We further tested

these DISC1 variants for their ability to rescue DISC1 shRNA-mediated inhibition of TCF/LEF reporter activity in mouse-derived P19 carcinoma selleckchem cell lines. We first tested the level of knockdown of DISC1 mRNA transcript to confirm that transfection of multiple plasmids into N2A cells does indeed result in DISC1 downregulation (Figure S1C). In this assay, WT human DISC1 completely rescues the reduction in TCF/LEF reporter activity after DISC1 knockdown (Figure 1B, data not shown). In line with the results from the previous assay, we found that all of the rare variants, including A83V, and the common R264Q and L607F DISC1 variants could not rescue this reduction. However, the S704C variant PI3K inhibitor completely rescued DISC1 shRNA-mediated reduction in TCF/LEF luciferase reporter activity. These results were not due to differences in the expression levels of the hDISC1 variants (Figure S1B). Taken together, these experiments demonstrate DISC1 variants significantly inhibit Wnt signaling in vitro. Wnt signaling is a well-known regulator of cell proliferation, particularly in neural progenitor cells

(Shimizu et al., 2008 and Wexler et al., 2007). Given that our results implicate DISC1 as a modulator of Wnt signaling, we tested the impact of the human DISC1 variants on the proliferation of N2A cells in vitro. Cells were transfected with the different DISC1 variant constructs and were then stained 48 hr later with phosphohistone-3 (PH3), which labels the nuclei of dividing cells. Using this assay, we found that overexpression of WT-DISC1 resulted in an almost 3-fold increase in the number PH3-positive cells (Figure 1C). When comparing the other DISC1 variants, we observed similar trends as tuclazepam the TCF/LEF reporter assays. The A83V, R264Q, and L607F variants did not significantly increase PH3 staining in transfected cells, while the S704C significantly

increased PH3 staining similar to WT-DISC1 (Figure 1C). We next directly tested the impact of hDISC1 variants on the number of proliferating cells. N2A cells were plated vitro, transfected with GFP, WT-DISC1, or the different DISC1 variants, stimulated with Wnt3a and the number of cells were then counted after a 24 hr period. Here, we found that WT-DISC1 expression caused a significant increase in cell number compared with GFP controls (Figure 1D). However, the A83V, R264Q and L607F variants did not significantly increase cell number, while the S704C variant increased the cell number similar to WT-DISC1 (Figure 1D). We then asked whether these effects required downstream activation of Wnt signaling. Using a similar experimental paradigm, N2A cells were cotransfected in the same manner but also with dominant-negative LEF and then stimulated with Wnt3a.