Fig. 4. Myc maintains self-renewal ability of differentiating NPCs. (A) Illustration of the experimental set-up for the re-sphering assay. (B) A light microscopy image of a `re-sphering' Myc neurosphere in 2% FCS after 4 weeks of culture. (C) During differentiation-promoting culture conditions Myc overexpression induced maintenance of self-renewal, seen as re-sphering, in 1-2% of the 76,800 plated cells (800 cells per well in 150 µl in a 96-well culture plate in each experiment) during differentiation in 2% FCS (Myc 1 week 0.0%, s.d.=0.05, n=4; Myc 2 weeks 1.5%, s.d.=1.4, n=7; Myc 3 weeks 1.3%, s.d.=1.3, n=7; Myc 4 weeks 1.5%, s.d.=1.3, n=7). Thus, re-sphering was visible after 2 weeks in culture. The number of formed neurospheres did not increase with time (when kept in 2% FCS). All the control cells differentiated and did not ever form neurospheres in differentiation-promoting conditions (contr 1–4 weeks 0%, s.d.=0, n=7, 2 weeks Student's t-test *P=0.028; 3 weeks *P=0.043; 4 weeks *P=0.020).