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Fig. 8. NMDA regulates the activity of proneural bHLH, homeogene transcription factors and VEGF secretion in differentiated NPCs. (A) Diagram illustrating the experimental procedure. (B) Semiquantitative mRNA analysis of the transcription factors NeuroD, Ngn1 and Emx2. (C) Expression of NeuroD, Ngn1 and Emx2 was blocked by MK801. Cells were treated daily for 3 consecutive days and analyzed 24 hours after the last stimulation in B,C. (D) RT-PCR analysis of NeuroD. Excitation with NMDA (5 µM) in medium permissive of neuronal differentiation caused an increase in the expression of NeuroD as NMDA treatment was repetitive at an interval of 24 hours. (E) Western blot analysis of NeuroD in the presence of NMDA, and in the presence or absence of MK801. Cells were treated with a pulse of NMDA for 24 hours, two pulses over 48 hours or three pulses over 72 hours and analyzed 24 hours after the stimulation in D-E. (F) Western blot analysis of secretion of VEGF into the culture medium from the differentiated NPCs and astrocytes after the three pulses of each treatment over 72 hours. (G) Real-time PCR analysis of NeuroD and Id2 following various stimuli. Cells were daily treated with each stimulus at a single pulse for 24 hours or three pulses over 72 hours and analyzed 24 hours after the last stimulation. Values are the mean ± s.e.m. (n=5). Gene expression levels are depicted relative to the expression of GAPDH, and calculated relative to expression in control differentiating cells, which have been allowed to differentiate for corresponding periods of time. *P<0.05, **P<0.01, ***P<0.001.
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