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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.

FilenameFormatSizeDescription
EJN_3935_sm_FigS1.tif215KSupporting info item
EJN_3935_sm_FigS2.tif95KSupporting info item
EJN_3935_sm_FigS3.tif790KSupporting info item

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