Proliferation, adhesion to ECMs, and neurite outgrowth in neurosphere cultures.
(A) Proliferation in neurosphere cultures prepared from wild-type (wt, n = 3) and double knockout (DKO, n = 3) mice, from passage 1 to passage 3. (B–D) Cell number quantification in neurospheres prepared from wt (n = 3) and DKO (n = 3) mice that adhered to culture dishes coated with laminin (B), fibronectin (C), and collagen IV (D) at the concentrations indicated. (E) Neuronal cells, stained using anti-βIII-tubulin, differentiated from neurospheres from wt (upper) and DKO (lower) mice. Note that the neurite/axon length and number of branches were lower in DKO neuronal cells than in wt cells. (F) Distribution of the longest neurite length from an individual wt and DKO cell. (G) Mean length of the longest neurite of wt (n = 87) and DKO (n = 101) neuronal cells. (H) Mean of the total number of neuronal branching points per cell in wt (n = 23) and DKO (n = 20) neuronal cells. (I) Western blot analysis of neurospheres prepared from wt (n = 3) and DKO (n = 3) mice using anti-pTrkA (Y490ph), TrkA, pLyn (Y396ph), and Lyn antibodies. (J, K) The relative levels of pTrkA/TrkA (J) and pLyn/Lyn (K) of wt and DKO neurospheres are shown. The protein levels in wt neurospheres were normalized to 1. *, p<0.05; ***, p<0.001.