(A) Sample confocal images of immunostainining for GFP, phosphorylated AKT (pAKT), an immature neuronal marker DCX, and DAPI. Retroviruses co-expressing GFP and DISC1-shRNA, or a control-shRNA, were used to infect proliferating neural progenitors in the adult brain. GFP⁺ neurons were examined at 14 days post-viral injection. Scale bar: 25 μm.
(B) A summary of quantifications of pAKT levels in the cytosol of GFP⁺DCX⁺ immature neurons at different stages of development in the adult brain. The pAKT fluorescence intensity of individual GFP⁺DCX⁺ new neuron was first normalized to the GFP^-DCX⁺ immature new neurons in the same image. Values represent mean ± SEM (n = 10-33 cells from at least four animals; *: P < 0.01, ANOVA)
(C and D) Same as in (A and B), except that phosphorylated S6 (pS6) was examined.

(A) Western Blot analysis of AKT and S6 signaling. HEK293 cells transfected with expression vectors for HA-KIAA1212, myc-DISC1, or co-transfected with both, were serum starved overnight and then treated with insulin (100 nM) or saline for 15 mins. Cell lysates were subjected to Western Blot analysis for phosphorylated and total endogenous AKT, phosphorylated and total endogenous S6, myc-DISC1 (DISC1) and HA-KIAA1212 (KIAA), respectively. Shown are sample blots and summaries of quantifications of the levels of AKT and S6 phosphorylation. For each condition, a value was first obtained for the ratio of phosphorylated over total amount of AKT or S6, which was then normalized to those without overexpression and insulin treatment for each individual experiment. Values represent mean ± SEM (n = 5 experiments; *: P < 0.01, ANOVA).
(B) Inhibition of association between KIAA1212 and AKT by DISC1. HEK293 cells were transfected with expression constructs for HA-KIAA1212, or for both HA-KIAA1212 and myc-DISC1. Cultures were serum starved overnight and then treated with insulin (100 nM) or saline for 15 mins. Cell lysates were subjected to co-IP using antibodies against HA and followed by immunoblotting for AKT, or vice versa.
(C) Association between KIAA1212 and DISC1. Similar as (A), HEK293 cells were co-transfected with expression constructs for HA-KIAA1212 and DISC1. Cell lysates were subjected to co-IP using antibodies against HA and followed by immunoblotting for DISC1, or vice versa.
(D) Lack of strong association between DISC1 and AKT. Similar as in (A), HEK293 cells were transfected with expression constructs for DISC1, or for both DISC1 and KIAA1212. Cell lysates were subjected to co-IP using antibodies against AKT and followed by immunoblotting for DISC1, or vice versa.

(A) Sample projected confocal images of GFP⁺ new neurons in the adult brain. Same as in Figure 4, GFP⁺ neurons with retrovirus-mediated genetic manipulation were examined at 14 dpi. Scale bar: 50 μm.
(B) A summary of total dendritic length of GFP⁺ neurons at 14 dpi under different conditions. Shown is the cumulative plot with each symbol representing data from an individual GFP⁺ neuron from at least four animals for each condition. (*: P < 0.01, Kolmogorov-Smirnov test)
(C) Analysis of dendritic complexity of GFP⁺ neurons using the Sholl analysis. The same sets of GFP⁺ neurons as in (B) were used. Values represent mean ± SEM (*: P < 0.01, student t-test)

RTKs: receptor tyrosine kinases; GPCRs: G protein–coupled receptors; D2 receptor: Dopamine type 2 (D2) receptor.

(A and B) Association of DISC1 and KIAA1212 in hippocampal neurons. Lysates from primary hippocampal neurons in culture (A) and acutely dissected hippocampal tissues (B) were subjected to co-IP using antibodies against DISC1 or KIAA1212 (KIAA), or using control antibodies, followed by Western Blot analysis.
(C and D) Mapping of KIAA1212 domains involved in direct association with DISC1. Shown in (C) is a schematic diagram of different domains and regions of KIAA1212 used to generate GST-tagged recombinant fragments. Shown in (D) is a sample Western Blot of the in vitro pull-down of recombinant His-tagged DISC1 by different recombinant GST-KIAA1212 fragments.
(E) Direct association between DISC1 and the CT2 domain of KIAA1212. A fixed amount of 6xHis-DISC1 was mixed with various amount of GST-CT2. Amounts of bound and free DISC1 were quantified. The percentage of bound DISC1 was plotted against the concentration of CT2 and Kd value was calculated by the Scatchard analysis.

(A to C) Morphogenesis of new neurons in the adult brain. Engineered retroviruses were used for birth-dating, lineage tracing with GFP expression, and genetic manipulation with expression of the transgene or shRNA. Two different control viruses, control-shRNA and pCUXIE (vector for transgene expression), were used for comparison of expression of shRNAs and transgenes, respectively. GFP⁺ neurons were examined at 14 dpi. Shown in (A) are sample single section confocal images of GFP⁺ and DAPI under different manipulations. Scale bar: 25 μm. Also shown are summaries of the soma size (B) and number of primary dendrites (C) of GFP⁺ neurons under different conditions. Numbers associated with each bar graph indicate the number of neurons examined from at least four animals under each condition. Values represent mean ± SEM (*: P < 0.01, ANOVA).
(D and E) Positioning of newborn neurons in the dentate gyrus of the adult hippocampus. Shown in (D) is a schematic diagram of four areas defined for the dentate gyrus region. GL: granule cell layer; area 1: inner granule cell layer; area 2: middle granule cell layer; area 3: outer granule cell layer; ML: molecular layer (area 4). Shown in (E) is a summary of the percentile distribution of GFP⁺ neuron cell body within each domain as defined in (D). The same groups of GFP⁺ neurons as in (B) and (C) were used for quantifications.

(A) A schematic diagram of experimental design. Rapamycin (20 mg/kg body weight) or vehicle was i.p. injected.
(B) Quantification of pS6 levels in GFP⁺ neurons under different experimental conditions. Similar to Figure 1C, the pS6 fluorescence intensity of individual GFP⁺DCX⁺ new neuron was normalized to the GFP^-DCX⁺ immature new neurons in the same image. Values represent mean ± SEM (n = 42-49 cells from at least four animals; *: P < 0.01, ANOVA).
(C-G) Development of new neurons in the dentate gyrus of the adult hippocampus. GFP⁺ neurons were examined at 14 dpi. Shown are summaries of the soma size (C) and number of primary dendrites (D), neuronal positioning (E), total dendritic length (F) and Sholl analysis (G) of GFP⁺ neurons under different conditions. Numbers associated with bar graph (in C) indicate the number of neurons examined from at least four animals under each condition. Values represent mean ± SEM (*: P <0.01, ANOVA).