Figure 5.

Effects of LCMT-1 knockdown on transformation are rescued by expression of an shRNA-resistant LCMT-1. (A) Rescue of the LCMT-1 knockdown line. Lysates from VC and L3 cells stably expressing an empty control plasmid (VC-control and L3-control, respectively) and L3 LCMT-1 knockdown cells stably expressing an LCMT-1 rescue plasmid (L3-rescue) were analyzed by Western blot analysis for LCMT-1 protein expression. (B and C) PP2Ac methylation is rescued in the L3 rescue cell line. (B) Equal volumes of lysates from VC-control, L3-control, and L3-rescue were either treated with preneutralized base solution (- lanes; show unmethylated PP2Ac levels in cells) or base treated and then neutralized (+ lanes; 100% demethylated controls) before being analyzed by Western blot analysis for the level of unmethylated PP2Ac and α-tubulin (loading control). (C) Graph depicts the average percent methylation of PP2Ac in the lysates, calculated as described in the legend to Figure 2C. (D) Anchorage-independent growth of VC-control, L3-control, and L3-rescue cells in soft agar. Photographs show small, single, representative fields within the agars. Average colony numbers (E) and average colony volumes (F) were determined, and data are shown in graphs as fold change relative to VC-control. Error bars in all panels represent SD of three independent experiments. *P ≤ .05. **P ≤ .01.