Figure 4.

Ack1 promotes differentiation in PC12 cells and this effect is enhanced by neurotrophins. (a) Phase-contrast morphology of PC12, PC12-pRK5 (empty vector), and PC12-Ack1 cells at 3 days of culture in NGF-free medium. Scale bar: 50 μm. (b) Western blot analysis of Ack1 (top) and tubulin (bottom) in non-transfected PC12 cells (wild type), PC12 cells transfected with an empty vector (PC12-pRK5), and Ack1-overexpressing cells (Ack1#1 and Ack1#2) (lanes 3 and 4). (c) Densitometric analysis of western blot shows an increase in the immunoreactivity of PC12-Ack1 cells to Ack1 antibody. (d) Effects of Ack1 overexpression on cellular differentiation. PC12 lines were plated and grown for the times indicated, and neurite outgrowth was analyzed. The number of differentiated cells is markedly increased upon Ack1 overexpression. Data are represented as mean±S.E.M. Each group is compared with its corresponding control at day 1 of treatment (***P<0.001). (e–h) Ack1 overexpression exerts a differentiation effect upon NGF treatments. PC12 lines were plated and grown in medium containing 50 ng/ml of NGF for the times indicated. The number of differentiated cells (f) and the total neuritic length (g) was strikingly increased upon Ack1 overexpression. Phase-contrast morphology of PC12, PC12-pRK5 (empty vector), and PC12-Ack1 cells at several days of culture is shown in (e). Scale bar: 50 μm. At day 10 in NGF medium, control-PC12 and PC12-Ack1 cells had distinct morphologies, as illustrated in phase-contrast micrographs (e). Ack1 overexpression induced a prominent increase in the number of branching points (h) and the neurites were significantly shorter. Data are represented as mean±S.E.M. (**P<0.05; ***P<0.001). Each group was compared with its corresponding control at day 1. In (f–h), PC12-Ack1 and PC12-pRK5 (empty vector) are compared with PC12 wild-type cells. (j) Western blot analysis showing that Ack1 RNAi (shRNA) reduces the expression of endogenous Ack1. The expression of Ack1 was normalized by the analysis of actin expression (bottom panel). (k) Densitometric analysis of western blots reveal a reduction of 74% in one case and 67% in a second case in the immunoreactivity to Ack1 antibody in the PC12-shRNA cells compared with scrambled-RNAi ones. (i, l, and m) Depletion of endogenous Ack1 partially counteracted NGF-induced differentiation. (i) Phase-contrast micrographs of PC12 cells stably transfected with scrambled RNAi, or two shRNAs against Ack1 illustrate the morphology of these lines after culture in NGF for 5 days in comparison with wild-type PC12 cells. Knock-down of endogenous Ack1 inhibited the neuritic differentiation phenotype and neuritic arborization (l) promoted by NGF treatment for different periods. (m) Quantification of the number of branching points of PC12, PC12-scrambled RNAi, and PC12-RNAi lines submitted to NGF treatment for 10 days. PC12-RNAi1 and PC12-RNAi-2 cells were compared with PC12-scrambled using the T-test (*P<0.01; **P<0.05). (**P<0.05; ***P<0.001). Scale bar: 50 μm