Figure 2.

Effects of butyrate, doxorubicin or their combination on caspase 3 and 7 activation and AIF release in myeloma cells. (A, B) Effects of butyrate, doxorubicin and their combination on caspase 3 and 7 activation. (A) NCI H929, RPMI 8226 and U266 cells were treated with butyrate (SB; 300 μM for NCI H929 and 600 μM for RPMI 8226 and U266), doxorubicin (Dox; 40 nM) or with their combination. After 24 h treatments, fold change in caspase 3 and 7 activity relative to untreated cells was assessed by caspase 3 and 7 glo kit (Promega Inc.). TRAIL (50 ng ml⁻¹, Peprotech Inc., Rocky Hill, NJ, USA) was used as a positive control. Each data point in the bar graph is mean±s.e.m. of three independent experiments performed in triplicate. (B) Caspase 3 cleavage was assessed after 16, 24 or 36 h by subjecting 30 μg of whole-cell lysates (WCL) of RPMI 8226 cells to immunoblot analysis with a caspase 3-specific antibody. TRAIL-treated sample was used as a positive control and β-Actin as a loading control. (C, D) Effects of caspase 3 inhibitor DEVD-CHO on butyrate- and doxorubicin-induced apoptosis of myeloma cells. RPMI 8226 cells (1 × 10⁶) were pretreated with either vehicle (DMSO) or 1 μM of cell permeable caspase 3-specific inhibitor DEVD-CHO (Biomol Inc.) for 2 h. Then the cells were left untreated or treated with TRAIL (50 ng ml⁻¹) or butyrate (600 μM) plus doxorubicin (40 nM). Caspase 3 and 7 activity was determined as in Figure 3A, and percentage of cells undergoing apoptosis was determined 48 h post-treatment by TUNEL assay as in Figure 2. Scatter plot shown is one of two independent experiments with similar results, in which 10 000 events were collected (top panel). Each bar on the graph is mean±s.e.m. of two independent experiments, and P-values of significantly different treatments are provided. (E) Butyrate plus doxorubicin combination results in nuclear translocation of AIF in RPMI 8226 and NCI H929 cells. RPMI 8226 or NCI H929 cells were left untreated or treated with indicated concentrations of butyrate, doxorubicin or their combination for 48 h. The localisation of AIF was assessed by indirect immunofluorescence staining with an AIF antibody followed by Alexa Flour-488-conjugated secondary antibody (Green staining). Nuclei of the cells were stained with DAPI (blue). Merged images were produced by superimposing both images. Results shown are representative of three independent experiments with similar results.