The S158C mutant C2GnT-I does not act in a dominant-negative fashion. BW5147 cells expressing WT C2GnT-I (WT clone 6, from Figure 5A) were transfected again with EGFP alone (WT + vector-EGFP), WT C2GnT-I–EGFP (WT + WT-EGFP), or mutant C2GnT-I–EGFP (WT + mut-EGFP). (A) Relative expression of C2GnT-I in WT or mutant C2GnT-I–EGFP analyzed by real-time RT-PCR. Clones with similar levels of C2GnT-I mRNA, calculated as C2GnT-I–actin ratios, were selected for further analysis. *P < .001 for WT or mutant C2GnT-I–EGFP compared with vector alone. Values are mean ± SD of triplicates from 1 of 3 independent experiments. (B) Mutant C2GnT-I–EGFP does not decrease enzyme activity in WT clone 6 cells. Cells expressing WT C2GnT-I–EGFP had increased activity compared with WT clone 6 cells transfected with EGFP-vector alone or mutant C2GnT-I–EGFP. *P < .01. Values are mean ± SD of duplicates from 1 of 5 independent experiments. (C) Mutant C2GnT-I–EGFP does not reduce the susceptibility of WT clone 6 cells to galectin-1–induced death. (Left) Annexin V binding of WT clone 6 cells (dotted line) versus WT clone 6 cells transfected with WT C2GnT-I–EGFP (solid line). (Center) Annexin V binding of WT clone 6 cells (dotted line) versus WT clone 6 cells expressing mutant C2GnT-I–EGFP (solid line). (Right) Cell death was determined by annexin-V–allophycocyanin binding and 7-aminoactinomycin D uptake. Percentage of cell death was similar for WT clone 6 cells expressing mutant C2GnT-I–EGFP and EGFP-vector alone (P, NS), indicating that the mutant C2GnT-I–EGFP did not reduce the susceptibility to galectin-1. Cell death was significantly higher for WT clone 6 cells expressing WT C2GnT-I–EGFP (*P < .01). Values are mean ± SD for triplicates from 1 of 6 independent experiments.