Removal of endogenous gal-3 from CD45 increases phosphatase activity to potentiate DLBCL cell death. (A) Removal of cell-surface gal-3 alters CD45 clustering. SUDHL-6 cells were treated with 75 μg/mL GCS-100 or buffer control for 1 hour and cell-surface glycoproteins were immediately cross-linked with the nonreducible cross-linker BS3. CD45 was immunoprecipitated from cell lysates and resolved by 3%-8% Tris-acetate SDS-PAGE. In control-treated cells, high-molecular-weight clusters of CD45 are present (arrowheads), which are diminished when cells are treated with GCS-100. (B) Removal of cell-surface gal-3 increases phosphatase activity. SUDHL-6 and SUDHL-9 cells were treated with 75 μg/mL GCS-100 and phosphatase activity measured (***P < .0001, values are mean ± SD of triplicates from 1 representative of 6 independent replicate experiments). (C) Removal of cell-surface gal-3 alters downstream signaling regulated by via CD45 phosphatase. SUDHL-6 cells were treated with 75 μg/mL GCS-100 or buffer control for 30 minutes before treatment with 10 μg/mL anti-IgM for 1 minute. Lysates were immunoblotted with anti-phospho-Erk and anti-Erk. The levels of Erk and phospho-Erk were determined by densitometry and the levels of phospho-Erk were normalized to Erk expression. Increased Erk phosphorylation after anti-IgM cross-linking was observed in GCS-100–treated cells. (D-E) GCS-100 sensitization to cell death requires phosphatase activity. SUDHL-6 cells were treated with the phosphatase inhibitor 12.5nM bp(V)phen for 1 hour, followed by 75 μg/mL GCS-100 for 1 hour, and (D) dex or (E) rituximab for 24 hours. Viability was determined as described in “Methods.” **P < .001, ***P < .0001, values are mean ± SD of triplicates from 1 representative of 3 independent replicate experiments for each treatment.
