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Fig. 6. Myc-induced effects on self-renewal are mediated through Miz-1 binding. (A) Western blot of the control, WT Myc and mutated MycV394D neurosphere lysates shows that the level of Myc expression is approximately the same in both Myc populations. Staining for nucleolin verifies the same result. Staining for β-actin shows that the amount of the cell lysate per lane is similar. (B) Overexpression of the Myc mutant, deficient in Miz-1 binding (MycV394D), increased proliferation of NPCs to the same level as wild-type Myc, as measured by BrdU labelling (mutant 86.4%, s.d.=27.2, n=5; Myc 76.7%, s.d.=5.3, n=5; control 28.9%, s.d.=5.6, n=4; Student's t-test for mutants vs control, **P=0.008). (C) The total number of cells was counted during 7 days of stem cell culture (mutant 6510, s.d.=1250, n=3, Student's t-test for mutants vs control, *P=0.0457). (D) Self-renewal is not increased by Myc overexpression if Miz-1 binding is inhibited. Self-renewal was measured by the ability of single cells to form new neurospheres in EGF- and FGF-containing stem cell culture medium. (MycV394D 9.8%, s.d.=8.2, n=9; wt Myc 24.0%, s.d.=8.7, n=10; control 4.2%, s.d.=2.2, n=13; Student's t-test for mutants vs Myc **P=0.0021). (E) Overexpression of Myc does not induce maintenance of self-renewal if Miz-1 binding is inhibited, as measured by the ability of cells to form neurospheres in conditions (2% FCS) that promote differentiation in the re-sphering assay (mycV394D mutant 1-4 weeks 0%, s.d.=0, n=5; control 1-4 weeks 0%, s.d.=0, n=7; 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%, s.d.=1.3, n=7; Student's t-test for mutants vs Myc 2 weeks Student's t-test *P=0.028; 3 weeks *P=0.043; 4 weeks *P=0.020).
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