Figure 6.

Effect of the lathyrane ELAC on stemness and self‐renewal capacity. (A) Scheme of the procedure followed to test the effect of different compounds on the expression of the transcription factor Sox2 (an NSC marker) and nestin (an intermediate neural progenitor marker) in neurospheres formed during 72 h in the presence of these compounds (primary neurospheres); see Methods for details. (B) Primary neurospheres grown for 72 h in the presence of different compounds were disaggregated, and single cells were cultured for another 72 h in the absence of any treatment, in order to determine the capacity of these cells to generate new secondary neurospheres after treatment withdrawal. (C) Examples of fluorescence images of primary neurospheres formed after treatment with ELAC (5 μM), Ac‐ELAC (5 μM) or none (control), immunostained for nestin (red) and Sox2 (green), as indicated; nuclei are counterstained with DAPI (cyan or blue). Scale bar indicates 50 μm. (D) Quantification of the percentage of neurosphere cells expressing nestin and Sox2 after 72 h of treatment with ELAC (5 μM), Ac‐ELAC (5 μM) or none (control). (E) Number of secondary neurospheres formed from disaggregated single cells that had previously been treated for 72 h with the indicated compounds, as explained in (A and B). Results are the mean ± SEM of five independent experiments. Each individual experiment was performed in triplicates; *P < 0.05 when compared with control in a Student's t‐test performed for paired samples.