CD154 induces p73 in CLL cells with or without functional p53. CLL cells were CD40-activated as described in “Materials and methods” and protein lysates were prepared at the indicated time points. Immunoblot membranes prepared from such samples were probed for p73, β-tubulin, or β-actin to monitor for protein loading, as indicated at the right of each immunoblot. (A) The immunoblot of a representative time course of p73 expression is shown. (B) Two independent immunoblots from 2 different CLL samples were examined by densitometry using NIH Image software. The value before CD40-ligation (before) was subtracted from all time points as background value and set to 0%. The maximum value on day 3 after CD40-ligation was set to 100%. All other values were calculated relative to the maximum value [(intensity on day x / maximum intensity) × 100]. The graph shows mean values plus or minus the standard deviation of signal intensity on the y-axis relative to the time after CD154 treatment (eg, day 0 [D0], day 1 [D1], or day 3 [D3]), as indicated on the x-axis. (C) CLL cells were classified as having functional (p53 +) or nonfunctional p53 (p53-) by DNA sequencing of the p53 gene and/or by the functional assay of their response to γ-irradiation. Two patient samples each of CLL cells that were p53+ (samples from patients 1 and 2) or p53-(samples from patients 3 and 4) were analyzed 24 hours after treatment with HeLa cells (-) or HeLa-CD154 cells (+), as indicated at the top of each lane of the immunoblots.