of γ-T3 for the indicated days, and then subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. E, γ-T3 potentiates the apoptotic effect of thalidomide (Thal) and Velcade (Val). U266 cells (1 × 106/ml) were treated with 25 μm γ-T3 and 10 ng/ml thalidomide or 20 nm bortezomib alone or in combination for 24 h at 37 °C. Cells were stained with a Live/Dead assay reagent for 30 min, then analyzed under a fluorescence microscope, and 20 random fields were counted. C, Control; T, Thalidomide; V, Velcade; *, p < 0.01; **; p < 0.05 versus control.