CHP1 downregulation improves neurite outgrowth in Smn-depleted NSC34 motor neuron-like cells and in zebrafish. (A) Western blot analysis of siRNA-mediated knockdown of Chp1 and Smn in NS34 cells. At 72 h post-transfection and retinoic acid-induced differentiation, CHP1 and SMN are significantly reduced (ACTB: loading control). *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-tailed Student’s t-test. Error bars represent SEM. (B) Representative images and quantification of neurite length of NSC34 cells, transfected with the respective siRNA and differentiated with retinoic acid for 72 h. The reduced neurite length in Smn-depleted NSC34 cells was restored to control level by Chp1 knockdown (N = 3, n = 110). F-actin was stained with phalloidin. Scale bar = 50 µm. *P < 0.05, ***P < 0.001, unpaired two-tailed Student’s t-test and Holm correction for multiple comparison. Error bars represent SD. (C) Representative images of motor axons posterior to the yolk globule of 34 hpf zebrafish embryos injected with the respective morpholinos (MOs) and labelled with α-Znp1 antibody. Zebrafish embryos were injected at the 1–4 cell stage with 2 ng chp1, smn morpholino, or both and further analysed 34 hpf. Motor axons of smn-depleted fish show severe truncations (absent or shortened axons) and branching phenotypes, which are ameliorated upon co-injection with chp1 morpholino. Scale bar = 50 µm. Solid arrowheads indicate truncated and absent axons, open arrowheads indicate terminal branching. (D) Western blotting demonstrates the morpholino-mediated knockdown of smnand chp1, respectively. Actb = loading control. (E) Quantitative analysis of caudal primary motor neurons shows that co-injection of smn and chp1 morpholinos ameliorates the frequencies of reduced motor axon length (absent or shortened axons) of smn morphants (n > 150 analysed motor axons). ***P < 0.001, Chi-square test. n.s. = not significant.