Fig 2: DISC1 knockdown and rescue in striatal neurons.(A) Example immunoblot showing depletion of endogenous DISC1 detected in PC12 cells using the C-terminal antibody (see Materials and Methods ). The immunoreactive band corresponding to the major full length DISC1 species is shown. (B) Example of DISC1 depletion in striatal cultures, detected using the C-terminal (C-term) antibody and verified using the midi antibody, as indicated. (C) Quantification of knockdown and rescue phenotypes, based on count of cilia number compiled from 8 independent experiments. (D) Examples of the phenotypes observed in the indicated conditions (indicated above each column). The rescue condition (right column of images) shows the localization of DISC1-GFP rather than GFP. Insets indicate relevant regions of the cell body at higher magnification.

Fig 3: DISC1 knockdown reduces cilia number.(A) NIH3T3 cells were transfected with the indicated RNA duplexes and then stained with anti-acetylated tubulin (to mark primary cilia, left panels) and DAPI (to mark nuclei, middle panels). Merged images (right panels) show acetylated tubulin and DAPI staining in green and blue, respectively. Cilia were prominently observed in the majority of cells transfected with control (non-silencing) RNA duplexes (top row of images, an example is indicated by arrow). In cells transfected with either siRNA targeting DISC1, the vast majority of cells did not project a detectable primary cilium. Depleting IFT88 (bottom row of images) produced a similar effect. (B) Quantification of cilia loss by counting the number of cells extending a primary cilium, as marked by acetylated tubulin immunostaining, observed in blinded analysis of the indicated populations of siRNA-transfected cells. Bars represent the mean fraction of cells with a visible cilium, averaged across 5 experiments counting =250 cells/condition in each. Error bars represent the s.e.m. calculated across the experiments (***, p<0.0001).

Fig 4: Depletion of endogenous DISC1 and IFT88 by RNA interference.NIH3T3 cells were transfected with the indicated siRNA duplexes (sequences are listed in Materials and Methods). Total cell extracts were prepared 72 hours later, and levels of DISC1 (A) or IFT88 (B) protein were assessed by immunoblot using antibodies recognizing the endogenous proteins. Representative immunoblots are shown. Densitometric scanning across multiple experiments verified >80% depletion of both proteins by the respective siRNA duplexes.

Fig 5: PAK7 and DISC1 interact in mouse brain synapses. (A) Immunoprecipitation of PAK7 and DISC1 from synaptoneurosomal extracts of adult mouse brains. The extract was immunoprecipitated for PAK7 and the eluate was immunostained for DISC1. (B) Reciprocal experiment described in A: immunoprecipitation of DISC1 followed by immunostaining of the eluate with antiPAK7. In both A and B, the samples immunoprecipitated with control IgG showed no signal, showing the specificity of the PAK7–DISC1 interaction. An additional control in (C) shows that the brain extracts before immunoprecipitation have the same amount of loading control (ß-actin). (D–N) Immunostaining for PAK7 (green) and DISC1 (red) in brain sections containing Hippocampus and Cortex of P7 (D–I) and Adult (J–O) mice. The experiment shows an overlapping (yellow) of PAK7 and DISC1 in puncta, confirming a synaptic interaction between the two proteins. The co-localization is particularly evident in the adult (J–O) preparation.