Figure 6.

N-Acetyl-𝒟-glucosamine (GlcNAc) protects transformed cells from glucose depletion-dependent cell death. Normal (a) and transformed (b) cells, grown in HG and LG, were subjected to western blot analysis with anti O-glycosylation antibody (O-GlcNAc). As loading control the expression of vinculin and Ponceau staining (data not shown) was analyzed. Quantitative analysis of O-glycosylation status was performed by densitometric analysis of western blot films of normal (c) and transformed (d) cells. The values obtained for O-GlcNAc were normalized to the corresponding vinculin values and plotted as fold change over the normal sample 0 h (0 h=1) both in HG and LG. Normal (e) and transformed (f) cells, grown in LG, were counted at 72 and 96 h after 24 h of treatment with different concentrations of GlcNAc or 1 mM glucose. Data represent the average of at least three independent experiments (±S.D.). (g and h) UPR activation after GlcNAc or glucose (Glc) treatment was followed through the expression analysis of Grp78 and CHOP proteins. (i and j) FACS analysis of Annexin-V plus PI-labeled normal (i) and transformed (j) cells, grown until 96 h in HG (left panels), LG (middle panels) and LG+10 mM GlcNAc (24 h of treatment). Figures are representative of three independent experiments. (k and l) Analysis of the expression of p-JNK in normal (k) and transformed (l) cells at 96 h of culture. The values obtained for p-JNK, shown in the right histograms, were normalized to the corresponding total JNK and vinculin values and plotted as fold changes over nt samples. Data represent the average of at least three independent experiments (±S.E.M.); *P<0.05, Student's t-test. As loading control the expression of vinculin was used