(A) 293T cells were transfected with a control vector (CONsh), an shRNA vector targeting Pcdh20 mRNA (PC20sh), or PC20sh_mut (which harbours point mutations in PC20sh) together with an HA-tagged Pcdh20 expression vector and a GFP expression vector. The cells were subjected to immunoblotting with antibodies to HA and GFP. (B) CONsh or PC20sh vector together with GFP vector was introduced on E14.0 cortices by in utero electroporation. Two days later, the cortices were removed, dissociated and cultured for 4 days in vitro. The GFP-positive cells were FACS sorted, and the amounts of Pcdh20 mRNA were then analyzed by RT-qPCR. The Pcdh20 levels were normalized by the expression of β-actin. Values are means ± SEM of three biological replicates. (C) CONsh or PC20sh vector together with GFP vector was introduced in dissociated E15.5 cortical cells by electroporation. Four days later, the cells were subjected to immunoblotting with antibodies to Pcdh20 and GFP. (D, E) CONsh, PC20sh, PC20sh_m, or PC20UTRsh vector together with a GFP vector was electroporated into the lateral ventricle of E14.0 embryos, then, the P7 brains were fixed and analyzed. The sections were counterstained with propidium iodide (PI, magenta). Most GFP-positive cells in the control experiment were located in L4, while the cells carrying the PC20sh or PC20UTRsh vector were located mainly in L2/3. Results of quantitative analyses of D are presented in E (n = 6 CONsh, n = 6 PC20sh, n = 5 PC20sh_mut, n = 5 PC20UTRsh). Details are described in Materials and Methods. (F) 293T cells were transfected with CONsh or PC20sh together with wild-type Pcdh20 (wtPcdh20) or a resistant form of Pcdh20 harboring mutations in the PC20sh-targeting site (resPcdh20). The cells were subjected to immunoblotting with antibodies to Pcdh20 and GFP. (G) PC20sh vector together with resPcdh20 was injected, and the brains were analyzed as in E. Results of quantitative analyses of Figure 2—figure supplement 1A are presented in G (n = 4 PC20sh+pCAGGS, n = 5 PC20sh+resPcdh20). (H–K) CONsh (H, J) or PC20sh (I, K) vector together with a GFP vector was electroporated into E14.0 brains, then, E18.0 (H, I) and P2 (J, K) brains were analyzed. The sections were counterstained with PI (magenta). The boxed regions are shown at higher magnification in the insets (J, K). Note that most GFP-positive cells with the PC20sh vector migrated normally, but were malpositioned in the P2 brains. (L, M) Quantitative data from E18.0 (H, I) (n = 4 CONsh, n = 4 PC20sh) and P2 (J, K) (n = 4 CONsh, n = 4 PC20sh) brains are presented. Scale bars, 200 µm (D, H, J).