Fig 2: 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 3: Localization of DISC1-GFP near the base of primary cilia.(A) NIH3T3 cells transfected with DISC1-GFP were fixed and immunolabeled for acetylated tubulin to mark primary cilia. An example of such a double-labeled cell is shown, with DISC1-GFP in green and acetylated tubulin in red in the merged image. Arrow indicates indicates the concentration of DISC1-GFP observed near the ciliary base. (B) The same experiment conducted using DISC1-HA. (C) Triple localization of DISC1-GFP, endogenous PCM1, and acetylated tubulin verifying localization of DISC1 in a centrosomal region near the ciliary base. (D) Merged image from the triple localization with DISC1-GFP in green, PCM1 in red, and acetylated tubulin in blue. (E) The region indicated in panel D displayed at higher magnification. Scale bar, 10 µm.

Fig 4: Novelty recognition of immune challenged mice with whole-brain and HP-confined DISC1 suppression. (A) Photograph of the arena used for NOR and RR tasks. (B) (i) Schematic diagrams of the protocol for NOR task. (ii) Violin plots displaying the relative interaction time spent by CON, GHPE, and GE mice with the objects during the NOR test trial. The dotted line indicates chance level. (iii) Violin plots displaying the NOR discrimination ratio when averaged for CON, GHPE and GE mice. (C) (i) Schematic diagrams of the protocol for RR task. (ii) Violin plots displaying the relative interaction time spent by CON, GHPE, and GE mice with the objects during the RR test trial. The dotted line indicates chance level. (iii) Violin plots displaying the RR discrimination ratio when averaged for CON, GHPE, and GE mice. (D) (i) Schematic diagrams of the protocol for OF task. (ii) Violin plots displaying the distance covered in 10 min by CON, GHPE, and GE mice during the OF task. (iii) Violin plots displaying the distance covered in the outer circle and the inner circle by CON, GHPE, and GE mice during OF task. (E) (i) The Pearson’s correlation between gPDC and discrimination ratio in NOR task. (ii) Same as (i) for Pearson’s correlation between gPDC and RR. Single data points are represented as dots and the red horizontal bars in violin plots represent the median and the 25th and 75th percentiles. *P < 0.05, ***P < 0.001.

Fig 5: 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.