Figure 2.
The combined Len/VD3 treatment activated caspase 9, induced mitochondrial depolarization and involved Bax. (a) The Len/VD3 treatment induced caspase 9 activation. MCL cells (2 × 105 cells/ml) were incubated for 4 days with or without 1 μM Len, 100 nM VD3 or Len/VD3. Cells were then lysed and activation of caspase 9 was assessed by western blotting. A representative experiment out of three is shown. (b) The Len/VD3 treatment induced mitochondrial depolarization. Z-138 cells (2 × 105 cells/ml) were incubated for 4 days with or without 1 μM Len and 100 nM VD3, and then stained with JC-1. A representative experiment out of three is shown. (c–e) Silencing of BAX prevented cell death induced by Len/VD3 combination. JEKO-1 cells (5 × 105/ml) were seeded for 48 h with or without the Len/VD3 combination (1 μM Len and 100 nM VD3) before transfection with sicontrol (siCt) or siBAX RNA. Then, transfected cells were reseeded (5 × 105/ml) for additional 3 days with or without the Len/VD3 combination. (c) Western blotting analysis of Bax expression. Bax expression was assessed in siCt- and siBAX-transfected JEKO-1 cells treated or not with Len/VD3 combination. (d) Len/VD3 induced a Bax-dependent decrease in cellularity. The cellular density was measured by a direct counting. The data represent five independent experiments. (e) Len/VD3 induced a Bax-dependent cell death. Cells were stained with Annexin V and fluorescence was analyzed on a FACSCalibur. The data represent five independent experiments. *P<0.05, **P<0.01