DEV002154 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. (A) Diagrams illustrating experimental procedures. (B) BrdU incorporation (green) after repetitive treatment with NMDA for 3 consecutive days in the presence of bFGF. Cell cultures (6 DIV) were exposed daily to NMDA (1-20 μM) for 3 days, followed by a 1-hour pulse of BrdU (50 μM) and were fixed on 9 DIV. NMDA reduced BrdU incorporation and induced cell death, as indicated by a drop in total cell numbers. (C) Effect of different doses of NMDA on proliferation. (D) Comparison of BrdU incorporation following treatment of cells (6 DIV) with a single pulse of NMDA (5 μM) over 24 hours in the presence of bFGF followed by BrdU treatment and fixation 24 hours (7 DIV), 48 hours (8 DIV) or 72 hours (9 DIV) after stimulation. Cells treated with NMDA and fixed 24 hours or 48 hours later did not show an increased proportion of BrdU-positve cells. (E) Apoptosis assay for the comparison of cell death following treatment with a single pulse of NMDA (5 μM) over 1 day (NMDA 1’), two pulses over 2 days (NMDA 2’), or three pulses over 3 days (NMDA 3′) in the presence of bFGF. Green fluorescent nuclei correspond to apoptotic cells in which the terminal transferase has incorporated fluorescent dUTP (Bodipy-dUTP) into fragmented DNA. TUNEL-positive cells are shown as a fraction of the total cells defined by DAPI staining of nuclei. Each value represents the mean ± s.e.m. of three (A,B) and eight (E) independent experiments. CTL, control; Student’s t-test, ***P<0.001, **P<0.01, *P<0.05. Bars, 100 μm.

• Supplemental Figure 2 -

  Fig. S2. Immunocytochemical analysis of hippocampal NPCs during proliferation. Widespread cell-surface staining of Lex (red); DAPI staining is in blue.