L-plastin knock-down in prostate carcinoma cells. (A) L-plastin is expressed in PC3M cells and gets phosphorylated following PMA stimulation. Phosphorylation of L-plastin in untreated PC3M or PC3M preincubated with 10-8 M PMA for 30 min was determined by Western blot with a phospho-Ser5 L-plastin specific antibody (upper panel). Total expression of L-plastin (lower panel). (B) ShRNA mediated downregulation of L-plastin in PC3M. PC3M infected with lentivirus encoding L-plastin (LPL) or non-targeting (nt) shRNA were analysed by Western blot for LPL protein (upper part), and β-actin as loading control. Quantification by densitometry of three independent experiments is shown in the lower part. For each experiment L-plastin levels were normalized for β-actin. The amount of L-plastin in untreated PC3M was set as 100%. Results were analyzed using one-way ANOVA (n = 3) (* = p < 0,05). (C) No influence of L-plastin knock-down on contact dependent in vitro proliferation of PC3M. Data represent means ± SEM, experiments were repeated at least 3 times with samples in triplicate. (D) Unchanged anchorage independent in vitro proliferation of L-plastin knock-down PC3M. PC3M (as indicated) were allowed to grow in soft agar for 21 days. Cell culture plates with the colonies were scanned using a transmission light scanner (upper panel). Single colonies were acquired with a 20x objective (lower panel). Bars indicate the colony numbers in three optical fields. Data represent means ± SEM. Results were analyzed using one-way ANOVA (n = 4; ns = not significant). ( E/F ) Knock-down of L-plastin in PC3M leads to reduced tumor volumes in vivo. PC3M (as indicated) were injected subcutaneously into mice. (E) Representative images of excised tumors at day 42. (F) Tumor volumes calculated weekly (n = 10; *p < 0.05 for PC3M vs. PC3M LPL shRNA and PC3M nt shRNA vs. PC3M LPL shRNA).