Brief exposure to NMDA produces long-term protection of cerebellar granule cells from apoptosis
Article first published online: 9 MAR 2005
European Journal of Neuroscience
Volume 21, Issue 4, pages 827–840, February 2005
How to Cite
Xifro, X., Malagelada, C., Miñano, A. and Rodríguez-Álvarez, J. (2005), Brief exposure to NMDA produces long-term protection of cerebellar granule cells from apoptosis. European Journal of Neuroscience, 21: 827–840. doi: 10.1111/j.1460-9568.2005.03935.x
- Issue published online: 9 MAR 2005
- Article first published online: 9 MAR 2005
- Received 27 May 2004, revised 23 November 2004, accepted 2 December 2004
Fig. S1. MK-801 only blocks NMDA-mediated neuroprotection, but not K25, when added together with NMDA at 2 DIV. Cerebellar granule cells were grown in medium containing serum and K5 and were exposed to NMDA (100 ?m), K25 or MK-801 (10 ?m; MK) at 2 DIV. A, MK-801 was coadministered with NMDA at 2 DIV or was added 24 h (3 DIV) or 48 h (4 DIV) after addition of NMDA. B, MK-801 was coadministered with K25 at 2 DIV or was added 24 h (3 DIV) or 48 h (4 DIV) after addition of K25. Cell viability was measured by MTT assay at 7 DIV. Values are shown as percentage vs. control cultures (CT) plated at K25 and represent the mean ± SEM of 3 independent experiments performed in sixtuplicates. *P < 0.05 vs. K5 and +P < 0.05 vs. NMDA.
Fig. S2. Inhibition of caspase-3 prevents K5-mediated cell death. The inhibitor of caspase-3, Z-DEVD-cmk (DEVD), was added at 2 DIV in cultures grown at K5 or K25 (CT) and viability was monitored by MTT assay at 7 DIV. Results are shown as percentage vs. control (CT) and represent the mean ± SEM of 3 independent experiments performed in sixtuplicates.+ indicate a significal difference vs. control cultures (P < 0.05) and * indicate a significal difference vs. K5 cultures (P < 0.05).
Fig. S3. Protein kinases inhibitors do not affect K5-mediated caspase-3 activation. Cultures grown at K5 were exposed to wortmannin (wort; 0.5 ?m; inhibitor of PI-3 kinase), KN-62 (KN; 3 ?m; inhibitor of CaM kinase II), PD 98059 (PD; 10 ?m; inhibitor of MEK) or genistein (General; 10 ?m; inhibitor of tyrosine kinases) at 2 DIV. A, Cells were collected 12 h after addition of protein kinases inhibitors. Cellular lysates (40 ?g protein) were subjected to SDS-PAGE and Western blotting was performed with an antibody to caspase-3 (see material and methods). The p17 fragment and ?-tubulin levels were quantified by computer assisted densitometry. Results represents the ratio between p17 vs. ?-tubulin levels and is expressed in arbitrary units vs. K5. Values are the mean ± SEM of 3 independent experiments performed in duplicate. B, A representative Western blot is shown. C, Cleaved caspase-3 was determined by inmunohistochemistry 12 h after the addition of protein kinases inhibitors.
|EJN_3935_sm_FigS1.tif||215K||Supporting info item|
|EJN_3935_sm_FigS2.tif||95K||Supporting info item|
|EJN_3935_sm_FigS3.tif||790K||Supporting info item|
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.