Fig. 9

Analysis of cellular differentiation induced by cobimetinib, cis-RA or combination of the two agents. Exponentially growing NB cells were treated with either agent (1 μM cobimetinib or 10 μM cis-RA) or in combination. Cells were then washed and fixed with 4 % paraformaldehyde and permeabilized with 0.05 % Triton X-100. The cells were then evaluated by conventional immunohistochemistry using antibodies to Nestin (1:1000), GFAP (1:1000) and MAP2 (1:800) and fluorescent labelled secondary antibodies. Cells were also counterstained with DAPI and visualized by fluorescence microscopy and photographed. Presented are randomly picked microscopic fields for each experimental condition. Staining for GFAP and MAP2 expression shows an increase and while Nestin expression decreases with differentiation. Changes in morphology with elongated processes are also visible with increased differentiation of the cells. Data are representative of three separate experiments. Scale bar, 34 μM