Honokiol down-regulates the survival protein Mcl-1. (A) B-CLL cells were incubated with 40 μM honokiol for 24 hours. Cells were lysed, and Western analysis was performed using antibodies that recognize Mcl-1. Blots were stripped and reprobed for β-actin as a loading control. Three representative patients' samples are shown out of 6 examined. (B) Films from the immunoblots were scanned, and mean intensities were calculated using Kodak Digital Science 1D image analysis software (Eastman Kodak, New Haven, CT). The ratio of Mcl-1 to β-actin was then calculated for cells treated with or without honokiol, and data are shown as mean ± standard deviation. ^*P < .02 when compared with cells treated with medium alone. (C) B-CLL cells treated for 3 hours with or without 40 μM honokiol were analyzed for Mcl-1 mRNA expression by real-time PCR using SYBR green technology Mcl-1 levels were normalized to β-actin. (D) B-CLL cells were incubated with 40 μM honokiol for the indicated times and then lysed and analyzed for Mcl-1 expression by Western blotting as described above.