Figure 4.

tBid is a key mediator of KCZ sensitization of BTZ-induced apoptosis. (A) (upper) A schematic diagram for the inhibition of truncated Bid (tBid) using a small molecule inhibitor of tBid (BI-6C9) and RNA interference of Bid (shBid) in MCL Jeko-1 and Granta-519 cells. (middle) Inhibition of Bid by shBid diminishes the sensitizing effect of KCZ on BTZ-induced apoptosis. shCON and shBID cells were similarly treated with BTZ (0–7 nM) in the presence or absence of KCZ (50–75 μM) for 24 h and apoptosis was determined by Hoechst 33342 assay. Data are mean ± s.d. (n=3) *P < 0.05 vs. non-treated cells; two-sided Student’s t-test. ^#P < 0.05 vs. BTZ-treated cells; two-sided Student’s t-test. (lower) Inhibition of tBid reverses the sensitizing effect of KCZ on BTZ-induced apoptosis. Cells were co-treated with BTZ (6 nM) and KCZ (75 μM) in the presence or absence of the tBid inhibitor BI-6C9 (10–20 μM) and analyzed for apoptosis by Hoechst 33342 assay. Data are mean ± s.d. (n=3). *P < 0.05 vs. non-treated cells; two-sided Student’s t-test. ^#P < 0.05 vs. BTZ-treated cells; two-sided Student’s t-test. ^§P < 0.05 vs. BTZ-treated cells in the presence of KCZ; two-sided Student’s t-test. NTX, non-treatment. (B) KCZ increases tBid stability in BTZ-treated cells. Jeko-1 and Granta-519 cells were treated with BTZ and KCZ in the presence or absence of the protein translation inhibitor cycloheximide (CHX; 10 μg/ml) for various times (0–16 h) to follow the degradation of tBid protein. Cell lysates were prepared and tBid expression was determined by Western blotting. Representative immunoblots of tBid and loading control β-actin are shown. Expression-time profiles of tBid in Jeko-1 (solid lines) and Granta-519 (dashed lines) cells treated with CHX and BTZ alone (circle) or with KCZ (square) were plotted and tBid half-lifves were calculated and shown. Data are mean ± s.d. (n=3). *P < 0.05 vs. BTZ and CHX co-treated cells; two-sided Student’s t-test.