Figure 3
Inhibition of APC/C causes an increase in glutaminase, glutamate and apoptosis.
(a–d) Western blot images of glutaminase (gls) and cyclin B1 in neurons at different time points with proTAME (pT) treatment (12 μM). Blots were quantified by densitometry and normalized against α-tubulin levels; mean values ± SD of three independent experiments are shown and are significant after 10 h (p < 0,01), 16 and 24 h (p < 0,05) for glutaminase. Cyclin B1 levels are significant after 22 and 30 h (p < 0,05) compared to 3 h treatment with pT. (e) Mean values ± SD of glutamate levels after 0, 4, 10, 16 and 24 h with pT (12 μM) of three independent experiments (p < 0,01). (f) Mean values ± SD of glutamate levels in controls, or with pT, or pT + glutaminase inhibitor (GI) for 24 h of three independent experiments (p < 0,01). (g) Mean values ± SD of glutamate levels in controls in standard medium (SM) containing glutamine (gln) (C + Gln), without glutamine (C − Gln), in SM treated with Aβ (Aβ + Gln) or in medium without gln treated with Aβ (Aβ − Gln) (p < 0,01), in SM treated with pT (pT + Gln) or in medium without gln treated with pT (pT − Gln) for 20 h, of five independent experiments (p < 0,01). (h–j) Western blot of cdh1 and gls with control siRNA, or with cdh1 siRNA, each with or without Aβ. The blots were quantified by densitometry and normalized against α-tubulin; mean ± SD of three independent experiments are shown (p < 0,001; p < 0,05). (k–m) Flow cytometry analysis for apoptosis in neurons. (k) Mean fold change ± SD of three independent experiments of apoptotic cells upon a time-course of pT treatment at 0, 4, 10, 16 or 24 h is shown (p < 0,01). (l,m) Representative assays of controls, or treated with pT or pT + GI for 24 h. Three independent experiments were quantified and the fold change ± SD of apoptotic cells (Q3, Q4) is shown (p < 0,01; p < 0,05).
