Figure 1

Inhibition of BACE1 stabilizes ST6Gal I and increases cell surface sialylation. A. SW48 (ST6Gal I-negative) colon cancer cells were transiently transfected with an N-terminally Flag-tagged ST6Gal I expression plasmid, and 24 hours later, cells were exposed to IR (10 Gy). Cell lysates were assessed for expression of ST6Gal I by immunoblotting with an ST6Gal I antibody. B. SW480 vector-control cells and cells stably expressing ST6Gal I were treated with inhibitors of β-secretase, γ-secretase, or cathepsin B&L at a concentration of 20 μM. After incubating for 24 hours, cells were harvested and ST6Gal I levels were analyzed by immunoblotting with an anti-ST6Gal I antibody. C. Cell surface α2, 6 sialylation (SNA-FITC) and α2, 3 sialylation (MAA-FITC) were analyzed by FACS after 24 hours of treatment with 20 μM β-secretase or γ-secretase inhibitors. D. SW480 cells were transfected with siRNA against BACE1, and the levels of BACE1, ST6Gal I, and integrin β1 protein were assessed by immunoblotting. Sialylation of integrin β1 was assayed by SNA lectin affinity assay. E. SW480 vector-control cells and cells stably expressing ST6Gal I were treated with Si-BACE1 or transfected with a BACE1 expression plasmid. Then, α2, 6 sialylation (SNA-FITC) of the cell surface was analyzed by FACS. Data are presented as means ± SDs of three replicates (*p < 0.05 vs. the corresponding control).