Figure 2
Figure 2. Bak, Bik, and Noxa protein levels increase following bortezomib treatment of resistant cells. (A) Western blot analysis of whole cell lysates collected 24 hours following bortezomib (25nM) treatment of sensitive (Raji, RL) or resistant (Raji 2R, Raji 4RH, RL 4RH) cells demonstrated a striking increase in the expression of Bak, Bik, and Noxa in resistant but not sensitive, cells. Blots shown are from a representative experiment repeated more than 3 times. (B) Kinetic analysis of Bak, Bik, and Noxa expression following bortezomib treatment showed that their induction occurred before caspase activation (indicated by PARP cleavage) in resistant cells (Raji 2R shown here). Induction of Bak, Bik, and Noxa was not seen following bortezomib treatment of sensitive cells (Raji shown here), yet PARP cleavage occurred more rapidly than in resistant cells. Data shown are from a representative experiment repeated twice. Other bortezomib-treated sensitive and resistant cell lines displayed similar kinetics with respect to PARP cleavage along with Bak, Bik, and Noxa expression (data not shown). (C) Resistant cells (Raji 2R shown here) expressing a Bak transgene rapidly undergo apoptosis as indicated by caspase-3/7 activity. Eighteen hours following transfection of pIRES2-EGFP containing either a BAK1, BIK, or PMAIP1 (Noxa) transgene, or empty vector as a control, resistant cells were assayed for caspase-3/7 activity. Data shown are from a representative experiment repeated at least 3 times. Bars represent average caspase-3/7 activity from triplicate wells ± SD. Asterisks (*) denote a significant (P < .01) increase in caspase-3/7 activity compared with vector transfected cells. (D) Western blot confirmed expression of Bak, Bik, and Noxa transgenes. (E) Mutation of the BH3 domain of Bak abrogates its ability to induce apoptosis as indicated by caspase-3/7 activity. Data shown are from a representative experiment repeated twice. Bars represent average caspase-3/7 activity from triplicate wells ± SD. Asterisks (*) denote a significant (P < .01) decrease in caspase-3/7 activity compared with wild-type Bak transfected cells.

Bak, Bik, and Noxa protein levels increase following bortezomib treatment of resistant cells. (A) Western blot analysis of whole cell lysates collected 24 hours following bortezomib (25nM) treatment of sensitive (Raji, RL) or resistant (Raji 2R, Raji 4RH, RL 4RH) cells demonstrated a striking increase in the expression of Bak, Bik, and Noxa in resistant but not sensitive, cells. Blots shown are from a representative experiment repeated more than 3 times. (B) Kinetic analysis of Bak, Bik, and Noxa expression following bortezomib treatment showed that their induction occurred before caspase activation (indicated by PARP cleavage) in resistant cells (Raji 2R shown here). Induction of Bak, Bik, and Noxa was not seen following bortezomib treatment of sensitive cells (Raji shown here), yet PARP cleavage occurred more rapidly than in resistant cells. Data shown are from a representative experiment repeated twice. Other bortezomib-treated sensitive and resistant cell lines displayed similar kinetics with respect to PARP cleavage along with Bak, Bik, and Noxa expression (data not shown). (C) Resistant cells (Raji 2R shown here) expressing a Bak transgene rapidly undergo apoptosis as indicated by caspase-3/7 activity. Eighteen hours following transfection of pIRES2-EGFP containing either a BAK1, BIK, or PMAIP1 (Noxa) transgene, or empty vector as a control, resistant cells were assayed for caspase-3/7 activity. Data shown are from a representative experiment repeated at least 3 times. Bars represent average caspase-3/7 activity from triplicate wells ± SD. Asterisks (*) denote a significant (P < .01) increase in caspase-3/7 activity compared with vector transfected cells. (D) Western blot confirmed expression of Bak, Bik, and Noxa transgenes. (E) Mutation of the BH3 domain of Bak abrogates its ability to induce apoptosis as indicated by caspase-3/7 activity. Data shown are from a representative experiment repeated twice. Bars represent average caspase-3/7 activity from triplicate wells ± SD. Asterisks (*) denote a significant (P < .01) decrease in caspase-3/7 activity compared with wild-type Bak transfected cells.

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