Combined exposure of MM cells to PD184352 and ATO modulates the p53 family proteins. (Ai) HMCLs were seeded at 2.5 × 105 cells/mL in the presence of DMSO (vehicle) or PD (1 μM) for 3 hours and then were incubated for 24 hours with ATO (2 μM). Endogenous TA-p73α, TA-p73β, ΔN-p73, and p53 proteins were revealed by immunoblotting analysis using mouse monoclonal anti-p73 (clone 5B429) and mouse monoclonal anti-ΔN-p73 (clone 38C674), all provided by Imgenex (San Diego, CA) and mouse monoclonal anti-p53 (DO-1, sc-126) provided by Santa Cruz Biotechnology. Antiactin immunoblotting was performed as loading control. (Aii) TA-p73α, TA-p73β, ΔN-p73, and β-actin bands were subjected to densitometric scanning using the TINA 2 software, and the TA-(p73α + p73β)/ΔN-p73 ratio was calculated. (B) MM cells were cultured as described in panel A, and the expression of Bax and PUMA was revealed after 48 hours of treatment by immunoblotting using rabbit polyclonal anti-Bax and rabbit polyclonal anti-PUMA, all provided by Cell Signaling Technology. (Ci) Transfection of p53 (003329, Dharmacon RNA Technologies, Lafayette, CO) or pan-p73 (003331, Dharmacon RNA Technologies) siRNA, but not the unrelated nonspecific control siRNA (001206, Dharmacon RNA Technologies), led to a decrease in p53 or p73 protein expression in HMCLs without affecting the levels of the unrelated protein actin: 48 hours after siRNA transfection, the HMCLs were treated with PD and/or ATO for 24 hours and then lysed for Western blot analysis to monitor the expression of p53, p73, cleaved caspase-3, and cleaved PARP. (Cii) The percentages of sub-G1 apoptotic cells were monitored after 48 hours of treatment. Values are mean plus or minus SD of 4 independent experiments (*P < .05; **P < .01 vs PD/ATO cont; Dunnett test). CTR indicates control; PD, PD184352 (1 μM); ATO, arsenic trioxide (2 μM); cont, nonspecific control siRNA; C3, caspase-3.
