Stimulation of nestin expression in RG cultures by thrombin and TRAP6. RG were cultured for 2 days in the presence of serum (A) or were maintained for the same time in serum-free medium to suppress their nestin expression (B), or in serum-free medium plus 900 pM Thrombin (C) or 100 μM TRAP6 (D). Then, the cells were fixed and labelled with anti-nestin (green) and anti-GFAP (red) antibody. Nuclei were counterstained with TOPRO-3 (blue). In the presence of thrombin or TRAP6, the expression of nestin is maintained together with that of GFAP which is co-expressed in a varying proportion of the cells. Additional cell cultures that were treated in parallel with varying concentrations of thrombin (90 to 900 pM, E) or TRAP6 (0.3 to 100 μM, F) were collected and total protein extracts were prepared. Nestin concentrations in those extracts was then measured by Western blots as described in Material and Methods. Scale bar (in A = 40 μm) applies to all images.