JCS044891 Supplementary Material

Files in this Data Supplement:

• Supplemental Tables S1 -

• Supplemental Figure S1 -

  Fig. S1. Double immunolabeling of adherent neural precursors for nestin and pERK1/2. This figure provides individual channels corresponding to the overlays shown in Fig. 1A. Adherent undifferentiated cells cultured for 4 days after dissociation of primary neurospheres were immunostained for nestin (green) and pERK1/2 (red) and labeled with DAPI (blue). Cultures were stimulated for 5 minutes with ADPβS (50 µM) (Ba-c), UTP (50 µM) (Ca-c) or EGF (20 ng/ml) (Da-c). Carrier was added to controls (Aa-c). The scale bar in (Dc) applies to all images.

• Supplemental Figure S2 -

  Fig. S2. Double immunolabeling of adherent neural precursors for nestin and pCREB. This figure provides individual channels corresponding to the overlays shown in Fig. 1B. Adherent undifferentiated cells cultured for 4 days after dissociation of primary neurospheres were immunostained for nestin (green) and pCREB (red) and labeled with DAPI (blue). Cultures were stimulated for 5 minutes with ADPβS (50 µM) (Ba-c), UTP (50 µM) (Ca-c) or EGF (20 ng/ml) (Da-c). Carrier was added to controls (Aa-c). The arrow indicates a highly pCREB immunopositive mitotic cell. The scale bar in (Dc) applies to all images.

• Supplemental Figure S3 -

  Fig. S3. Astrocytes formed following differentiation of progenitors coexpress GFAP and nestin. Cells initially grown as neurospheres for 7 days were dissociated and transferred into adherent culture for 4 days, followed by growth-factor withdrawal-induced differentiation for 6 days. Nuclei were labeled with DAPI (A, blue) and cells were immunostained for GFAP (B, green) and nestin (C, red); (D) merged images. Note the heterogeneity in intermediate filament labeling between cells. The scale bar in (D) applies to all images. Representative image of three independent experiments.

• Supplemental Figure S4 -

  Fig. S4. Double immunolabeling for nestin and pERK1/2 of astrocytes formed from neural precursors. This figure provides individual channels corresponding to the overlays shown in Fig. 2A. Cells initially grown as neurospheres for 7 days were dissociated and transferred into adherent culture for 4 days, followed by growth factor withdrawal-induced differentiation for 6 days. Cultures were stimulated for 5 minutes with ADPβS (50 µM) (Ba-c), UTP (50 µM) (Ca-c) or EGF (20 ng/ml) (Da-c). Carrier was added to controls (Aa-c). Cells were immunostained for GFAP (green) and pERK1/2 (red) and labeled with DAPI (blue). The scale bar in (Dc) applies to all images.

• Supplemental Figure S5 -

  Fig. S5. Double immunolabeling for nestin and pERK1/2 of astrocytes formed from neural precursors. This figure provides individual channels corresponding to the overlays shown in Fig. 2D. Cells initially grown as neurospheres for 7 days were dissociated and transferred into adherent culture for 4 days, followed by growth factor withdrawal-induced differentiation for 6 days. Cultures were stimulated for 5 minutes with ADPβS (50 µM) (Ba-c), UTP (50 µM) (Ca-c) or EGF (20 ng/ml) (Da-c). Carrier was added to controls (Aa-c). Cells were immunostained for GFAP (green) and pCREB (red) and labeled with DAPI (blue). The scale bar in (Dc) applies to all images.

• Supplemental Figure S6 -

  Fig. S6. UDP does not induce ERK1/2 and CREB phosphorylation. Experiments were performed with adherent undifferentiated cells 4 days after dissociation of primary neurospheres. (A) ERK1/2 phosphorylation. (B) CREB phosphorylation. UDP and UTP were applied for 5 minutes at 50 µM. Carrier was added to controls. GAPDH served as a loading control. Values are means ± s.e.m. (n=3). *P<0.05, **P<0.01 relative to control; ANOVA, Tukey Kramer multiple comparisons test (unpaired).