FIGURE 2.

Pretreatment with ganetespib enhanced cell cycle arrest and cell apoptosis induced by ibrutinib. Jeko-1 and Granta-519 cells were treated with ganetespib (15 nM for Jeko-1 and 45 nM for Granta-519 cells, respectively) for 12 h, and then treated with either ibrutinib at 6 μM or vehicle for 12 h before cell cycle assays (A–D) or subjected to WB (E–F). (A,B) Representative flow cytometry plots and statistics of cell cycle distribution for Jeko-1 cells. (C–D) Representative flow cytometry plots and statistics of cell cycle distribution for Granta-519 cells. (E,F). Protein lysates of Jeko-1 (E) and Granta-519 cells (F) were subjected to immunoblot analysis with the indicated antibodies of cell-cycle markers. (G–J) Jeko-1 and Granta-519 cells were treated with ganetespib (15 nM for Jeko-1 and 45 nM for Granta-519 cells, respectively) for 12 h, and then treated with either ibrutinib at 6 μM or vehicle for 60 h before apoptosis assays. Representative flow cytometry plots and statistics of apoptosis distribution for Jeko-1 (G,H) and Granta-519 cells (I,J). Cells were stained with annexin V–FITC/propidium iodide and analyzed for the presence of annexin V (+)/PI (-) (early apoptosis) and annexin V (+)/PI (+) (late apoptosis). (K–O) Jeko-1 and Granta-519 cells were treated with ganetespib (15 nM for Jeko-1 and 45 nM for Granta-519 cells, respectively) for 12 h, and then treated with either ibrutinib at 6 μM or vehicle for 12 h before TUNEL assays (K–N) or subjected to WB (O). Positive TUNEL cells show green fluorescence in combination with blue fluorescence DAPI (4′,6-diamidino-2-phenylindole) in the nuclei. (K,L) Representative pictures and statistics of TUNEL assays of Jeko-1 cells. (M,N) Representative pictures and statistics of TUNEL assays of Granta-519 cells. (O) The protein levels of apoptosis-associated markers cleaved caspase 9, caspase 9, and BCL-2 in Jeko-1 and Granta-519 cells treated as mentioned. Mean ± SD, n = 3.